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UNIVERSITY - MADHYA PRADESH BHOJ OPEN UNIVERSITY BHOPAL (M.P.) PROGRAMME - M.Sc.Chemistry (Previous) PAPER - v (A-II) TITLE OF PAPER - BIOLOGY FOR CHEMIST BKOCK NO . - I UNIT WRITER - UNIT - I Smt. Shikha Mandloi Asst. Prof. Microbiology Sri Sathya Sai College for Women EDITOR - Dr.(Smt.) Renu Mishra, HOD, Botany & Microbiology, Sri Sathya Sai College for Women, Bhopal COORDINATION COMMITTEE - Dr. Abha Swarup, Director, Printing & Translation Major PradeepKhare, Consultant, Printing & Translation POST GRADUATE PROGRAMME M.Sc.CHEMISTRY (PREVIOUS) DISTANCE EDUCATION SELF INSTRUCTIONAL MATERIAL Paper-V(A-II) BIOLOGY FOR CHEMISTS BLOCK :I UNIT—I : CELL STRUCTURE AND FUNCTION MADHYA PRADESH BHOJ OPEN UNIVERSITY BHOPAL (M.P.) 1 Cell structure and function Introduction The cell is the basic unit of life. There are millions of different types of cells. There are cells that are organisms onto themselves, such as microscopic amoeba and bacteria cells. And there are cells that only function when part of a larger organism, such as the cells that make up your body. The cell is the smallest unit of life in our bodies. In the body, there are brain cells, skin cells, liver cells, stomach cells, and the list goes on. All of these cells have unique functions and features. And all have some recognizable similarities. All cells have an outer covering called the plasma membrane, protecting it from the outside environment. The cell membrane regulates the movement of water, nutrients and wastes into and out of the cell. Inside of the cell membrane are the working parts of the cell. At the center of the cell is the cell nucleus. The cell nucleus contains the cell's DNA, the genetic code that coordinates protein synthesis. In addition to the nucleus, there are many organelles inside of the cell - small structures that help carry out the day-to-day operations of the cell. One important cellular organelle is the ribosome. Ribosomes participate in protein synthesis. The transcription phase of protein synthesis takes places in the cell nucleus. After this step is complete, the mRNA leaves the nucleus and travels to the cell's ribosomes, where translation occurs. Another important cellular organelle is the mitochondrion. Mitochondria (many mitochondrion) are often referred to as the power plants of the cell because many of the reactions that produce energy take place in mitochondria. Also important in the life of a cell are the lysosomes. Lysosomes are organelles that contain enzymes that aid in the digestion of nutrient molecules and other materials There are many different types of cells. One major difference in cells occurs between plant cells and animal cells. While both plant and animal cells contain the structures discussed above, plant cells have some additional specialized structures. Many animals have skeletons to give their body structure and support. Plants have a unique cellular structure called the cell wall. The cell wall is a rigid structure outside of the cell membrane composed mainly of the polysaccharide cellulose. The cell wall gives the plant cell a defined shape which helps support individual parts of plants. In addition to the cell wall, plant cells contain an organelle called the chloroplast. The chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the chloroplast (including the common green pigment chlorophyll) absorb sunlight and use this energy to complete the chemical reaction. 2 UNIT-I Cell structure and function 1.0 Introduction 1.1 Objectives 1.2 Structure of prokaryotic and eukaryotic cells 1.3 Intracellular organelles and their functions 1.4 Comparision of plant and animal cells 1.5 Over view of metabolism: catabolism and anabolism 1.6 ATP- the biological energy currency 1.7 Origin of life;-unique properties of carbon, chemical evolution and rise of living systems 1.8 Introduction to biomolecules 1.9 Building blocks of biomacromolecules 2.0 Let us sum up 2.1Check your progress The key 2.2 Assignment/ Activity 2.3 References 3 1.0 Introduction The cell is the basic unit of life. There are millions of different types of cells. There are cells that are organisms onto themselves, such as microscopic amoeba and bacteria cells. And there are cells that only function when part of a larger organism, such as the cells that make up your body. The cell is the smallest unit of life in our bodies. In the body, there are brain cells, skin cells, liver cells, stomach cells, and the list goes on. All of these cells have unique functions and features. And all have some recognizable similarities. All cells have an outer covering called the plasma membrane, protecting it from the outside environment. The cell membrane regulates the movement of water, nutrients and wastes into and out of the cell. Inside of the cell membrane are the working parts of the cell. At the center of the cell is the cell nucleus. The cell nucleus contains the cell's DNA, the genetic code that coordinates protein synthesis. In addition to the nucleus, there are many organelles inside of the cell - small structures that help carry out the day-to-day operations of the cell. One important cellular organelle is the ribosome. Ribosomes participate in protein synthesis. The transcription phase of protein synthesis takes places in the cell nucleus. After this step is complete, the mRNA leaves the nucleus and travels to the cell's ribosomes, where translation occurs. Another important cellular organelle is the mitochondrion. Mitochondria (many mitochondrion) are often referred to as the power plants of the cell because many of the reactions that produce energy take place in mitochondria. Also important in the life of a cell are the lysosomes. Lysosomes are organelles that contain enzymes that aid in the digestion of nutrient molecules and other materials. Below is a labelled diagram of a cell to help you identify some these structures. 4 There are many different types of cells. One major difference in cells occurs between plant cells and animal cells. While both plant and animal cells contain the structures discussed above, plant cells have some additional specialized structures. Many animals have skeletons to give their body structure and support. Plants have a unique cellular structure called the cell wall. The cell wall is a rigid structure outside of the cell membrane composed mainly of the polysaccharide cellulose. The cell wall gives the plant cell a defined shape which helps support individual parts of plants. In addition to the cell wall, plant cells contain an organelle called the chloroplast. The chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the 5 chloroplast (including the common green pigment chlorophyll) absorb sunlight and use this energy to complete the chemical reaction: 6 CO2 + 6 H2O + energy (from sunlight) C6H12O6 + 6 O2 In this way, plant cells manufacture glucose and other carbohydrates that they can store for later use. 1.1 Objectives 1. This unit will fulfill the basic introduction , function, origin and chemical composition of some organelles. 2. Students will find the text useful as it will help in understanding some molecular diagnostic technique. 1.3 Structure of prokaryotic and eucaryotic cell Organisms contain many different types of cells that perform many different functions There are two types of cells: eukaryotic and prokaryotic. Prokaryotic cells are usually independent, while eukaryotic cells are often found in multicellular organisms. Prokaryotic cells The prokaryote cell is simpler, and therefore smaller, than a eukaryote cell, lacking a nucleus and most of the other organelles of eukaryotes. There are two kinds of prokaryotes: bacteria and archaea; these share a similar structure. 6 Nuclear material of prokaryotic cell consist of a single chromosome which is in direct contact with cytoplasm. Here the undefined nuclear region in the cytoplasm is called nucleoid. A prokaryotic cell has three architectural regions: On the outside, flagella and pili project from the cell's surface. These are structures (not present in all prokaryotes) made of proteins that facilitate movement and communication between cells; Enclosing the cell is the cell envelope – generally consisting of a cell wall covering a plasma membrane though some bacteria also have a further covering layer called a capsule. The envelope gives rigidity to the cell and separates the interior of the cell from its environment, serving as a protective filter. Though most prokaryotes have a cell wall, there are exceptions such as Mycoplasma (bacteria) and Thermoplasma (archaea). The cell wall consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell from expanding and finally bursting (cytolysis) from osmotic pressure against a hypotonic environment. Some eukaryote cells (plant cells and fungi cells) also have a cell wall; Inside the cell is the cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. A prokaryotic chromosome is usually a circular molecule (an exception is that of the bacterium Borrelia burgdorferi, which causes Lyme disease). Though not forming a nucleus, the DNA is condensed in a nucleoid. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular. Plasmids enable additional functions, such as antibiotic resistance. 7 Structures outside the cell wall Capsule A gelatinous capsule is present in some bacteria outside the cell wall. The capsule may be polysaccharide as in pneumococci, meningococci or polypeptide as Bacillus anthracis or hyaluronic acid as in streptococci Capsules are not marked by ordinary stain and can be detected by special stain. The capsule is antigenic. The capsule has antiphagocytic function so it determines the virulence of many bacteria. It also plays a role in attachment of the organism to mucous membranes. Flagella Flagella are the organelles of cellular mobility. They arise from cytoplasm and extrude through the cell wall. They are long and thick thread-like appendages, protein in nature. Are most commonly found in bacteria cells but are found in animal cells as well. Fimbriae (pili) They are short and thin hair like filaments, formed of protein called pilin (antigenic). Fimbriae are responsible for attachment of bacteria to specific receptors of human cell (adherence). There are special types of pili called (sex pili) involved in conjunction. 8 BACTERIAL CELL:-AN EAXMPLE OF PROCARYOTIC CELL Eukaryotic cells Eukaryotic cells are about 15 times wider than a typical prokaryote and can be as much as 1000 times greater in volume. The major difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is a cell nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA. 9 This nucleus gives the eukaryote its name, which means "true nucleus." Other differences include: The plasma membrane resembles that of prokaryotes in function, with minor differences in the setup. Cell walls may or may not be present. The eukaryotic DNA is organized in one or more linear molecules, called chromosomes, which are associated with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a membrane. Some eukaryotic organelles such as mitochondria also contain some DNA. Many eukaryotic cells are ciliated with primary cilia. Primary cilia play important roles in chemosensation, mechanosensation, and thermosensation. Cilia may thus be "viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation."[7] Eukaryotes can move using motile cilia or flagella. The flagella are more complex than those of prokaryotes. Table 1: Comparison of features of prokaryotic and eukaryotic cells Typical organisms Typical size Type of nucleus DNA Prokaryotes Eukaryotes bacteria, archaea protists, fungi, plants, animals ~ 1–10 µm nucleoid region; no real nucleus circular (usually) ~ 10–100 µm (sperm cells, apart from the tail, are smaller) real nucleus with double membrane linear molecules (chromosomes) with histone 10 proteins RNA-/protein- coupled synthesis cytoplasm protein synthesis in cytoplasm Ribosomes 50S+30S 60S+40S Cytoplasmatic structure Cell movement in RNA-synthesis very few structures flagella made flagellin nucleus highly structured by endomembranes and a cytoskeleton lamellipodia and filopodia containing actin one to several thousand (though some lack none Chloroplasts none Organization usually single cells Binary the of flagella and cilia containing microtubules; Mitochondria Cell division inside mitochondria) in algae and plants single cells, colonies, higher multicellular organisms with specialized cells fission Mitosis (simple division) (fission or budding) Meiosis The cells of eukaryotes and prokaryotes . All cells, whether prokaryotic or eukaryotic, have a membrane that envelops the cell, separates its interior from its environment, regulates what moves in and out (selectively permeable), and maintains the electric potential of the cell. Inside the membrane, a salty cytoplasm takes up most of the cell volume. All cells possess DNA, the hereditary material of genes, and RNA, containing the information necessary to build various proteins such as enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells. This article will list these primary components of the cell, then briefly describe their function. 11 Cell membrane The cytoplasm of a cell is surrounded by a cell membrane or plasma membrane. The plasma membrane in plants and prokaryotes is usually covered by a cell wall. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of lipids (hydrophobic fat-like molecules) and hydrophilic phosphorus molecules. Hence, the layer is called a phospholipid bilayer. It may also be called a fluid mosaic membrane. Embedded within this membrane is a variety of protein molecules that act as channels and pumps that move different molecules into and out of the cell. The membrane is said to be 'semi-permeable', in that it can either let a substance (molecule or ion) pass through freely, pass through to a limited extent or not pass through at all. Cell surface membranes also contain receptor proteins that allow cells to detect external signaling molecules such as hormones.. The cytoskeleton acts to organize and maintain the cell's shape; anchors organelles in place; helps during endocytosis, the uptake of external materials by a cell, and cytokinesis, the separation of daughter cells after cell division; and moves parts of the cell in processes of growth and mobility. The eukaryotic cytoskeleton is composed of microfilaments, intermediate filaments and microtubules. There is a great number of proteins associated with them, each controlling a cell's structure by directing, bundling, and aligning filaments. The prokaryotic cytoskeleton is less well-studied but is involved in the maintenance of cell shape, polarity and cytokinesis. Genetic material 12 Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms use DNA for their long-term information storage, but some viruses (e.g., retroviruses) have RNA as their genetic material. The biological information contained in an organism is encoded in its DNA or RNA sequence. RNA is also used for information transport (e.g., mRNA) and enzymatic functions (e.g., ribosomal RNA) in organisms that use DNA for the genetic code itself. Transfer RNA (tRNA) molecules are used to add amino acids during protein translation. Prokaryotic genetic material is organized in a simple circular DNA molecule (the bacterial chromosome) in the nucleoid region of the cytoplasm. Eukaryotic genetic material is divided into different, linear molecules called chromosomes inside a discrete nucleus, usually with additional genetic material in some organelles like mitochondria and chloroplasts. A human cell has genetic material contained in the cell nucleus (the nuclear genome) and in the mitochondria (the mitochondrial genome). In humans the nuclear genome is divided into 23 pairs of linear DNA molecules called chromosomes. The mitochondrial genome is a circular DNA molecule distinct from the nuclear DNA. Although the mitochondrial DNA is very small compared to nuclear chromosomes, it codes for 13 proteins involved in mitochondrial energy production and specific tRNAs. Foreign genetic material (most commonly DNA) can also be artificially introduced into the cell by a process called transfection. This can be transient, if the DNA is not inserted into the cell's genome, or stable, if it is. Certain viruses also insert their genetic material into the genome. . 13 1.3 INTRACELLULAR ORGANELLES AND THEIR FUNCTIONS The plasma membrane is followed by the cytoplasm which is distinguished in to A. cytosol and B cytoplasmic structures. A The cytosol is the gelatinous fluid that fills the cell and surrounds the organelles. B cytoplasmic structures:- In the cytoplasmic matrix certain living and non living structures remain suspended.The non living structures are called inclusions while living structures are called organelles. The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs," called organelles, that are specialized for carrying out one or more vital functions. There are several types of organelles in a cell. Some (such as the nucleus and golgi apparatus) are typically solitary, while others (such as mitochondria, peroxisomes and lysosomes) can be numerous (hundreds to thousands). Golgi Apparatus An Italian neurologist (i.e., physician) Camillo Golgi in 1873 discovered, them which is commonly known as the Golgi bodies. For the performance of certain important cellular functions such as biosynthesis of polysaccharides, packaging (compartmentalizing) of cellular synthetic products (proteins), production of exocytotic (secretory) vesicles and differentiation of cellular membranes, there occurs a complex organelle called Golgi complex or Golgi apparatus in the in the cytoplasm of animal and plant cells. The Golgi apparatus, like the endoplasmic reticulum, is a canalicular system with sacs, but unlike the endoplasmic reticulum it 14 has parallely arranged, flattened, membrane-bounded vesicles which lack ribosomes and stainable by osmium tetraoxide and silver salts. Occurence The Golgi apparatus occurs in all cells except the prokaryotic cells (viz., mycoplasmas, bacteria and blue green algae) and eukaryotic cells of certain fungi, sperm cells of bryophytes and pteridiophytes, cells of mature sieve tubes of plants and mature sperm and red blood cells of animals. Their number per plant cell can vary from several hundred as in tissues of corn root and algal rhizoids (i.e., more than 25,000 in algal rhizoids, Sievers, 1965), to a single organelle in some algae. (Certain algal cells such as Pinularia and Microsterias, contain largest and most complicated Golgi apparatuses. In higher plants, Golgi apparatuses are particularly common in secretory cells and in young rapidly growing cells. In animal cells, there usually occurs a single Golgi apparatus, however, its number may vary from animal to animal and from cell to cell. Thus Paramoeba species has two Golgi apparatus and nerve cells, liver cells and chordate oocytes have multiple Golgi apparatuses, there being about 50 of them in the liver cells. Distribution In the cells of higher plants, the Golgi bodies or dictyosomes are usually found scattered throughout the cytoplasm and their distribution does not seem to be ordered or localized in any particular manner (Hall et al., 1974). However, in animal cells the Golgi apparatus is a localized organelle. For example, in the cells of ectodermal or endodermal origin, the Golgi apparatus remains polar and occurs in between the nucleus and the periphery (e.g., thyroid cells, exocrine pancreatic cells and mucusproducing goblet cells of intestinal epithelium) and in the nerve cells it occupies a circum-nuclear position. Structure 15 The Golgi apparatus is morphologically very similar in both plant and animal cells. However, it is extremely pleomorphic: in some cell types it appears compact and limited, in others spread out and reticular (net-like). Its shape and form may very depending on cell type. Typically, however, Golgi apparatus appears as a complex array of interconnecting tubules, vesicles and cisternae.The simplest unit of the Golgi apparatus is the cisterna. This is a membrane bound space in which various materials and secretions may accumulate. Numerous cisternae are associated with each other and appear in a stack like (lamellar) aggregation. A group of these cisternae is called the dictyosome, and a group of dictyosomes makes up the cells Golgi apparatus. All dictyosomes of a cell have a common function. 1. Flattened Sac or Cisternae Cisternae (aboute 1m in diameter) are central, flattened, plate-like or saucerlike closed compartments which are help in parallel bundles or stacks on above the other. In each stack, cisternae, are separated by a space of 20 to 30 nm which may contain rod-like elements or fibres. Each stack of cisternae forms a dictyosome which may contain 5 to 6 Golgi cisternae in animal cells or 20 or more cisternae in plant cells. Each cisterna is bounded by a smooth unit membrane (7.5 nm thick), having a lumen varying in width from about 500 to 1000 nm The margins of each cisterna are gently curved so that the entire dictyosome of Golgi apparatus takes on a bow like appearance. The cisternae at the convex end of the dictyosome comprise proximal forming or cis-face and the cisternae at the concave end of the dictyosome comprise the distal, maturing or trans-face. The forming or cis-face of Golgi is located next to either the nucleus or a specialized portion of rough ER that lacks bound ribosomes and is called ''transitional'' ER. Trans face of Golgi is located near 16 the plasma membrane. This polarization is called cis-trans axis of the Golgi apparatus. 2. Tubules A complex array of associated vesicles and anastomosing tubules (30 to 50nm diameter) surround the dictyosome and radiate from it. In fact, the peripheral area of dictyosome is fenestrated (lace-like) in structure. 3.Vesicles The vesicles (60 nm in diameter) are of three types : (i) Transitional vesicles are small membrane limited vesicles which are through to form as blebs from the transitional ER to migrate and converge to cis face of Golgi, where they coalesce to form new cisternae. (ii) Secretory vesicles are varied-sized membrane-limited vesicles which discharge from margins of cisternae of Golgi. They, often, occur between the maturing face of Golgi and the plasma membrane. (iii) Clathrin-coated vesicles are spherical protuberances, about 50m in diameter and with a rough surface. They are found at the periphery of the organelle, usually at the ends of single tubules, and are morphologically quite distinct from the secretory vesicles. The clathrincoated vesicles are known to play a role in intra-cellular traffic or membranes and of secretory products. i.e., between ER and Golgi, as well as between GELR region and the endosomal and lysosomal compartments. Function Golgi vesicles are often, referred to as the ''traffic police" of the cell (Dernell et al., 1986. They play a key role in sorting many of cell's proteins and membrane constituents, and in directing them to their proper destinations. To perform this 17 function, the Golgi vesicles contain different sets of enzymes in different types of vesicles-cis, middle and trans cisternae- that react with and modify secretory proteins passing through the Golgi lumen or membrane proteins and glycoprotein's in the Golgi membranes as they are on route to their final destinations. For example a Golgi enzyme may add a "signal" or "tag" such as a carbohydrate or phosphate residues to certain proteins to direct them to their proper sites in the cell. Or, a proteolytic Golgi enzyme may cut a secretory or membrane protein into two or more specific segments (E.g., molecular processing involved in the formation of pancreatic hormone insulin: preproinsulinproinsulininsulin). Recently, in the function of Golgi apparatus, sub compartmentalization with a division of labour has been proposed between the cis region (in which proteins of RER are sorted and some of them are returned back possibly by coated vesicles), and the trans region in which the most refined proteins are further separated for their delivery to the various cell compartments (e.g., plasma membrane, secretory granules and lysosomes). Thus, Golgi apparatus is a centre of reception, finishing, packaging, and dispatch for a variety of materials in animal and plant cells : 1. Golgi Functions in Plants 18 In plants, Golgi apparatus is mainly involved in the secretion of mainly involved in the secretion of materials of primary and secondary cell walls (e.g., formation and export of glycoprotein, lipids, pectins and monomers for hemicellulose, cellulose, lignin, etc). During cytokinesis of mitosis or meiosis, the vesicles originating from the periphery of Golgi apparatus, coalesce in the phragmoplast area to form a semisolid layer, called cell plate. The unit membrane of Golgi vesicles fus During early electron microscopic studies, rounded dense bodies were observed in rat liver cells. These bodies were initially described as "perinulclear dense bodies", C. de Duve, in 1955, renamed these organelles as "lysosomes" to indicate that the internal digestive enzymes only became apparent when the membrane of these organelles was lysed (See Reid and Leech, 1980). However, the term lysosome means lytic body having digestive enzymes capable of lysis (viz., dissolution of a cell or tissue es during cell plate formation and becomes part of plasma membrane of daughter cells . Lysosomes The lysosomes (Gr. lyso=digestive + soma = bodies) are tiny membrane-bound vesicles involved in intracellular digestion. They contain a variety of hydrolytic enzymes that remain active under acidic conditions. The lysosomal lumen is maintained at an acidic pH (around 5) by an ATP-driven proton pump in the membrane. Thus, these remarkable organelles are primarily meant for the digestion of a variety of biological materials and secondarily cause aging and death of animal cells and also a variety of human diseases such as cancer, gout Pompe's silicosis and I-cell disease. 19 Occurence The lysosomes occur in most animal and few plant cells. They are absent in bacteria and mature mammalian ertythrocytes. Few lysosomes occur in muscle cells or in cells of the pancreas. Leucocytes, especially granulocytes are a particularly rich source of lysosomes. Their lysosomes are so large sized that they can be observed under the light microscope. Lysosomes are also numerous in epithelial cells of absorptive, secretory and excretory organs (e.g., intestine, liver, kidney, etc.) They occur in abundance in the epithelial cells of lungs and uterus. Lastly phagocytic cells and cells of reticuloendothelial system (e.g., bone marrow, spleen and liver) are also rich in lysosomes. Structure The lysosomes are round vacuolar structure which remain filled with dense material and are bounded by single unit membrane. Their shape and density vary greatly. Lysosmes are 0.2 to 0.5m in size. Since, size and shape of lysosomes vary from cell to cell and time to time (i.e. they are polymorphic), their identification becomes difficult. However, on the basis of the following three criteria, a cellular entity can be identified as a lysosome : (1) It should be bound by a limiting membrane (2) It should contain two or more acid hydrolases ; and (3) It should demonstrate the property of enzyme latency when treated in away that adversely affects organelle's membrane structure. Lysosomes are very delicate and fragile organelles. Lysosomal fraction have been isolated by sucrose-density centrifugation (or Isopycnic centrifugation) after mild methods of homogenization. Lysosomes tend to accumulate certain dyes (vital stains such as Neutral red, Niagara, Evans blue) and drugs such as anti-malarial drug chloroquine. Such 'loaded' 20 lysosomes can be demonstreated by fluorescence microscopy. The location of the lysosomes in the cell can also be pinpointed by various histochemical or cytochemical methods. Certain lysosomal enzymes are good histochemical markers. For example, acid phosphatase is the principal enzyme which is used as a marker for the lysosomes by the use of Gomori staining technique (Gomori, 1952). Specific stains are also used for other lysosomal enzymes such as B-glucuronidase, aryl sulphatatase, N-acetyl-B-glucosaminidase and 5-bromo-4-chloroindolacetate esterase. Lysosomal Enzymes According to a recent estimate, a lysosome may contain up to 40 types of hydrolytic enzymes (see Alberts et al., 1989). they include proteases (e.g., cathespsin for protein digestion), nucleases, glycosidases (for digestion of polysaccharides and glycosides ), lipase, phospholipases, phosphatases and sulphatases (table 8-2). All lysosomal enzymes are acid hydrolases, optimally active at the pH5 maintained within lysosomes. The lysosomal enzymes latent and out of the cytoplasmic matrix or cytosol (whose pH is about ~ 7.2), but the acid dependency of lysosmal enzymes protects the contents of the cytosol (cytoplasmic matrix) against any damage even if leakage of lysosomal enzymes should occur. The so-called latency of the lysosomal enzymes is due to the presence of the membrane which is resistant to the enzymes that it encloses. Most probably this is to the face that most lysosomal hydrolases are membrane-bound, which may prevalent the active centre of enzymes to gain access to susceptible groups in the membrane ( Reid and Leech, 1980). 21 Kind of Lysosomes Lysosomes are extremely dynamic organelles, exhibiting polymorphism in their morphology. Following four types of lysosomes have been recognized in different types of cells or at different time in the same cell. Of these, only the first is the primary lysosome, the other three have been grouped together as secondary lysosomes. 1. Primary Lysosomes These are also called storage granules, protolysosomes or virgin lysosomes. Primary lysosomes are newly formed organelles bounded by a single membrane and typically having a diameter of 100nm. They contain the degradative enzymes which have not participated in any digestive process. Each primary lysosome contains one type of enzyme or another and it is only in the secondary lysosome that the full complement of acid hydrolases is present. 2. Heterophagosomes They are also called heterophagic vacuoles, heterolysosomes or phagolysosomes. Heterophagosomes are formed by the fusion of primary lysosomes with cytoplasmic vacuoles containing extracellular particles into the cell by any of a variety of endocytic processes (e.g., pinocytosis, phagocytosis or receptor mediated endocytosis. The digestion of engulfed substances takes place by the enzymatic activities of the hydrolytic enzymes of the secondary lysosomes. The digested material has low molecular weight and readily passes through the membrane of the lysosomes to become the part of the matrix. 22 3. Autophagosomes They are also called autophagic vacuole, cytolysosomes or autolysosomes. Primary lysosomes are able to digest intracellular structures including mitochondria, ribosomes, peroxisomes and glycogen granules. Such autodigestion (called autophagy) of cellular organelles is a normal event during cell growth and repair and is especially prevalent in differentiating and de-differentiating tissues (e.g., cells undergoing programmed death during meta-morphosis or regeneration) and tissue under stress. Autophagy takes several forms. In some cases the lysosome appears to flow around the cell structure and fuse, enclosing it in a double membrane sac, the lysosomal enzymes being initially confined between the membrane. The inner membrane then breaks down and the enzymes are able to penetrate to the enclosed organelle. In other cases, the organelle to be digested is first encased by smooth ER, forming a vesicle that fuses with a primary lysosome. Lysosomes also regularly engulf bits of cytosol (cytoplasmic matrix) which is degraded by a process, called microautophagy. As digestion proceeds, it becomes increasingly difficult to identify the nature of the original secondary lysosome (i.e., heterophagosome or autophagosome) and the more general term digestive vacuole is used to describe the organelle at this stage. 4. Residual Bodies They are also called telolysosomes or dense bodies. Residual bodies are forced if the digestion inside the food vacuole is incomplete. Incomplete digestion may be due to absence of some lysosomal enzymes. The undigested food is 23 present in the digestive vacuole as the residues and may take the form of whorls of membranes. grains, amorphouse masses, ferritin-like or myelin figures. Residual bodies are large, irregular in shape and are usually quite electrondense. In some cells, such as Amoeba and other potozoa, these residual bodies are eliminated defecation. In other cells, residual bodies may remain for a long time and may load the cells to result in their aging. For example, pigment inclusions (age pigment or lipofuscin granules) found in nerve cells (also in liver cells, heart cells and muscle cells) of old animals may be due to the accumulation of residual bodies. Origin The biogenesis (origin) of the lysosomes requires the synthesis of specialized lysosomal hydrolases and membrane proteins. Both classes of proteins are synthesized in the ER and transported through the Golgi apparatus, then transported from the trans Golgi network to an intermediate compartment (an endolysosome) by means of transport vesicles (which are coated by clathrin protein). The lysosmal enzymes are glycol proteins, containing N-linked oligosaccharides that are processed in a unique way in the cis Golgi so that their mannose residues are phophorylated. These transport vesicles containing the M6P-receptors act as shuttles that move the receptors back and forth between the Golgi network and endolysosomes. They low pH in the endolysosome dissociates the lysosomal hydrolases from this receptor, making the transport of the hydrolases unidirectional. Function of Lysosomes The important functions of lysosomes are as follow : 24 1. Digestion of large extracellular particles. The lysosomes digests the food contents of the phoagosomes or pinosomes. 2. Digestion of intracellular substance. During the starvation, the lysosomes digest the stored food contents viz proteins, lipids and carbohydrates (glycogen) of the cytoplasm and supply to the cell necessary amount of energy. 3. Autolysis. In certain pathological conditions the lysosomes start to digest the various organelles of the cells and this process is known as autolysis or cellular autophagy. When a cell dies, the lysosome membrane ruptures and enzymes are liberated. These enzymes digest the dead cells. In the process of metamorphosis of amphibians and / tunicates many embryonic tissues, e.g., gills, fins, tail, etc., are digested by the lysosomes and utilized by the other cells. 4. Extracellular Digestion. The lysosomes of certain cells such as sperms discharge their enzymes outside the cell during the process of fertilization. The lysosomal enzymes digest the limiting membrane of the ovum and form penetration path in ovum for the sperms. Acid hydrolases are released from osteoclasts and break down bone for the reabsorption ; these cells also secrete lactic acid which makes the local pH enough for optimal enzyme activity. Likewise, preceding ossification (bone formation), fibroblasts release cathepsin D enzyme to break down the connective tissue. 25 Lysosomes in plants Plants contain several hydrolases, but they are not always as neatly compartmentalized as they are in animal cells. Many of these hydrolases are found bound to and functioning within the vicinity of the cells wall and are not necessarily contained in membrane bound vacuoles at these sites. Many types of vacuoles and storage granules of plants are found to contain certain digestive enzymes and these granules are considered as lysosomes of plant cell (Gahan, 1972). According to Matile (1969) the plant lysosomes can be defined as membrane bound cell compartments containing hydrolytic digestive enzymes. Matile (1975) has divided vacuoles of plants into following three types : 1. Vacuoles The vacuole of a mature plant cell is formed from the enlargement and fusion of smaller vacuoles present in meristematic cells; these provacuolse, which are 26 believed to be derived from the ER and possibly the Golgi and contain acid hydrolases. These lysosomal enzymes are associated with the tonoplast of large vacuole of differentiating cells. Sometimes, mitochondria and plastids are observed inside the vacuole suggesting autophagy in plants (Swanson and Webster,1989). 2. Spherosomes The spherosomes are membrane bounded, spherical particles of 0.5 to 2.5 m diameter, occurring in most plant cells. They have a fine granular structure internally which is rich in lipids and proteins. They originate from the endoplasmic reticulum (ER). Oil accumulates at the end of a strand of ER and a small vesicle is then cut off by contribution to form particles, called prospherosomes. The prospherosomes grow in size to form spherosoms. Basiocally, the spherosomes are involved in lipid synthesis and storage. But, the spherosomes of maize root tips (Matile, 1968) and spherosome of tobacco endosperm tissue (Spichiger, 1969) have been found rich in hydrolytic digestive enzymes and so have been considered as lysosomes. Like lysosomes they are not only responsible for the accumulation and mobilization of reserve lipids, but also for the digestion of other cytoplasmic components incorporated by phagocytosis. 3. Aleurone Grain The aleurone grains or protein bodies are spherical membrane-bounded storage particle occurring in the cells of endosperm and cytoledons of seeds. They are formed during the later stages of seed ripening and disappear in the early stages of germination. They store protein (e.g., globulins) and phosphate 27 in the form of phytin. Matile (1968) has demonstrated that aleurone grains from pea seed contain a wide range of hydrolytic enzymes including protease and phosphatase which are required for the mobilization of stored protein and phosphate, although the presence of other enzymes such as -amylase and RNAase suggest that other cell constituents may also be digested. Thus like spherosomes, aleurone grains store reserve materials, mobilize them during germination and in addition form a compartment for the digestion of other cell components (Hall et al., 1974). The aleurone grains are derived from the strands of the endoplasmic reticulum. During germinating of barley seed, the activity of hydrolases is found to be controlled by hormones such as gibberellic acid. Gibberellic acid, a plant growth hormone, is released by the embryo to the aleurone layer where, in turn, the hyrolases are released to the endosperm. This hormone operates by derepressing appropriate genes in the aleurone cells, which then begin to crank out new hydrolytic proteins . Endoplasmic Reticulum The cytoplasmic matrix is traversed by a complex network of inter-connecting membrane bound vacuoles or cavities. often remain concentrated in the endoplasmic portion of the cytoplasm ; therefore, known as endoplasmic reticulum, a name derived from the fact that in the light microscope it looks like a "net in the cytoplasm." (Eighteenth-century European ladies carried purses of netting called reticules). The name "endoplasmic reticulum" was coined in 1953 by Porter, who in 1945 had observed it in electron micrographs of liver cells. Fawcet and Ito (1958), 28 Thiery (1958) and Rose and Pomerat (1960) have made various important contributions to the endoplasmic reticulum. The occurrence of the endoplasmic reticulum varies from cell to cell. The erythrocytes (RBC), egg and embryonic cells lack in endoplasmic reticulum. The spermatocytes have poorly developed endoplasmic reticulum. The adipose tissues, brown fat cells and adrenocortical cells, interstitial cells of testes and cells of corpus luteum of ovaries, sebaceous cells and retinal pigment cells contain only smooth endoplasmic reticulum (SER). The cells of those organs which are actively engaged in the synthesis of proteins such as chinar cells of pancreas, plasma cells, goblet cells and cells of some endocrine glands are found to contain rough endoplasmic reticulum (RER) which is highly developed. The presence of both SER and RER in the hepatocytes (liver cells) is reflective of the variety of the roles played by the liver in metabolism. Morphology Morphologically, the endoplasmic reticulum may occur in the following three forms : 1. Lamellar form or cisternae (A closed, fluid-filled sac, vesicle or cavity is called cisternae) 2. vesicular form or vesicle and 3. tubular form or tubules. 1. Cisternae. The cisternae are long, flattened, sac-like, unbranched tubules having the diameter of 40 to 50 m. They remain arranged parallely in bundles or stakes. PER usually exists as cisternae which occur in those cells of pancreas, notochord and brain. 2. Vesicles. The vesicles are oval, membrane bound vacuolar structures having the diameter of 25 to 500m. They often remain isolated in the cytoplasm and occur in most cells but especially abundant in the SER. 29 3. Tubules. The tubules are branched structures forming the reticular system along with the cisternae and vesicles. They usually have the diameter from 50 to 190m and occur almost in all the cells. Tubular form of ER is often found in SER and is dynamic in nature, i.e., it is associated with membrane movements, fission and fusion between membranes of cytocavity network (see Thorpe, 1984). Ultra structure The cavities of cisternae, vesicles and tubules of the endoplasmic reticulum are bounded by a thin membrane of 50 to 60 A0 thickness. The membrane of endoplasmic reticulum is fluid-mosaic like the unit membrane of the plasma membrane, nucleus, Golgi apparatus, etc. The membrane, thus, is composed of a bimolecular layer of phospholipids in which 'float' proteins of various sorts. The membrane of endoplasmic reticulum remains continuous with the membranes of plasma membrane, nuclear membrane and Golgi apparatus. The cavity of the endoplasmic reticulum is well developed and acts as a passage for the secretory products. Palade (1956) has observed secretory granules in the cavity of endoplasmic reticulum. Sometimes, the cavity of RER is very narrow with two membranes closely apposed and is much distended in certain cells which are actively engaged in protein sysnthesis (e.g., acinar cells, plasma cells and goblet cells). Weibel et al. , 1969, have calculated that the total surface of ER contained in 1ml of liver tissue is about 11 square metres, two-third of which is or rough types (i.e., RER). Types of Endoplasmic reticulum 30 Two types of endoplasmic reticulum have been observed in same or different types of cells which are as follows : 1. Agranular or Smooth Endoplasmic Reticulum This type of endoplasmic reticulum possesses smooth walls because the ribosomes are not attached with its membranes. The smooth type of endoplasmic reticulum occurs mostly in those cells, which are involved in the metabolism of lipids (including steroids) and glycogen. The smooth endoplasmic reticulum is general found in adipose cells, interstitial cells, glycogen storing cells of the liver, conduction fibres of heart, spermatocytes and leucocytes. The muscle cells are also rich in smooth type of endoplasmic reticulum and here it is known as sarcoplasmic reticulum. In the pigmented retinal cells it exists in the form of tightly packed vesicles and tubes known as myeloid bodies. Glycosomes. Although the SER forms a continuous system with RER, it has different morphology. For example, in liver cells it consists of a tubular network that pervades major portion of the cytoplasmic matrix. These fine tubules are present in regions rich in glycogen and can be observed as dense particles, called glycosomes, in the matrix. Glycosomes measure 50 to 200 mm in diameter and contain glycogen along with enzymes involved in the synthesis of glycogen (Rybicka, 1981). Many glycosomes attached to the membranes of SER have been observed by electron microscopy in the liver and conduction fibre of heart. 2. Granular of Rough Endoplasmic Reticulum The granular or rough type of endoplasmic reticulum possesses rough walls because the ribosomes remain attached with its membranes. Ribosomes play a vital role in the process of protein synthesis. The granular or rough type of endoplasmic reticulum is found abundantly in those cells which are active in protein sysnthesis such as pancreatic cells, plasma cells, goblet cells, and liver cells. The granular type 31 of endoplasmic reticulum takes basiophilic stain due to its RNA content of ribosomes. The region of the matrix containing granular type of endoplasmic reticulum takes basiophilic stain and is names as ergastoplasm, basiophilic bodies, chromophilic substances or Nissl bodies by early cytologists. In RER, ribosomes are often present as polysomes held together by mRNA and are arranged in typical, "rosettes" of spirals. RER contains two transmembrane glycoproteins (called ribophorins I and II of 65,000 and 64,000 dalton MW, respectively), to which are attached the ribosomes by their 60S subunits. endoplasmic reticulum in the intact cell was established by Palade and Siekevitz 1956. Enzymes of the ER membranes The membranes of the endoplasmic reticulum are found to contain many kinds of enzymes which are needed for various important synthetic activities. Some of the most common enzymes are found to have different transverse distribution in the ER membranes (Table 6-1). The most important enzymes are the stearases, NADHcytochrome C reductase, NADH diaphorase, glucose-6-phosphotase and Mg++ activated ATPase. Certain enzymes of the endoplasmic reticulum such as nucleotide diphosphate are involved in the biosynthesis of phospholipids, ascorbic acid, glucuronide, steroids and hexose metabolism. The enzymes of the endoplasmic reticulum perform the following important functions : 1. Synthesis of glycerides, e.g., triglycerides, phospholipids, glycolipids and plasmalogens. 2. Metabolism of plasmalogens. 3. Sythesis of fatty acids. 32 4. Biosynthesis of the steroids, e.g., cholesterol biosynthesis, steroid hydrogenation of unsaturated bonds. 5. NADPH2+O2- requiring steroid transformations : Aromatization and hydroxylation. 6. NADPH2+O2-requireing steroid transformations : Aromatization hydroxylation's side-chain oxidation, thio-ether oxidations, desulphuration. 7. L-ascorbic acid metabolism. 8. UDP-glucose dephosphorylation. 9. Ary1- and steroid sulphatase. Function of Endoplasmic reticulum The endoplasmic reticulum acts as secretory, storage, circulatory and nervous system for the cell. performs following important functions : A. Common Functions of Granular and Agranular Endoplasmic Reticulum 1. The endoplasmic reticulum provides and ultrastructural skeletal framework to the cell and gives mechanical support to the colloidal cytoplasmic matrix. 2. The exchange of molecules by the process of osmosis, diffusion and active transport occurs through the membranes of endoplasmic reticulum. Like plasma membrane, the ER membrane has permeases and carries. 3. The endoplasmic membranes contain many enzymes which perform various synthetic and metabolic activities. Further the endoplasmic reticulum provides increase surface for various enzymatic reactions. 33 4. The endoplasmic reticulum acts as an intracellular circulatory or transporting system. Various secretory products of granular endoplasmic reticulum are transported to various organelles as follows : Granular ERagranular ERGolgi membranelysosomes, transport vesicles or secretory granules. Membrane flow many also be an important mechanism for carrying particles, molecules and ions into and out of the cells. Export of RNA and nucleoproteins from nucleus to cytoplasm may also occur by this type of flow (see De Robertis and De Robertis, Jr., 1987). 5. The ER membranes are found to conduct intra-cellular impulses. For example, the sarcoplasmic reticulum transmits impulses from the surface membrane into the deep region of the muscle fibres. 6. The ER membranes form the new nuclear envelope after each nuclear division. 7. The sarcoplasmic reticulum plays a role in releasing calcium when the muscle is stimulated and actively transporting calcium back into the sarcoplasmic reticulum when the stimulation stops and the muscle must be relaxed. B. Functions of Smooth Endoplasmicreticulum Smooth ER performs the following functions of the cell : 1. Synthesis of lipids. SER perform synthesis of lipids (e.g., phospholipids, cholesterol, etc.) and lipoproteins. Studies with radioactive precursors have indicated that the newly synthesized phospholipids are rapidly transferred to other cellular membranes by the help of specific cytosolic enzymes, called phospholipids exchange proteins. 34 2. Glycogenolysis and blood glucose homeostasis. This process of glycogen synthesis (glycogenesis) occurs in the cytosol (in glycosomes). The enzyme UDPG-glycogen transferase, which is directly involved in the synthesis of glycogen by addition of uridine diphosphate glucose (UDPG) to primer glycogen is bound to the glycogen particles or glycosomes. SER is found related to glycogenolysis or breakdown of glycogen. An enzyme, called glucose-6-phosphatase (a marker enzyme) exists as an integral protein of the membrane of SER (e.g., liver cell). Generally, this enzyme acts as a glycogenic phosphorhydrolase that catalyzes the release of free glucose molecule in the lumen of SER from its phosphorylated form in liver. Thus, this process operates to maintain homeostatic levels of glucose in the blood for the maintenance of functions of red blood cells and nerve tissues. 3. Sterol metabolism. The SER contains several key enzymes that catalyze the synthesis of cholesterol which is also a precursor substance for the biosynthesis of two types of compounds- the steroid hormones and bile acids : (i) Cholesterol biosynthesis. The cholesterol is synthesized from the acetate and its entire biosynthetic pathway involve about 20 steps, each step catalyzed by an enzyme. Out of these twenty enzymes, eleven enzymes are bounded to SER membranes, rest nine enzymes are the soluble enzymes located in the cytosol and mitochondria. Examples of SER-bound enzyme include HMG-Co A reductase and squalene synthetase (see Thorpe, 1984). 35 (ii) Bile acid synthesis. The biosynthesis of the bile acids represents a very complex pattern of enzymes and products. Enzymes involved in the biosynthetic pathway of bile acids are hydroxylases, monooxygenases, dehydrogenases, isomerases and reductases. For example, by the help of the enzyme cholesterol 7-hydroxylase, the cholesterol is first converted into 7-hydroxyl cholesterol, which is then converted into bile acids by the help of hydroxylase enzymes. The latter reaction requires NADPH and molecular oxygen and depends on the enzymes of Electron transport chains of SER such as cytochrome P-450 and NADPH-cytochrome-c reductase . (iii) Steroid hormone biosynthesis. Steroid hormones are synthesized in the cells of various organs such as the cortex of adrenal gland, the ovaries, the testes and the placenta. For example, cholesterol is the precursor for both types of sex hormones-estrogen and testosterone-made in the reproductive tissues, and the adrenocorticoids (e.g., corticosterone, aldosterone and cortisol) formed in the adrenal glands. Many enzymes (e.g., dehydrogenase,s isomerases and hydroxylases) are involved in the biosynthetic pathway of steroid hormones, some of which are located in SER membranes and some occur in the mitochondria 4. Detoxification. Protectively, the ER chemically modifies xenobiotics (toxic materials of both endogenous and exogenous origin), making them more hydrophilic, hence, more readily excreted. Among these materials are drugs, aspirin (acetyl-salicylic-acid), insecticides, anaesthetics, petroleum products, pollutant and carcinogens (i.e., inducers of cancer ; e.g., 3-4-benzophrene and 3-methyl cholantherene). 36 The enzymes involved in the detoxification of aromatic hydrocarbonds are aryhydraoxylases. It is now know that benzophyrene (found in charcoalbroiled meat) is not carcinogenic, but under the action of aryl hydroxylase enzyme in the liver, it is converted into 5,6-epoxide, which is a powerful carcinogen (see De Robertis and De Robertis, Jr., 1987) A wide variety of drugs (e.g., Phenobarbital), when administrated to animals, they bring about the proliferation of the ER membranes (first RER and then SER) and /or enhanced activity of enzymes related to detoxification (Thorpoe, 1984). C. Functions of Rough Endoplasmic Reticulum The major function of the rough ER is the synthesis of protein. It has long been assumed that proteins destined for secretion (i.e., export) from the cell or proteins to be used in the synthesis of cellular membranes are synthesized on rough DR-bound ribosomes, while cytoplasmic proteins are translated for the most part on free ribosomes. In fact, the array of the rough endoplasmic reticulum provides extensive surface area for the association of metabolically active enzymes, amino acids and ribosomes. There is more efficient functioning of these materials to synthesize proteins when oriented on a membrane surface than when they are simply in solution, mainly because chemical combinations between molecules can be ccomplished in specific 37 geometric patterns. The membrane-bound ribosomes are attached with specific binding sites or receptors of rough ER membrane by their large 60S subunit, with small or 40S subunit sitting on top like a cap. These receptors are membrane proteins which extend well into and possibly through the lipid bilayer. The receptor proteins with bound ribosomes can float laterally like other membrane proteins and may facilitate formation of the polysome and probably translation which requires that mRNA and ribosome move with respect to each other. 38 Further, the secretory proteins, instead of passing into the cytoplasm, appear to pass instead into the cisternae of the rough ER and are, thus, protected from protease enzymes of cytoplasm. It is calculated that about 40 amino acid residues long segment at the - COOH end of the nascent protein remains protected inside the tunnel of 'free' or 'bond' ribosomes and rest of the chain, with-NH2 end, is protected by the lumen of RER. The passage of nascent polypeptide chain into the ER cisterna take place during translation leaving only a small segment exposed to the cytoplasm at any one time. Protein glycosylation. The covalent addition of sugars to the secretory proteins (i.e., glycosylation) is one of the major biosynthetic functions of rough ER. Most of the proteins that are isolated in the lumen of RER before being transported to the Golgi apparatus, lysosomes, plasma membrane or extracellular space, are glycoproteins (A notable exception is albumin). In contrast, very few proteins in the cytosol (Cytoplasmic matrix) are glycosylated and those that carry them have a different sugar modification. The process of protein glycosylation in RER lumen is one of the most well understood cell biological phenomena. During this process, a single species of loligosaccharide (Which comprises N-acetyl-glucosamine, mannose and glucose, containing a total of 14 sugar residues) is transferred to proteins in the ER. Microbodies : Structure and Types Microbodies are spherical or oblate in form. They are bounded by a single membrane and have an interior or matrix which is amorphous or granular Micrbodies are most easily distinguished from other cell organelles by their content of catalase 39 enzyme. Catalase can be visualized with the electron microscope when cells are treated with the stain DAB (i.e., 3,3'-diaminobenzidine). The product is electron opaque and appears as dark regions in the cell where catalase is present The technique of isolation of microbodies of plant tissues includes the following steps : (1) Tissues are group very carefully to save microbodies from disruption. (2) The homogenate is with differential centrifugation to obtain a fraction of the cell homogenate which is rich in microbodies. (3) The enriched fraction is subjected to isopycnic ultra-centrifugation on discontinuous or continuous sucrose density gradient. Recent biochemical studies have distinguished two types of microbodies, namely peroxisomes and glycoxysomes. These two organelles differ both in their enzyme complement and in the type of tissue in which they are found. Peroxisomes are found in animal cells and the leaves of higher plants. They contain catalases and oxidases (e.g., D-amino oxidase and urate oxidase). In both they participate in the oxidation of substrates, producing hydrogen peroxide which is subsequently destroyed by catalase activity. In plant cells, peroxisomes remain associated with ER. chloroplasts and mitochondria and are involved in photorespiration. Gloxysomes occur only in plant cells and are particularly abundant in germinating seeds which store fats as a reserve food material. They contain enzymes of glyoxylate cycle besides the catalases and oxidases Peroxisomes Peroxisomes occur in many animal cells and in a wide range of plants. They are present in all photosynthetic cells of higher plants in etiolated leaf tissue, in coleoptiles and hypocotyls, in tobacco stem and callus, in ripening pear fruits and also in Euglenophyta, Protozoa, brown algae, fungi liverworts, mosses and ferns. 40 Peroxisomes are variable in size and shape, but usually appear circular in cross section having diameter between 0.2 and 1.5m (0.2 and 0.25 m diameter in most mammalian tissues : 0.5m diameter in rat liver cells). They have a single limiting unit membrane of lipid and protein molecules, which encloses their granular matrix. In some cases (e.g., in the festuciod grasses) the matrix contains numerous threads or fibrils, while in others they are observed to contain either an amorphous nucleoid or a dense inner core which in many species shows a regular crystalloid structure (e.g., tobacco leaf cell, Newcomb and Frederick, 1971). Little is known about the function of the core, except that it is the site of the enzyme urate oxidase in rat liver peroxisomes and much of the catalase in some plants (see Hall et al., 1974). Recently, a possible relationship has been stressed between peroxides and free radicals (such as superoxide anion -O2-) with the process of aging. These radicals may act on DNA molecule to produce mutations altering the transcription into mRNA and the translation into proteins. In addition, free radicals and peroxides can affect the membranes by causing peroxidation of lipids and proteins. For these reasons reducing compounds such as vitamin E or enzymes such as superoxide dismutase could play a role in keeping the healthy state of a cell. . Biogenesis of Peroxisomes At one time it was thought that the membrane 'shell' of the peroxisomes is formed by building of the endoplasmic reticulum (ER), while the 'content' or matrix is imported from the cytosol (cytoplasmic matrix). However, there is now evidence suggesting that new peroxisomes always arise from pre-existing ones, being formed by growth and fission of old organelles similar to mitochondria and chloroplasts. Thus, peroxisomes are a collection of organelles with a constant membrane and a variable enzymatic content. All of their proteins (both structural and 41 enzymatic) are encoded by nuclear genes and are synthesized in the cytosol (cytoplasmic matrix) (i.e., on the free ribosomes). The proteins present in either lumen or membrane of the peroxisome are taken up post-translationally from the cytosol (cytoplasmic matrix) as the haeme-free monomer; the monomers are imported into the lumen of peroxisomes, where they assemble into tetramers in the presence of haeme./ Catalase and many peroxisomal proteins are found to have a signal sequence (comprising of three amino acids) which is located near their carboxyl ends and directs them to peroxisome (Gould, Keller and Subramani,1988). Peroxisomes contain receptors exposed on their cytosolic surface to recognize the signal on the imported proteins. All of the membrane proteins of the peroxisomes. Including signal receptor proteins, are imported directly from the cytosol (cytoplasmic matrix). The lipids required to make new peroxisomal membrane are also imported from the cytosol (cytoplasmic matrix), possibly being carried by phospholipids transfer proteins from sites of their synthesis in the DR membranes (Affe and Kennedy, 1983). Glyoxysomes Glyoxysomes are found to occur in the cells of yeast, Neurospora, and oil rich seeds of many higher plants. They resemble with peroxisomes in morphological details, except that, their crystalloid core consists of dense rods of 6.0 m diameter. They have enzymes for fatty acid metabolism and gluconeogenesis, i.e. conversion of stored lipid molecules of spherosomes of germinating seeds into the molecules of carbohydrates. Functions Glyoxysomes perform following biochemical activities of plants cells : (1) Fatty acid metabolism. During germination of oily seeds, the stored lipid molecules of spherosomes are hydrolysed by the enzyme lipase (glycerol ester 42 hydrolase) to glycerol and fatty acids. The phospholipids molecules are hydrolysed by the enzyme phospholipase. The long chain fatty acids which are released by the hydrolysis are then broken down by the successive removal of two carbon or C2 fragments in the process of -oxidation. During -oxidation process, the fatty acid is first activated by enzyme fatty acid thiokinase to fatty acyl-CoA which is oxidized by a FAD-linked enzyme fatty acyl-CoA dehydrogenase into-2-enoyl-CoA. Trans-2-enoyl-CoA is hydrated by an enzyme enoyl hydratase or crotonase to produce the L-3-hydroxyacyl-CoA, which is oxidized a NAD Linked L-3-hydratase or crotonase to produce the L-3-hydroxyacylCoA, which is oxidized by a NAD linked L-3-hydroxyacyl-CoA dehydrogenase to produce 3-Ketoacly-CoA. The 3-keto acyl-CoA looses a two carbon fragment under the action of the enzyme thiolase to generate an acetyl-CoA and a new fatty acylCoA with two less carbon atoms thatn the original. This new fatty acyl-CoA is then recycled thought the same series of reactions until the final two molecules of acetylCoA are produced. In plant seeds -oxidation occurs in glyoxysomes (Cooper and Beevers, 1969). But in other plant cells -oxidation occurs in glyxysomes and mitochondria. The glyoxysomal -oxidation requires oxygen for oxidation of reduced flavorprotien produced as a result of the fatty-acyl-CoA dehydrogenase activity. In animal cells oxidation occurs in mitochondria. In plant cells, the acetyl-CoA, the product of -oxidation chain is not oxidized by Krebs cycle, because it remains spatially separated from the enzymes of Krebs cycle, instead of it, acetyl-CoA undergoes the glyoxylate cycle to be converted into succinate. (2) Glyoxylate cycle. The glyoxylate pathway occurs in glyoxysomes and it involve some of the reactions of the Krebs cycle in which citrate is formed 43 from oxaloacetate and acetyl-CoA under the action of citrate synthetase enzymes. The citrate is subsequently converted into isocitrate by aconitase enzyme. The cycle then involves the enzymatic conversion of isocitrate to glyoxylate and succinate by isocitratase enzyme : Isocitratase Isocitrate Glyoxylate + Succinate The glyoxylate and another mole of acetyl-CoA form a mole of malate by malate synthesis ; Malate synthetase Acetyl CoA+Glyoxylate Malate This malate is converted to oxaloacetate by malate dehydrogenase for the cycle to be completed. Thus, overall, the glyoxylate pathway involves : 2 Acetyl-CoA+NAD+ Succintiate + NADH+ H+ Succinate is the end product of the glyoxysomal metabolism of fatty acid and is not further metabolized within this organelle. The synthesis of hexose or gluconeogenesis involves the conversion of succinate to oxaloacetate, which presumably takes place in the mitochondria, since the glyoxsomes do not contain the enzymes fumarase and succinic dehydrogenase. Two molecules of oxaloacetate are formed from four molecules of acetyl-CoA without carbon loss. This oxaloacetate is converted to phosphoenol pyruvate in the phosphoenol pyruvate caboxykinase reaction with the loss of two molecules of CO2 : 2 Oxaloacete + 2ATP 2 Phosphoenol pyruvate + 2CO2 + 2ADP 44 MITOCHONDRIA In cell biology, a mitochondrion (plural mitochondria) is a membraneenclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 10 micrometers (μm) in diameter. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον or chondrion, granule.Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria, whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported. The mitochondrial proteome is thought 45 to be dynamically regulated Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes. Structure A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins.[6] The two membranes, however, have different properties. Because of this double-membraned organization, there are five distinct compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the 46 inner mitochondrial membrane, the cristae space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane). Outer membrane The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral proteins called porins. These porins form channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death. The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondriaassociated ER-membrane). This is important in ER-mitochondria calcium signaling and involved in the transfer of lipids between the ER and mitochondria ] Intermembrane space The intermembrane space is the space between the outer membrane and the inner membrane. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c. 47 Inner membrane The inner mitochondrial membrane contains proteins with five types of functionsThose that perform the redox reactions of oxidative phosphorylation 1. ATP synthase, which generates ATP in the matrix 2. Specific transport proteins that regulate metabolite passage into and out of the matrix 3. Protein import machinery. 4. Mitochondria fusion and fission protein It contains more than 151 different polypeptides, and has a very high protein-tophospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn't contain porins and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane formed by the action of the enzymes of the electron transport chain. 48 The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria the area of the inner membrane is about five times greater than the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function.One recent mathematical modeling study has suggested that the optical properties of the cristae in filamentous mitochondria may affect the generation and propagation of light within the tissue. Matrix The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly-concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycleMitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins). Organization and distribution Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. A single mitochondrion is often found in unicellular 49 organisms. Conversely, numerous mitochondria are found in human liver cells, with about 1000–2000 mitochondria per cell making up 1/5 of the cell volume.[6] The mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum.[6] Often they form a complex 3D branching network inside the cell with the cytoskeleton. The association with the cytoskeleton determines mitochondrial shape, which can affect the function as well. Recent evidence suggests vimentin, one of the components of the cytoskeleton, is critical to the association with the cytoskeleton. Functions The most prominent roles of mitochondria are to produce ATP (i.e., phosphorylation of ADP) through respiration, and to regulate cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. However, the mitochondrion has many other functions in addition to the production of ATP. Mitochondrial DNA. The human mitochondrial genome is a circular DNA molecule of about 16 kilobases. It encodes 37 genes: 13 for subunits of respiratory complexes I, III, IV and V, 22 for mitochondrial tRNA (for the 20 standard amino acids, plus an extra gene for leucine and serine), and 2 for rRNA. One mitochondrion can contain two to ten copies of its DNA. As in prokaryotes, there is a very high proportion of coding DNA and an absence of repeats. Mitochondrial genes are transcribed as multigenic transcripts, which are cleaved and polyadenylated to yield mature mRNAs. Not all proteins necessary for mitochondrial function are encoded by the mitochondrial genome; most are coded by genes in the cell nucleus and the corresponding proteins are 50 imported into the mitochondrion. The exact number of genes encoded by the nucleus and the mitochondrial genome differs between species. In general, mitochondrial genomes are circular, although exceptions have been reported In general, mitochondrial DNA lacks introns, as is the case in the human mitochondrial genome;] however, introns have been observed in some eukaryotic mitochondrial DNA such as that of yeast] and protists including Dictyostelium discoideum. In animals the mitochondrial genome is typically a single circular chromosome that is approximately 16-kb long and has 37 genes. The genes while highly conserved may vary in location. Curiously this pattern is not found in the human body louse (Pediculus humanus). Instead this mitochondrial genome is arranged in 18 minicircular chromosomes each of which is 3–4 kb long and has one to three genes. This pattern is also found in other sucking lice but not in chewing lice. Recombination has been shown to occur between the minichromosomes. The reason for this difference is not known. 51 1.4 COMPARISION OF PLANT AND ANIMAL CELLS Comparision of plant and animal cells. Cilia: Shape: Animal Cell Plant Cell Present It is very rare Round (irregular Rectangular shape) shape) Plant Chloroplast: (fixed cells Animal cells don't have chloroplasts chloroplasts have because they make their own food One or more small Vacuole: vacuoles smaller (much than plant cells). Centrioles: One, large vacuole taking up 90% of cell volume. Present in all animal Only present in lower cells plant forms. Plastids: Absent Present Cell wall: Absent Present Plasma Membrane: only cell membrane Lysosomes: central cell wall and a cell membrane Lysosomes occur in Lysosomes usually not cytoplasm. evident. Plant and animal cells have some structural differences. 52 PlantCell Animal Cell Comparison of structures between animal and plant cells 53 Cell walls in animal cells vs. plant cells.A notable difference between animal cells and plant cells is that animal cells do not have a cell wall where as plant cells do. Typical animal cell Organelles Typical plant cell Nucleus o Nucleolus (within o nucleus) Nucleus Nucleolus (within nucleus) Rough endoplasmic reticulum Rough ER (ER) Smooth ER Smooth ER Ribosomes Ribosomes Cytoskeleton Cytoskeleton Golgi apparatus (dictiosomes) Golgi apparatus Cytoplasm Cytoplasm Mitochondria Mitochondria Plastids and its derivatives Vesicles Vacuole(s) Lysosomes Cell wall Centrosome o Centrioles Both plant and animal cells have plasma membranes. 54 CHECK YOUR PROGRESS 1 Note: Write your answer in the space given below. Check your answer with the one at the end of the unit. Fill in the blanks. 1. Prokaryotic cell shave ----------------- chromosomes. 2. An example of prokaryotic cell is -------------------. 3. Eukaryotic cells have------------------- and ------------------------------------. CHECK YOUR PROGRESS-1 4. Mitochondria are called as --------------------------------------------------- of the cell. 5. Lysosomes are ------------------------------------- sacs of the cell. 6. Plant cell wall is made up of -----------------------------. 7. Animal cell have ----------------------- as reserve food material 8. Plastids are--------------------- in animal cells. OVERVIEW OF METABOLISM;-CATABOLISM AND ANABOLISM During synthesis of metabolites energy is required and such constructive reactions are called anabolism.Example photosynthesis Co2 +h2o+Ribulose 1,5, diphospate- Rudp carbooxylase--------- 2 mol of 3 PGA Where as reactions in which break down of certain metabolites occurs and energy is released are called catabolism.Example Glycolysis,krebs cycle.. The process of respiration is basically an oxidation- reduction process, where electrons are with 55 drawn from substrate (glucose) are accepted by various components of etc and reducing powers, and leads to generation of precursor metabolites reducing power + NADPH+H +ATP. To recall, all living organisms respire to produce energy needed to perform all vital activities. The energy required for biological activities is obtained from organic compounds available in food. Plants synthesize their own food through photosynthesis. Defination : ― Respiration is a process by which organic food materials such as sugar, fats, etc get successively oxidized to produce CO2, H2O and energy.‖ C6H12O6 + 6O2 6CO2 + 6H2O + 673Kcal energy The overall reaction of cellular repiration is given as C6H12O6 + 6O2 + 38Adp +38iP 6CO2 + 6H2O + 38ATP AN OVERVIEW OF RESPIRATION. a) You must bear a clear understanding in mind that both photosynthesis and respiration involves gaseous exchange but light reaction of photosynthesis requires sunlight whereas respiration occurs all the time. O2 utilized in the process &Co2 is released . b) The sites of respiration are cytoplasms and mitochondria. The organic compounds are broken down inside the cells by oxidation process, known as cellular respiration. The energy released is stored in pyrophosphate bonds of ATP. 56 ADP + H3PO4 ATP(ADP˜P) Energy stored in ATP is utilized for carying out different cellular and biological activites because of this, energy is called energy currency of the cell. c) The overall reaction is as follows: C6H12O6 + 6O2 + 38Adp +38iP 6CO2 + 6H2O + 38ATP The main features of respiration are: Oxidation of organic compounds occurs in under aerobic conditions Complete oxidation occurs End products are CO2 & H2O Higher amount of (673 Kcal )energy is liberated out Process occurs in cytoplasm and mitochondria Chlorophyll pigment is not essential Various respiratory substance are: glucose, fructose, fats, protein, etc. The ratio of volume of CO2 released to the volume of O2 absorbed during respiration is called respiratory ratio or R.Q. Volume of CO2 released R.Q. = Volume of O2 absorbed To develop a clear understanding of the process let us understand the mechanism of respiration MECHANISM OF RESPIRATION Cellular respiration is a complicated process which is completed in many steps. for every step, a particular enzyme is required which works in a sequential manner one after the another. 57 it is completed in 3 steps: a) Glycolysis / EMP pathway b) Oxidation of pyruvic acid c) ETC & oxidative phosphorylation a. GLYCOLYSIS/ EMP PATHWAY Greek, glucose – sugar, lysis – dissolution. If I say that glycolysis is a fermentive pathway would you agree? Reasons to support my statement are: a) It does not involves O2 intake b) ATP generated is through substrate level phosphorylation. c) Organic compound donates electrons and organic compound accepts it. This process was discovered by three German scientists Embden, meyerhof and Parnas. On their name the pathway is also called EMP pathway. All the reactions of glycolysis take place in the cytoplasm and through the glycolysis glucose is oxidized into pyruvic acid in presence of many enzymes present in the cytoplasm. Thus the process of sequential oxidation of glucose into pyruvic acid is known as glycolysis. 58 β- OXIDATION OF PYRUVIC ACID OR KREBS CYCLE All the chemical reaction of Kreb‘s cycle can be summarized in following steps: 1. Aerobic oxidation of P.A 2. Condensation of Acetyl-CoA with oxalo-acetic acid 3. Isomerisation of citric acid into isocitric acid ,( (a) dehydration and b) hydration) 59 4. Oxidative decarboxylation of isocitric acid ((a) dehydration and b) decarboxylation) 5. Oxidative decarboxylation of α-Keto glutaric acid. 6. Conversion of succinyl CoA into succinic acid. 7. Dehydrogenation of succinic acid into fumaric acid 8. Hydration of fumaric acid into malic acid 9. Dehydrogenation of malic acid in OAA. Overall reaction of respiration is: Glycolysis + Kreb‘s cycle = Glucose + 4ADP + 4H3PO4 + 8NAD+ + NADP+ +2FAD 6CO2 + 4 ATP + 8NADH + 10H+ +2NADPH + 2FADH2 Thus as a result of oxidation of pyruvic acid, one molecule of CO 2 in oxidative decarboxylation and two molecules of CO2 in Kreb‘s cycle are liberated. The total number of CO2 evolved becomes 3 which indicates that 3 carbon pyruvic acid has been completely oxidized in glycolysis. Because two molecules of P.A. which are formed by one molecule of glucose in glycolysis, enter into Kreb‘s cycle for oxidation, a total of 6CO 2 molecule will be evolved. 2PA * 3CO2 = 6CO2 All the NADH2 and FADH2 are oxidized to NAD and FAD through a chain of reaction c/a etc.in this process ATP molecules are released (1NADH2 = 3ATP, 1FADH2 = 2ATP). In the process of Kreb‘s cycle 8 molecules of NADH2 =24ATP , 2FADH2 = 4 ATP and two molecules of ATP are synthesized from 2GTP. 60 c ELECTRON TRANSPORT SYSTEM AND OXIDATIVE PHOSPHORYLATION Electron Transport System During repiration simple carbohydrates and intermediate compounds like acid and malie acid are oxidized. Each oxidative step involves release of a pair of hydrogen atoms which dissociates into two protons and two electrons. 2H 2H+ + 2e- These protons and electrons are accepted by various hydrogen acceptors like NAD,NADP, FAD etc. After accepting hydrogen atoms these acceptors get reduced to produce NADH2, NADPH2 and FADH2. The pairs of hydrogen atoms released a series of coenzymes and cytochromes which form electron transport system, before reacting with O2 to form H2O. ½ O + 2H+ + 2e- H2O 2NADH + O2 + 2H+ 2NAD++ 2H2O As you know that H ions and electrons removed from the respiratory substrate during oxidation do not directly react with oxygen. Instead they reduce acceptor molecules NAD and FAD to NADH2 and FADH2. These molecules then transfertheir electron to a system of electron acceptors and transfer molecules. The proteins of the inner mitochondrial membrane act as electron transporting enzymes. They are arranged in an ordered manner in the membrane and function in a specific sequence. This assembly of electron transport enzymes is known as mitochondrial respiratory 61 chain or the electron transport chain. Specific enzymes of this chain receive electrons from reduced prosthetic groups, NADH2 or FADH2 produced by glycolysis and the TCA cycle. The electrons are then transported successively from enzyme to enzyme, down a descending ‗stairway‘ of energy yielding reactions. This process takes place in mitochondrial cristae which contain all the components of E,T.S. Components of electron transport system: the electron transport system is made up of following enzymes and proteins: 1. Nicotinamide adenine dinucleotide (NAD). 2. Flavoproteins (FAD and FMN). 3. Fe-S protein complex. 4. Co-enzyme Q or ubiquinone. 5. Cytochome-b 6. Cytochrome-c1. 7. Cytochrome-c 8. Cytochrome-a 9. Cytochrome-a3. All the above enzymes are found in F1 particles of mitochondria. Mechanism of action of electron transport system: During respiration electron pairs liberated from respiratory compounds are accepted by coenzymes like NAD or NADP and FMN etc. The transfer of electrons in all compounds except succinic acid takes place first in NAD+ or NADP+ and later on in FAD. The transfer of electgrons from succinic acid takes place diretly to the FAD and not through NAD+ or NADP+. Due to this reason only two molecules of ATP are 62 formed in the formation of fumaric acid from succinic acid whereas in case of other compounds 3 ATP molecules are produced because these cases the electrons are first picked up by NAD. Different Steps of E.T.S. are as follows: 1. Hydrogne pairs released from different substrates of Krebs cycle except succinic acid reacts with NAD+. The electrons and proton are transferred to NAD causing its reduction and one proton is released in the medium. 2H+ 2H + 2e- (protons) (electrons) NAD + 2H+ + 2e- NADH + H+ (reduced) (ion pool) 2. Now, 2e- and one H+ are transferred from NADH to FAD causing oxidation of NADH to NAD and reduction of FMN into FMNH2. One H+ is picked up from hydrogen ion pool to complete this reaction. The free energy released at this step is stored during oxidative phophorylation and one molecule of ATP is generated fronm ADP and inorganic phosphate. The hydrogen pair from succinic acid is first transferred to FAD to form FADH2. The FADH2 transfers electrons to coenzyme Q throught Fe-S and CoQ. The electrons pass to cytochromes Cyt-b, Cyt-c1, Cyt-c, Cyt-a, Cyt-a3 and then to oxygen atoms. Oxygen atom accepts those electrons and reacts with hydrogen ions of the matrix to form water. O2 + 4e2(O--) + 4e+ 2(O--) 2H2O 63 Oxygen is thus the terminal electron acceptor of the mitochondrial respiratory chain. At each step of electron acceptor has a higher electron affinity than the electron donor from which it receives the electron. The energy from such electron transport is utilized in transporting protons from the matrix across the inner membrane to its outer side. This creates a higher proton concentration outside the inner membrane than in the matrix. The difference in proton concentration across the inner membrane is called proton gradient. The reduction of various cytochromes requires only electrons and no protons. Each cytochromes possesses an iron elements in the centre which functions for accepting (Fe3+ Fe2+) or donating (Fe2+ Fe3+) When a cytochrome accepts electrons, it is reduced and if it donates electrons, it is oxidized. Oxidative Phosphorylation In all living beings ATP generated during oxidative breakdown of complex food products. This process of synthesis of ATP molecules from ADP and inorganic phosphate by electron transport system of aerobic respiration called as oxidative phosphorylation. ADP + iP O 2 ATP E.T. Chain The process of oxidative phosphorylation takes place in mitochondrial crests through electron transport chain. Due to high proton concentration outside the inner membrane, protons return to the matrix down the proton gradient. Just as a flow of water from a higher to lower 64 level can be utilized to turn a water-wheel or a hydroelectric turbine, the energy released by the flow of protons down the gradient is utilized in synthesizing ATP. The return of proton occurs through the inner membrane particles. In the F 0-F1 complex the F1 head piece functions as ATP synthetase. The latter synthesizes ATP from ADP and inorganic phosphate using the energy from the proton gradient. Transport of two electrons from NADH2 by the electron transport chain simultaneously transfers three pairs of protons to the outer compartment. One high energy ATP bond is produced per pair of protons returning to the matrix through the inner membrane particles. Therefore, oxidative phosphorylation produces three ATP molecules per molecules of NADH2 oxidized. Since FADH2 donates its electrons further down the chain. Its oxidation can only produce two ATP molecules. During oxidative phosphorylation ATP molecules are produced during following steps: I. When NADH2 is oxidized to NAD by reacting with FAD. II. When the electron transfer from cytochrome-b to cytochrome-c1. III. When the electron transfer from cytochrome-a to cytochrome-a3. Now it is clear that oxidation of one molecule of reduced NADH2 or NADPH2 results in the formation of 3 molecules of ATP while oxidation of FADH 2 leads to the formation of 2 molecules of ATP. 65 1.6 ATP THE BIOLOGICAL ENERGY CURRENCY Defination : ― Respiration is a process by which organic food materials such as sugar, fats, etc get successively oxidized to produce CO2, H2O and energy.‖ C6H12O6 + 6O2 6CO2 + 6H2O + 673Kcal energy The overall reaction of cellular repiration is given as C6H12O6 + 6O2 + 38Adp +38iP 6CO2 + 6H2O + 38ATP Higher amount of (673 Kcal )energy is liberated out Objective: The main aim of this unit is to develop an understanding of the process of respiration. After learning this unit you should be able to Differentiate between various types of (respiration, fermentation) fueling reaction. Understand the significance of respiration in a) Generation of precursors b) Generation of reducing power c) Generation of ATP Realize the role and significance of various enzymes involved in the process. Understand the existence of alternative oxidation pathways The applications of fermentation and the basic difference between the process of aerobic respiration , anaerobic respiration & fermentation. Role of ATP : Adenosine - P ~ P ~ P + H2O adenosine - P~P + P 7.8Kcal 66 4° = - Adenosine - P ~ P + H2O adenosine ~P + P 4° = - adenosine + P 4° = - 7.3Kcal Adenosine ~ P + H2O 3.4Kcal High energy compounds other than ATP Compound cause action in Priosyn.of: GTP Protein(ribosome function) CTP Phospholipids UTP Peptidoglycan layer of bacterial wall Dcoxythymidine~ P~P~P lipopolysaccarid layer of bacterial wall dTTTP Acyl~SCoA Fatty acids 1.7 ORIGIN OF LIFE UNIQUE PROPERTIIES OF CARBON,CHEMICAL EVOLUTION AND RISE OF LIVING SYSTEMS The origin of cells has to do with the origin of life, which began the history of life on Earth. Origin of the first cell There are several theories about the origin of small molecules that could lead to life in an early Earth. One is that they came from meteorites Another is that they were created at deep-sea vents. A third is that they were synthesized by lightning in a reducing atmosphere); although it is not clear if Earth had such an atmosphere. There are essentially no experimental data defining what the first self-replicating forms 67 were. RNA is generally assumed to be the earliest self-replicating molecule, as it is capable of both storing genetic information and catalyzing chemical reactions (see RNA world hypothesis). But some other entity with the potential to self-replicate could have preceded RNA, like clay or peptide nucleic acid. Cells emerged at least 4.0–4.3 billion years ago. The current belief is that these cells were heterotrophs. An important characteristic of cells is the cell membrane, composed of a bilayer of lipids. The early cell membranes were probably more simple and permeable than modern ones, with only a single fatty acid chain per lipid. Lipids are known to spontaneously form bilayered vesicles in water, and could have preceded RNA. But the first cell membranes could also have been produced by catalytic RNA, or even have required structural proteins before they could form. Origin of eukaryotic cells The eukaryotic cell seems to have evolved from a symbiotic community of prokaryotic cells. DNA-bearing organelles like the mitochondria and the chloroplasts are almost certainly what remains of ancient symbiotic oxygen-breathing proteobacteria and cyanobacteria, respectively, where the rest of the cell seems to be derived from an ancestral archaean prokaryote cell – a theory termed the endosymbiotic theory. There is still considerable debate about whether organelles like the hydrogenosome predated the origin of mitochondria, or viceversa: see the hydrogen hypothesis for the origin of eukaryotic cells. Sex, as the stereotyped choreography of meiosis and syngamy that persists in nearly all extant eukaryotes, may have played a role in the transition from prokaryotes to eukaryotes. An 'origin of sex as vaccination' theory suggests that the eukaryote 68 genome accreted from prokaryan parasite genomes in numerous rounds of lateral gene transfer. Sex-as-syngamy (fusion sex) arose when infected hosts began swapping nuclearized genomes containing co-evolved, vertically transmitted symbionts that conveyed protection against horizontal infection by more virulent symbionts. 1.8 INTRODUCTION TO BIOMOLECULES The living organisms are composed of molecules which are intrinsically inanimate.These molecules confer a remarkable combination of characteristics called life.Many of he most important molecules in biological systems are polymers,that is large molecules made up of smaller subunits joined together by covalent bonds,and in some cases in a specific order. Most of the molecular constituents of living systems are composed of C-atoms joined with other carbon atoms and with hydrogen ,oxygen or nitrogen.Thus form a great variety of molecules such as aminoacids ,monosaccharides,nucleotides and fattyacids.These are called micromolecules.These micromolecules serve polysaccharides and as monomeric lipids subunits respectively of which proteins,nucleic are designated acids, as macromolecules.These macromolecules serve more than one functions in living cells and in all living organisms CARBOHYDRATES Carbohydrates are madeup of just threedifferent elements,carbon,hydrogen,and oxygen.the simplest carbohydrates are monosaccharides,,have the general formula (ch2o)n.they are classed as either aldoses or ketoses.example glucose.A disaccharide 69 is formed when two monosaccarides join together with a concomitant loss of water molecules.Further monosaccaharides can be added,giving chains of three, four ,five or more units.these are termed oligosaccarides and chain with many units are polysaccharides.The chemical bond joinig the monosaccharide units together is called a glycosidic linkage. Biologically important molecules such as starch ,cellulose and glycogenare all polysaccharides.serve as major components of the cell walls of bacteria and of the soft cell coats in animal tissues. The carbohydrates, or saccharides, are most simply defined as polyhydroxy aldehydes or ketones and their derivatives. Many have the empirical formula (CH2O)n, which originally suggested they were ―hydrates‖ of carbon. Monosaccharides, also called simple sugars, consist of a single polyhydroxy aldehyde or ketone unit. The most abundant monosaccharide is the six-carbon D-glucose; it is the parent monosaccharide from which most others are derived. D-Glucose is the major fuel for most organisms and the basic building block of the most abundant polysaccharides, such as starch and cellulose. Oligosaccharides (Greek oligo, ―few‖) contain from two to ten monosaccharide units joined in glycosidic linkage. Polysaccharides contain many monosaccharide units joined in long linear or branched chains. Most polysaccharides contain recurring monosaccharide units of only a single kind or two alternating kinds. 70 Polysaccharides have two major biological functions, as a storage form of fuel and as structural elements. In the biosphere there is probably more carbohydrate than all other organic matter combined, thanks largely to the abundance in the plant world of two polymers of D-glucose, starch and cellulose. Starch is the chief form of fuel storage in most plants, whereas cellulose is the main extracellular structural component of the rigid cell walls and the fibrous and woody tissues of plants. Glycogen, which resembles starch in structure, is the chief storage carbohydrate in animals. Others polysaccharides PROTEINS Proteins are the most abundant molecules in cells, consisting 50 percent or more of their dry weight. They are found in every part of every cell, since they are fundamental in all aspects of cell structure and function. There are many different kinds of proteins, each specialized for a different biological function. Moreover, most of the genetic information is expressed by proteins. The structure of protein molecules and its relationship to their biological function and activity are central problems in biochemistry today. Proteins consist of long chains, in which amino acids occur in specific linear sequences. Yet we know that in each type of protein the polypeptide chain is folded into a specific three-dimensional conformation, which is required for its specific biological function and activity. How is the linear, or one-dimensional, information inherent in the amino acid sequence of polypeptide chains translated into the threedimensional conformation of native protein molecules? The answer to this question comes from some of the most significant advances in modern biological research. These discoveries, made possible by the application of 71 physical-chemical measurements to pure proteins, have illuminated the function and comparative biology of proteins. In this chapter, we examine various aspects of the primary structure of proteins, which we have defined as the covalent backbone structure of polypeptide chains, including the sequence of amino acid residues. We begin by considering the properties of simple peptides. Then we examine three major aspects: (1) the determination of amino acid sequence in polypeptide chains, (2) the significance of variations in the amino acid sequences of different proteins in different species, and (3) the laboratory synthesis of polypeptide chains.(4) structure of proteins. LIPIDS Lipids are esters of higher fatty acids. Lipids are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents, e.g., choloroform, ether, or benzene. There are several different families or classes of lipids but all derive their distinctive properties from the hydrocarbon nature of a major portion of their structure. (1) as structural components of membranes, (2) as storage and trasport forms of metabolic fuel, (3) as a protective coating on the surface of many organisms, and (4) as cell-surface components concerned in cell recognition, species specificity, and tissue immunity. Some substances classified among the lipids have intense biological activity; they include some of the vitamins and hormones. Although lipids are a distinct class of biomolecules, we shall see that they often occur combined, either covalently or through weak bonds, with members of other classes of bio-molecules to yield hybrid molecules such as glycolipids, lipoproteins, 72 which contain both lipids and proteins. In such biomolecules the distinctive chemical and physical properties of their components are blended to fill specialized biological functions. NUCLEIC ACIDS DEOXYRIBONUCLEIC ACID - DNA AND RIBONUCLEIC ACID RNA Occurrence DNA is found in the cells of all living organisms except plant viruses, where RNA forms the genetic material and DNA is absent. In bacteriophages and viruses there is a single molecule of DNA, which remains coiled and is enclosed in the protein coat. In bacteria, mitochondria and plastids of eukaryotic cells DNA is circular and lies naked in the cytoplasm. In the nuclei of eukaryotic cells DNA occurs in the form long spirally coiled and unbranched threads. The number of DNA molecules is equivalent to the number of chromosomes per cell. In them DNA is found in combination with proteins forming nucleoproteins or the chromatin material and is enclosed in the nucleus. The nucleic acids are of considerable importance in biological systems.Two types of nucleic acids are found in the cells of all living organisms. These are: 1. Deoxyribonucleic acid - DNA 2. Ribonucleic acid - RNA The nucleic acid was first isolated by Friedrich Miescher in 1868 from the nuclei of pus cells and was named nuclein. At that time its biological significance was barely understood. The name nucleic acid was given to it after knowing its acidic property. 73 They are of two types; (1) Ribose nucleic acid, and(2) Deoxyribose nucleic acid . The basic chemical subunits of the nucleic acids are nucleotides. The nucleotides are made up of three components: (i) A heterocyclic ring containing nitrogen, known as a nitrogenous base, (ii) a five carbon pentose sugar, and (iii) A phosphate group. The bases found in nucleic acid are of two kinds- purines and pyrimidines.Adenine and guanine are purine and cytosine, uracil and thymine are pyrimidinebases.The nucleotides found in nucleic acids are much fewer in number than the α-amino acids. DNA is found in almost all the cells as a major component of chromosomes of the nucleus.. Certain viruses, including many of the bacterial viruses or bacteriophages, are DNA-protein particles. Mostly the plant viruses are RNA-protein particles. Ribose nucleic acid (RNA) is also of common occurrence in plants as well as animals. It is of three types- (i)ribosomal RNA (r-RNA); (ii) soluble RNA or transfer RNA (t-RNA) and (iii) messenger RNA (m-RNA). Ribosomal-RNA is found in small sub-cellular particles, the ribosomes. RNAs with sendimentation Coefficient value, 5S, 16S and 23S have been reported from 70S ribosomes, while 18S, 28S, 5.8S and 5S r-RNAs have been reported from 80S ribosomes. T-RNA is found in free from in the cytoplasm. M-RNA is found in small quantities in association with ribosomes. THE CHEMICAL BASIS OF HEREDITY DNAs are present mainly in the nucleus (in chromosomes) of the cell so they are the carrier of gnetic information because the DNA molecule can produce a copy of itself each going to one cell i.e. the parent DNA molecule gives rise to two identical daughter molecules each going to one cell and thus each daughter cell receives exactly the same complement of DNA ( both qualitatively and quantitatively as the parent cell. 74 DNA as the bearer of genetic information in the cell is strongly supported by the Watson-Crick structure for this compound which explains beautifully the phenomenon of replication (and hence genetic continuity) of DNA. This phenomenon of DNA replication can be explained as below: the double helix of DNA separates into two strands: the individual strands combine in sequence with their complementary free nucleotides (present in nuclear sap) through specific hydrogen bonding ( Adenine….Thymine, Guanine…Cytisine) and now phosphate ester linkages are formed between two nucleotides by the enzyme catalase. Thus DNA in animals, plants, bacterial cells, and some viruses maintain the genetic continuity. The direct evidence in favour of the genetic role of DNA is derived from the process of bacterial transformation. If an extract of the strain of pneumococcus, possessing capsules having specific polysaccharides, is added to the culture of strain of pneumococcus not having the above mentained capsules, the latter is transformed to the former. The active agent (transforming factor) is a DNA molecule which endows the bacterial cell with the capacity for synthesizing an enzyme or enzyme system not present previously in non-encapsulated strain. This enzyme in turn catayzes the formation of the specific capsular polysaccharide. 75 1.9 BUILDING BLOCKS OF BIOMACROMOLECULES The molecular constituents of living systems are composed of C-atoms joined with other carbon atoms and with hydrogen ,oxygen or nitrogen.They form a great variety of molecules suc nucleic aci ds h as aminoacids, monosaccharides, nucleotides and fattyacids.These are called micromolecules.These micromolecules serve as monomeric subunits of proteins, polysacchrides, nucleic acids and lipids. A. MICROMOLECULES OF CARBOHYDRATES Monosaccharides have the empirical formula (CH2O)n, where n=3 or some larger number. The carbon skeleton of the common monosaccharides is unbranched and each carbon atom except one contains a hydroxyl group; at the remaining carbon there is a carbonyl oxygen, which, as we shall see, is often combined in an acetal or ketal linkage. If the carbonyl groups is at the end of the chain, the monosaccharide is an aldehyde derivative and called an aldose; if it is at any other position, the monosaccharides is a ketone derivative and called ketose. Fig. 2 76 The simplest monosaccharides are the three carbon trioses glyceraldehydes and dihydrosyacetone. Glyceraldehyde is an aldotriose; dihydroxyacetone is a ketotriose. Also among the monosaccharides are the tetroses (four carbons), pentoses (five carbons), hexoses (six carbons), heptoses (seven carbons), and octoses (eight carbons). Each exists in two series, i.e., aldotetroses and ketotetroses, aldopentoses and ketopentoses, aldohexoses and ketohexoses, etc. The structures of D aldoses and D ketoses are shown. In both classes of monosaccharides the hexoses are by far the most abundant. However, aldopentoses are important components of nucleic acids and various polysaccharides; derivatives of trioses and heptoses are important intermediates in carbohydrate metabolism. All the simple monosaccharides are white crystalline solids that are free soluble in water but insoluble in nonpolar solvents. Most have sweet taste. CONFIGURATION OF MONOSACCHARIDES & THEIR FAIMILY First, we must clarify two terms often confused. Configuration denotes the arrangement in space of substituent groups in stereoisomers such structures cannot be inter-converted without breaking one or more covalent bonds. Conformation refers to the spatial arrangement of substituent groups that are free to assume many different positions, without breaking bonds, because of rotation about the single bonds in the molecule. In the hydrocarbon ethane, for example, one might expect complete freedom of rotation around the C-C single bond to yield an infinite number of conformations of the molecule. However, the staggered conformation is more stable than all others and thus predominates, whereas the eclipsed form is least stable. 77 All the monosaccharides expect dihydroxyacetone contain one or more asymmetric carbon atoms and thus are chiral molecules. Glyceraldehyde contains only one asymmetric carbon atom and therefore can exist as two different stereoisomers (Figure- 4). It will be recalled that C- and L- Glyceraldehyde are the reference, or parent, compounds for designating the absolute configuration of all stereoisomeric compounds. Aldotetroses have two asymmetric carbon atoms and aldopentoses have three. The aldohexoses have four asymmetric carbon atoms and thus exist in the form of 2n = 24 = 16 D different stereoisomers, 8 of which are Dshown in Figure- 2. As expected, the monosaccharides with asymmetric carbon atoms are optically active. For example, the usual form of glucose found in nature in 20 = +52.70), and the usual form of fructose is levorotatory ([α]20 = -92.40), but both are members of the D series since their absolute configurations are related to D-glyceraldehyde. For sugars having two or more asymmetric carbon atoms, the convention has been adopted that the prefixes D and L refer to the asymmetric carbon atoms farthest removed from carbonyl carbon atom. 78 Shows the projection formulas of the D aldoses. All have the same configuration at the asymmetric carbon atom farthest from the carbonyl carbon, but because most have two or more asymmetric carbon atoms, a number of isomeric D aldoses exist, most important biologically being D-glyceraldehyde. D-ribose, D-glucose, D-mannose, and Dgalactose. Figure-4 shows the projection formulas of the D ketoses; all share the same configuration at the asymmetric carbon atom farthest from the carbonyl group. Ketoses are sometimes designated by inserting ul into the name of the corresponding aldose; e.g., D-ribulose is the ketopentose corresponding to the aldopentose D-ribose. The most important The stereoisomers of glyceraldehyde, showing projection formulas (top) and perspective formulas (bottom). ketoses biologically and dihydroxyacetone. Dribulose, and D-fructose. Aldoses and ketoses of the L series are mirror images of their D counterparts. L sugars are found in nature, but they are not so abundant as D sugars. Among the most important are L-fucose, L-rhamnose , and L-sorbose. Two sugars differing only in the configuration around one specific carbon atom are called epimers of each other. Thus, D-glucose and D-mannose are epimers with respect to carbon atom 2, and D-glucose and D-galactose are epimers with respect to carbon atom 4. 79 B. . MICROMOLECULES OF AMINOACIDS A polypeptide is an unbranched chain of amino acids. Standard amino acids Amino acids are the structural units that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These polymers are linear and unbranched, with each amino acid within the chain attached to two neighboring amino acids. The process of making proteins is called translation and involves the step-by-step addition of amino acids to a growing protein chain by a ribozyme that is called a ribosome The order in which the amino acids are added is read through the genetic code from an mRNA template, which is a RNA copy of one of the organism's genes. Twenty-two amino acids are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids. Of these, 20 are encoded by the universal genetic code. The remaining 2, selenocysteine and pyrrolysine, are incorporated into proteins by unique synthetic mechanisms. Selenocysteine is incorporated when the 80 mRNA being translated includes a SECIS element, which causes the UGA codon to encode selenocysteine instead of a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG, which is normally a stop codon in other organisms. This UAG codon is followed by a PYLIS downstream sequence. Non-standard amino acids Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard. Those either are not found in proteins (for example carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery (for example, hydroxyproline and selenomethionine). Non-standard amino acids that are found in proteins are formed by post-translational modification, which is modification after translation during protein synthesis. These modifications are often essential for the function or regulation of a protein; for example, the carboxylation of glutamate allows for better binding of calcium cations, and the hydroxylation of proline is critical for maintaining connective tissues. Another example is the formation of hypusine in the translation initiation factor EIF5A, through modification of a lysine residue.[26] Such modifications can also determine the localization of the protein, e.g., the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane. 81 β-alanine and its α-alanine isomer Some nonstandard amino acids are not found in proteins. Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids — for example, ornithine and citrulline occur in the urea cycle, part of amino acid catabolism (see below)[ A rare exception to the dominance of α-amino acids in biology is the β-amino acid beta alanine (3-aminopropanoic acid), which is used in plants and microorganisms in the synthesis of pantothenic acid (vitamin B5), a component of coenzyme A. Essential Nonessential Isoleucine Alanine Leucine Asparagine Lysine Aspartic acid Methionine Cysteine* Phenylalanine Glutamic acid Threonine Glutamine* Tryptophan Glycine* Valine Proline* Selenocysteine* Serine* Tyrosine* Arginine* 82 Histidine* Ornithine* Taurine* The first few amino acids were discovered in the early 19th century. In 1806, the French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet isolated a compound in asparagus that proved to be asparagine, the first amino acid to be discovered. Another amino acid that was discovered in the early 19th century was cystine, in 1810, although its monomer, cysteine, was discovered much later, in 1884. Glycine and leucine were also discovered around this time, in 1820. General structure Lysine The carbon atom next to the carboxyl group is called the α–carbon and amino acids with a side-chain bonded to this carbon are referred to as alpha amino acids. These 83 are the most common form found in nature. In the alpha amino acids, the α–carbon is a chiral carbon atom, with the exception of glycine. In amino acids that have a carbon chain attached to the α–carbon (such as lysine, shown to the right) the carbons are labeled in order as α, β, γ, δ, and so on. In some amino acids, the amine group is attached to the β or γ-carbon, and these are therefore referred to as beta or gamma amino acids.Amino acids are usually classified by the properties of their side-chain into four groups. The side-chain can make an amino acid a weak acid or a weak base, and a hydrophile if the side-chain is polar or a hydrophobe if it is nonpolar.. Isomerism Of the standard α-amino acids, all but glycine can exist in either of two optical isomers, called L or D amino acids, which are mirror images of each other (see also Chirality). While L-amino acids represent all of the amino acids found in proteins during translation in the ribosome, D-amino acids are found in some proteins produced by enzyme posttranslational modifications after translation and translocation to the endoplasmic reticulum, as in exotic sea-dwelling organisms such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria, and D-serine may act as a neurotransmitter in the brain The L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotary). In alternative fashion, the (S) and (R) designators are used to indicate the absolute stereochemistry. Almost all of the amino acids in proteins are (S) at the α carbon, with cysteine being (R) and glycine nonchiral. Cysteine is unusual since it has a sulfur atom at the second position in its side-chain, which has a larger atomic mass than the groups attached to the first 84 carbon, which is attached to the α-carbon in the other standard amino acids, thus the (R) instead of (S). An amino acid in its (1) unionized and (2) zwitterionic forms Zwitterions The amine and carboxylic acid functional groups found in amino acids allow them to have amphiprotic properties. Carboxylic acid groups (-CO2H) can be deprotonated to become negative carboxylates (-CO2- ), and α-amino groups (NH2-) can be protonated to become positive α-ammonium groups (+NH3-). At pH values greater than the pKa of the carboxylic acid group (mean for the 20 common amino acids is about 2.2, see the table of amino acid structures above), the negative carboxylate ion predominates. At pH values lower than the pKa of the α-ammonium group (mean for the 20 common α-amino acids is about 9.4), the nitrogen is predominantly protonated as a positively charged α-ammonium group. Thus, at pH between 2.2 and 9.4, the predominant form adopted by α-amino acids contains a negative carboxylate and a positive α-ammonium group, as shown in structure (2) on the right, so has net zero charge. This molecular state is known as a zwitterion, from the German Zwitter meaning hermaphrodite or hybrid. Below pH 2.2, the predominant form will have a neutral carboxylic acid group and a positive α-ammonium ion (net charge +1), and above pH 9.4, a negative carboxylate and neutral α-amino group (net charge -1). The 85 fully neutral form (structure (1) on the right) is a very minor species in aqueous solution throughout the pH range (less than 1 part in 107). Amino acids also exist as zwitterions in the solid phase, and crystallize with salt-like properties unlike typical organic acids or amines. Isoelectric point At pH values between the two pKa values, the zwitterion predominates, but coexists in dynamic equilibrium with small amounts of net negative and net positive ions. At the exact midpoint between the two pKa values, the trace amount of net negative and trace of net positive ions exactly balance, so that average net charge of all forms present is zero. This pH is known as the isoelectric point pI, so pI = ½(pKa1 + pKa2). The individual amino acids all have slightly different pKa values, so have different isoelectric points. For amino acids with charged side-chains, the pKa of the sidechain is involved. Thus for Asp, Glu with negative side-chains, pI = ½(pKa1 + pKaR), where pKaR is the side-chain pKa. Cysteine also has potentially negative sidechain with pKaR = 8.14, so pI should be calculated as for Asp and Glu, even though the side-chain is not significantly charged at neutral pH. For His, Lys, and Arg with positive side-chains, pI = ½(pKaR + pKa2). Amino acids have zero mobility in electrophoresis at their isoelectric point, although this behaviour is more usually exploited for peptides and proteins than single amino acids. Zwitterions have minimum solubility at their isolectric point and some amino acids (in particular, with non-polar side-chains) can be isolated by precipitation from water by adjusting the pH to the required isoelectric point. (*) Essential only in certain cases. 86 C. MICROMOLECULES OF FATTY ACIDS Although fatty acids occur in very large amounts as building block components of the saponifiable lipids, only traces occur in free (unesterified) form in cells and tissues. Well over 100 different kinds of fatty acids have been isolated from various lipids of animals, plants, and microorganisms. All possess a long hydrocarbon chain and a terminal carboxyl group. The hydrocarbon chain may be saturated, as a in palmitic acid, or it may have one or more double bonds, as in oleic acid; a few fatty acids contain triple bonds. Fatty acids differ from each other primarily in chain length and in the number and position of their saturated bonds. They are often symbolized by a shorthand notation that designates the length of the carbon chain and the numbers, posistion, and configuration of the double bonds. Thus palmitic acid (16 carbons, saturated) is symbolized 16:0 and oleic acid [18 carbons and one double bond (cis) at carbons 9 and 10] is symbolized 18:19. it is understood that the double bonds are cis unless indicated otherwise. Some generalizations can be made on the different fatty acids of higher plants and animals. 1. The most abundant have an even number of carbon atoms with chains between 14 and 22 carbon atoms long, but those with 16 or 18 carbons predominate. 87 The most common among the saturated fatty acids are palmitic acid (C16) and stearic acid (C18). 2. Unsaturated fatty acids predominate over the saturated ones, particularly in higher plants and in animals living at low temperatures. 3. Unsaturated fatty acids have lower melting points than saturated fatty acids of the same chain length 4. In most monounsaturated (monoenoic) fatty acids of higher organisms there is a double bond between carbon atoms 9 and 10. In most polyunsaturated (polyenoic) fatty acids one double bond is between carbon atoms9 and 10; the additional double bonds usually occur between the 9, 10 double bond and the methy-terminal end of the chain. 5. In most types of polyunsaturated fatty acids the double bonds are seperated by one methylene group, for example, -CH=CH-CH2-CH=CH-; only in a few types of plant fatty acids are the double bonds in conjugation, that is, CH=CH-CH=CH-. ESSENTIAL FATTY ACID When weanling or immature rats are placed on a fat-free diet, they grow poorly, develop a scaly skin, lose hair, and ultimately die with many pathological signs. When linoleic acid is present in the diet, these conditions do not develop. Linolenic acid and arachidonic acid also prevent these symptoms. Saturated and monounsaturated fatty acids are inactive. It has been concluded that mammals can synthesize saturated and monounsaturated fatty acids from other precursors but are unable to make linoleic and -linoleic acids. Fatty acids required in the diet of mammals are called essential fatty acids. The most abundant essential fatty acid in 88 mammals is lionoleic acid, which makes up from 10 to 20 percent of the total fatty acids of their triacylglycerols and -linolenic acids cannot be synthesized by mammals but must be obtained from plant sources, in which they are very abundant. Linoleic acid is a necessary precursor in mammals for the biosynthesis of arachidonic acid, which is not found in plants. Although the specific functions of essential fatty acids in mammals were a mystery for many years, one function has been discovered. Essential fatty acids are necessary precursors in the biosynthesis of a group of fatty acid derivatives called prostaglandins, homonelike compounds which in trace amounts have profound effects on a number of important physiological activities. STRUCTURE AND FUNCTION OF TRIACYLGLYCEROLS (TRIGLYCERIDES) Fatty acid esters of the alcohol glycerol are called acylglycerols or glycerides; they are sometimes referred to as "neutral fats," a term that has become archaic. When all three hydroxyl groups of glycerol are esterified with fatty acids, the structure is called a triacylglycerol. Triacyglycerols are the most abundant family of lipids and the major components of depot or storage lipids in plant and animal cells. Triacyglycerols that are solid at room temperature are often referred to as "fats" and those which are liquid as "oils" Diacylglycerols (also 89 called diglycerides) and monoacylglycerols (or monoglycerides) are also found in nature, but in much samller amounts.Triacylglycerols occur in many different types, according to the identity and position of the three fatty acid components esterified to glycerol. 1. Those with a single kind of fatty acid in all three positions, called simple triacylglycerols, are named after the fatty acids they contain. Examples are tristearoylglycerol, tripalmitoylglycerol, and trialeoyglycerol; the trivial and more commonly used names are tristearin, tripalmitin, and triolein, respectively. 2. Mixed triacyglycerols contain two or more different fatty acids. 90 2.6PHOSPHOGLYCERIDES (GLYCEROPHOSPHOLIPIDS) The second large class of complex lipids consists of the phosphoglycerides, also called glycerol phosphatides. They are characteristic major components of cell membranes; only very small amounts of phosphoglycerides occur elsewhere in cells. Phosphoglycerides are also loosely referred to as phospholipids or phosphatides, but it should be noted that not all phosphorus-containing lipids are phosphoglycerides; e.g., sphingomyelin is a phospholipid because it contains phosphorus, but it is better classified as a sphingolipid because of the nature of the backbone structure to which the fatty acid is attached. In phosphoglycerides one of the primary hydroxyl groups of glycerol is esterifed to phosphoric acid; the other hydroxyl groups are esterfied to fatty acids. The parent compound of the series is thus the phosphoric ester of glycerol. This compound has an asymmetric carbon atom and can be designated as either D-glycerol 1-phosphate or L-glycerol 3-phosphate. Because phosphoglycerides possess a polar head in addition to their nonpolar hydrocarbon tails, they are called amphiphatic or polar lipids.The different types of phosphoglycerides differ in the size, shape, and electric charge of their polar head groups. Each type of phosphoglyceride can exist in many different chemical species differing in their fatty 91 acid substituents. Usually there is one saturated and one unsaturated fatty acid, the latter in the 2 position of glycerol. The parent compound of the phosphoglycerides is phosphatidic acid, which contains no polar alcohol head group. It occurs is only very small amounts in cells, but it is an importnat intermediate in the biosynthesis of the phosphoglycerides. The most abundant phosphoglycerides in higher plants and animals are phosphatidylethanolamine and phosphatidylcholine, which contain as head groups the amino alcohols ethanolamine and choline, respectively. Plasmologens differ from all the other phosphoglycerides. D MICROMOLECULES OF NUCLEIC ACIDS PURINE AND PYRIMIDINE BASES OF NUCLEIC ACIDS. Chemical composition The Chemical analysis has indicated that DNA is composed of three different types of compounds: 1. Sugar Molecule represented by a pentose sugar, the deoxyribose or 2‘deoxyribose. 2. Phosphoric Acid. 3. Nitrogenous Bases: These are nitrogen containing organic ring compounds. These are of the following four types: I. Adenine represented by –A II. Thymine represented by –T III. Cytosine represented by –C 92 IV. Guanine represented by –G V. These four nitrogenous bases are separated into two categories: (a) Purines: These are two-ringed nitrogen compounds. Adenine and guanine are the two purines found in DNA. Their structural formulae are represented in fig.2. (b) Pyrimidines: These are formed of one ring only and include cytosine and thymine. Chemical analysis of DNA further reveals three fundamental features described by Chargaff and is called Chargaff’s base ratio. Molecular Structure The constituents of DNA were isolated quite early but how these are arranged so as to carry out their cytological and genetical activities was not known for long. DNA is a long chain polymer was clearly understood in late 1930s. However, in 1953, D.S. Watson and F.H.C. Crick presented a working model of DNA. This model illustrates not only its chemical structure but also the mechanism by which it duplicates itself. 1.Nucleosides A nitrogenous base with a molecule of deoxyribose (without phosphate group) is known as nucleoside. The nitrogenous base is attached to first carbon atom C-1 of deoxyribose N-glycosidic bond. In all, there are four nucleosides in a DNA molecule. These are: 1. Adenosine – Adenine + Deoxyribose 2. Guanosine – Guanine + Deoxyribose 3. Cytidine – Cytosine + Deoxyribose 93 4. Thymidine – Thymine + Deoxyribose 2. Nucleotides ( The Monomers of DNA) A nucleotide is formed of one molecule of deoxyribose, one molecule of phosphoric acid and one of the four nitrogenous bases. Since there are four nitrogenous bases, there are four type of nucleotides namely: 1. Deoxyadenylic acid -Adenine + Deoxiribose + Phosphoric acid 2. Deoxyguanylic acid -Guanine + Deoxiribose + Phosphoric acid 3. Deoxycytidylic acid -Cytosine + Deoxiribose + Phosphoric acid 4. Deoxythymidylic acid -Thymine + Deoxiribose + Phosphoric acid 1.Polynucleotide Chain (Linking of Nucleotides in a DNA Molecule) DNA is a macromolecule formed by the linking of several thousand nucleotides. These are called monomers or building blocks of DNA. In a nucleotide the phosphate (phosphoric acid) molecule is attached to fifth carbon atom (C-5) of the deoxyribose molecule through a phosphodiester linkage. The adjacent nucleotides are connected together forming the sugar phosphate chain in which sugar and phosphate molecule are arranged in alternate fashion. The phosphate molecule of a nucleotide is joined to the third carbon atom of the deoxyribose. These are directed at right angles to the long axis of the polynucleotide chain and are stacked one above the other. Marked Ends of Polynculeotide Chain: Each polynucleotide chain has marked ends. Its top end has a sugar residue with free 5‘ carbon atom which is not linked to another nucleotide. The triphosphate group is still attached to it. 94 This end is called the 5’ or 5’-P terminus. The other end of the chain ends in a sugar residue with C-3 carbon atom not linked. It bears 3‘-OH group. This end of polypeptide chain is called 3’ end or 3’-OH terminus . It means the pplypeptide chains have direction and are marked as 3‘-5‘. 1.5 DOUBLE HELICAL MODEL OF DNA (WATSON & CRICK’S MODEL) In 1953, James Watson and Francis Crick deduced the three dimensional structure of DNA and immediately inferred its mechanism of replication, Watson and Crick analyzed X-ray diffraction photographs of DNA fibres taken by Rosalind Franklin and Maurice Klilkins and derived a structural model that has proved to be essentially correct. The salient features of their model are:1. Two helical polynucleotide chains are coiled around common axis, the chains run in the opposite directions. 2. The purine and pyrimidine bases are on the inside of the helix, where as the phosphate and deoxyribose units are on the outside the planes of the bases are perpendicular to the helix axis. The planes of the sugars are nearly at right angles to those of the bases. 3. The diameter of the helix is 20 A0, adjacent bases are separated by 34 A0 along the helix and related by a rotation of 360, hence the helical structure repeats after in residues on each chain, i.e., at interval of 34A0. 4. The two chains are held together by hydrogen bonds between the pairs of bases adenine is always paired with thymine guanine is always paired width cytosine. 5. The sequence of bases along a polynucleotide chain is not restricted in any way, the precise sequence of bases carries the genetic information. 6. The ratio of A+G/C+T always equals to one 95 7. In every organism, the sequence of nucleotides in constant. The ratio of A=T/G=C is also specific to organisms. 8. Each pitch of DNA has two major and two minor groves. Fig. DNA Structure The most important, aspect of the DNA double helix is the specificity of the pairing of the bases. Watson and Crick deduced that adenine must pair with thymine and guanine with cytosine. 96 CHECK YOUR PROGRESS 2 Note: Write your answer in the space given below. Check your answer with the one at the end of the unit. Fill in the blanks. 1 Lipids are ________ of higher fatty acids. 2 Fluid mosaic model of membrane was proposed by ________ 3 DNA and RNA are -------------------. 4 Adinine and guanine are------------------. 5 Term ATP stands for --------------------. Write short notes on A) glycolysis .b) building blocks of biomolecules ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1.10 LET US SUM UP The cell is the basic unit of life. There are millions of different types of cells. There are cells that are organisms onto themselves, such as microscopic amoeba and bacteria cells. And there are cells that only function when part of a larger organism, such as the cells that make up your body. The cell is the smallest unit of life in our bodies. In the body, there are brain cells, skin cells, liver cells, stomach cells, and the list goes on. All of these cells have unique functions and features. And all have some recognizable similarities. All cells have an outer covering called the plasma 97 membrane, protecting it from the outside environment. The cell membrane regulates the movement of water, nutrients and wastes into and out of the cell. Inside of the cell membrane are the working parts of the cell. At the center of the cell is the cell nucleus. The cell nucleus contains the cell's DNA, the genetic code that coordinates protein synthesis. In addition to the nucleus, there are many organelles inside of the cell - small structures that help carry out the day-to-day operations of the cell. One important cellular organelle is the ribosome. Ribosomes participate in protein synthesis. The transcription phase of protein synthesis takes places in the cell nucleus. After this step is complete, the mRNA leaves the nucleus and travels to the cell's ribosomes, where translation occurs. Another important cellular organelle is the mitochondrion. Mitochondria (many mitochondrion) are often referred to as the power plants of the cell because many of the reactions that produce energy take place in mitochondria. Also important in the life of a cell are the lysosomes. Lysosomes are organelles that contain enzymes that aid in the digestion of nutrient molecules and other materials There are many different types of cells. One major difference in cells occurs between plant cells and animal cells. While both plant and animal cells contain the structures discussed above, plant cells have some additional specialized structures. Many animals have skeletons to give their body structure and support. Plants have a unique cellular structure called the cell wall. The cell wall is a rigid structure outside of the cell membrane composed mainly of the polysaccharide cellulose. The cell wall gives the plant cell a defined shape which helps support individual parts of plants. In addition to the cell wall, plant cells contain an organelle called the chloroplast. The chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the chloroplast (including the common green pigment chlorophyll) absorb sunlight and use this energy to complete the chemical reaction 98 Eukaryotic cells are about 15 times wider than a typical prokaryote and can be as much as 1000 times greater in volume. The major difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is a cell nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA. This nucleus gives the eukaryote its name, which means "true nucleus." Other differences include: The plasma membrane resembles that of prokaryotes in function, with minor differences in the setup. Cell walls may or may not be present. The eukaryotic DNA is organized in one or more linear molecules, called chromosomes, which are associated with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a membrane. Some eukaryotic organelles such as mitochondria also contain some DNA. Many eukaryotic cells are ciliated with primary cilia. Primary cilia play important roles in chemosensation, mechanosensation, and thermosensation. Cilia may thus be "viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation."[7] Eukaryotes can move using motile cilia or flagella. The flagella are more complex than those of prokaryotes. 99 INTRACELLULAR ORGANELLS AND THEIR FUNCTIONS Golgi Apparatus An Italian neurologist (i.e., physician) Camillo Golgi in 1873 discovered, them which is commonly known as the Golgi bodies. The Golgi apparatus occurs in all cells except the prokaryotic cells (viz., mycoplasmas, bacteria and blue green algae) and eukaryotic cells of certain fungi, sperm cells of bryophytes and pteridiophytes, cells of mature sieve tubes of plants and mature sperm and red blood cells of animals. In animal cells, there usually occurs a single Golgi apparatus, however, its number may vary from animal to animal and from cell to cell. Thus Paramoeba species has two Golgi apparatus and nerve cells, liver cells and chordate oocytes have multiple Golgi apparatuses, there being about 50 of them in the liver cells. The Golgi apparatus is morphologically very similar in both plant and animal cells. However, it is extremely pleomorphic: in some cell types it appears compact and limited, in others spread out and reticular (net-like). Its shape and form may very depending on cell type. Lysosomes The lysosomes (Gr. lyso=digestive + soma = bodies) are tiny membrane-bound vesicles involved in intracellular digestion. They contain a variety of hydrolytic enzymes that remain active under acidic conditions. The lysosomes occur in most animal and few plant cells. They are absent in bacteria and mature mammalian ertythrocytes. Few lysosomes occur in muscle cells or in cells of the pancreas. Leucocytes, especially granulocytes are a particularly rich source of lysosomes. Their lysosomes are so large sized that they 100 can be observed under the light microscope. Lysosomes are also numerous in epithelial cells of absorptive, secretory and excretory organs (e.g., intestine, liver, kidney, etc.) They occur in abundance in the epithelial cells of lungs and uterus. Lastly phagocytic cells and cells of reticuloendothelial system (e.g., bone marrow, spleen and liver) are also rich in lysosomes.The lysosomes are round vacuolar structure which remain filled with dense material and are bounded by single unit membrane. , a lysosome may contain up to 40 types of hydrolytic enzymes . The socalled latency of the lysosomal enzymes is due to the presence of the membrane which is resistant to the enzymes that it encloses. Lysosomes are extremely dynamic organelles, Endoplasmic Reticulum The cytoplasmic matrix is traversed by a complex network of inter-connecting membrane bound vacuoles or cavities. often remain concentrated in the endoplasmic portion of the cytoplasm ; therefore, known as endoplasmic reticulum, a name derived from the fact that in the light microscope it looks like a "net in the cytoplasm Morphologically, the endoplasmic reticulum may occur in the following three forms : 1. Lamellar form or cisternae (A closed, fluid-filled sac, vesicle or cavity is called cisternae) 2. vesicular form or vesicle and 3. tubular form or tubules. The cavities of cisternae, vesicles and tubules of the endoplasmic reticulum are bounded by a thin membrane of 50 to 60 A0 thickness. The membrane of endoplasmic reticulum is fluid-mosaic like the unit membrane of the plasma membrane, nucleus, Golgi apparatus, etc. The membrane, thus, is composed of a bimolecular layer of phospholipids in which 'float' proteins of various sorts. 101 Two types of endoplasmic reticulum have been observed in same or different types of cells which are as follows :1.Agranular or Smooth Endoplasmic Reticulum and Granular of Rough Endoplasmic Reticulum The membranes of the endoplasmic reticulum are found to contain many kinds of enzymes which are needed for various important synthetic activities, steroids and hexose metabolism. The enzymes of the endoplasmic reticulum perform the following important functions : The endoplasmic reticulum acts as secretory, storage, circulatory and nervous system for the cell. performs following important functions : Microbodies : Structure and Types Microbodies are spherical or oblate in form. They are bounded by a single membrane and have an interior or matrix which is amorphous or granular Micrbodies are most easily distinguished from other cell organelles by their content of catalase enzyme. Catalase can be visualized with the electron microscope when cells are treated with the stain DAB (i.e., 3,3'-diaminobenzidine). The product is electron opaque and appears as dark regions in the cell where catalase is present Peroxisomes Peroxisomes occur in many animal cells and in a wide range of plants. They are present in all photosynthetic cells of higher plants in etiolated leaf tissue, in coleoptiles and hypocotyls, in tobacco stem and callus, in ripening pear fruits and also in Euglenophyta, Protozoa, brown algae, fungi liverworts, mosses and ferns. 102 Peroxisomes are variable in size and shape, but usually appear circular in cross section having diameter between 0.2 and 1.5m (0.2 and 0.25 m diameter in most mammalia Glyoxysomes. Glyoxysomes are found to occur in the cells of yeast, Neurospora, and oil rich seeds of many higher plants. They resemble with peroxisomes in morphological details, except that, their crystalloid core consists of dense rods of 6.0 m diameter. They have enzymes for fatty acid metabolism and gluconeogenesis, i.e. conversion of stored lipid molecules of spherosomes of germinating seeds into the molecules of carbohydrates. MITOCHONDRIA In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 10 micrometers (μm) in diameter. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. The most prominent roles of mitochondria are to produce ATP (i.e., phosphorylation of ADP) through respiration, and to regulate cellular metabolism.[7] The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. However, the mitochondrion has many other functions in addition to the production of ATP. . 103 COMPARISION OF PLANT AND ANIMAL CELLS Comparision of plant and animal cells. Cilia: Shape: Animal Cell Plant Cell Present It is very rare Round (irregular Rectangular shape) shape) Plant Chloroplast: (fixed cells Animal cells don't have chloroplasts chloroplasts have because they make their own food One or more small Vacuole: vacuoles smaller (much than plant cells). Centrioles: One, large vacuole taking up 90% of cell volume. Present in all animal Only present in lower cells plant forms. Plastids: Absent Present Cell wall: Absent Present Plasma Membrane: only cell membrane Lysosomes: central cell wall and a cell membrane Lysosomes occur in Lysosomes usually not cytoplasm. 104 evident. During synthesis of metabolites energy is required and such constructive reactions are called anabolism.Example photosynthesis Co2 +h2o+Ribulose 1,5, diphospate- Rudp carbooxylase--------- 2 mol of 3 PGA Where as reactions in which break down of certain metabolites occurs and energy is released are called catabolism.Example Glycolysis,krebs cycle.. Origin of the first cell There are several theories about the origin of small molecules that could lead to life in an early Earth. One is that they came from meteorites Another is that they were created at deep-sea vents. A third is that they were synthesized by lightning in a reducing atmosphere); although it is not clear if Earth had such an atmosphere. There are essentially no experimental data defining what the first self-replicating forms were. RNA is generally assumed to be the earliest self-replicating molecule, as it is capable of both storing genetic information and catalyzing chemical reactions (see RNA world hypothesis). But some other entity with the potential to self-replicate could have preceded RNA, like clay or peptide nucleic acid. The living organisms are composed of molecules which are intrinsically inanimate.These molecules confer a remarkable combination of characteristics called life.Many of he most important molecules in biological systems are polymers,that is large molecules made up of smaller subunits joined together by covalent bonds,and in some cases in a specific order. Most of the molecular constituents of living 105 systems are composed of C-atoms joined with other carbon atoms and with hydrogen ,oxygen or nitrogen.Thus form a great variety of molecules such as aminoacids ,monosaccharides,nucleotides and fattyacids.These are called micromolecules.These micromolecules serve polysaccharides and as monomeric lipids subunits respectively of which proteins,nucleic are designated acids, as macromolecules.These macromolecules serve more than one functions in living cells and in all living organisms. 2.1 CHECK YOUR PROGRESS ;THE KEY KEY 1 1 single 2 Bacteria E. coli 3Membrane bound organelles and nucleus 4 power house of the cell. 5 suicidal sacs 6 cellulose 7 glycogen 8 absent KEY2 1 esters 2Sanger and Nicholsan 3Nucleic acid 4 Purines 5Adinine tri phosphate 106 2.2 ASSIGNMENT / ACTIVITY 1 Explain in detail the structure of prokaryotic cell. 2 Compare the structure of prokaryotic and eukaryotic cells. 3 Describe the structure of plant and animal cell. 4 Write short notes on cell organelles,origin of life, biomolecules, ATP . 2.3 REFERENCES 1 BIOCHEMISTRY BY LEHININGER 2 BIOCHEMISTRY BY STRYER 3 CELL BIOLOGY BY RASTOGI 4 CELL ANDMOLECULAR BIOLOGY BY AJOY PAUL 5 CELL BIOLOGY BY S. CHANDRA ROY 6 MOLECULAR BIOLOGY BY ROBERTIES DE ROBERTIES. 107 UNIVERSITY - MADHYA PRADESH BHOJ OPEN UNIVERSITY BHOPAL (M.P.) PROGRAMME - M.Sc.Chemistry (Previous) PAPER - TITLE OF PAPER - BIOLOGY FOR CHEMIST BKOCK NO . - II UNIT WRITER - UNIT - I Smt. Shikha Mandloi Asst. Prof. Microbiology Sri Sathya Sai College for Women UNIT – II Smt. Shikha Mandloi Asst. Prof. Microbiology Sri Sathya Sai College for Women EDITOR - COORDINATION COMMITTEE V (A-II) Dr.(Smt.)Renu Mishra,HOD,Botany & Microbiology,Sri Sathya Sai College for Women, Bhopal - Dr. Abha Swarup, Director, Printing & Translation Major Pradeep Khare, Consultant, Printing & Translation POST GRADUATE PROGRAMME M.Sc. CHEMISTRY( PREVIOUS) DISTANCE EDUCATION SELF INSTRUCTIONAL MATERIAL 108 Paper-V(A-II) BIOLOGY FOR CHEMISTS BLOCK :II MADHYA PRADESH BHOJ OPEN UNIVERSITY BHOPAL (M.P.) UNIT II Carbohydrates Introduction The carbohydrates, or saccharides, are most simply defined as polyhydroxy aldehydes or ketones and their derivatives. Monosaccharides, consist of a single polyhydroxy aldehyde or ketone unit. The most abundant monosaccharide is the six-carbon D-glucose; it is the parent monosaccharide from which most others are derived. D-Glucose is the major fuel for most organisms and the basic building block of the most abundant polysaccharides, such as starch and cellulose.Oligosaccharides (Greek oligo, ―few‖) contain from two to ten monosaccharide units joined in glycosidic linkage. Polysaccharides contain many monosaccharide units joined in long linear or branched chains. Most polysaccharides contain recurring monosaccharide units of only a single kind or two alternating kinds.Polysaccharides have two major biological functions, as a storage form of fuel and as structural elements. In the biosphere there is probably more carbohydrate than all other organic matter combined. Starch is the chief form of fuel storage in most plants, whereas cellulose is the main extracellular structural component of the rigid cell walls and the fibrous and woody tissues of plants. Glycogen, which resembles starch in structure, is the chief storage carbohydrate in animals. Others polysaccharides serve as major components of the cell walls of bacteria and of the soft cell coats in animal tissues. Lipids are esters of higher fatty acids. Lipids are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents, e.g., choloroform, ether, or benzene. 109 There are several different families or classes of lipids but all derive their distinctive properties from the hydrocarbon nature of a major portion of their structure. (1) as structural components of membranes, (2) as storage and transport forms of metabolic fuel, (3) as a protective coating on the surface of many organisms, and (4) as cell-surface components concerned in cell recognition, species specificity, and tissue immunity. Some substances classified among the lipids have intense biological activity; they include some of the vitamins and hormones.Although lipids are a distinct class of biomolecules, we shall see that they often occur combined, either covalently or through weak bonds, with members of other classes of bio-molecules to yield hybrid molecules such as glycolipids, lipoproteins, which contain both lipids and proteins. In such biomolecules the distinctive chemical and physical properties of their components are blended to fill specialized biological functions. CARBOHYDRATES 1.0 Introduction 1.1 Objectives 1.2 Configuration of Monosaccharides and their family 1.3 Structure and function of important derivatives of monosaccharides 1.3.1 Glycosides 1.3.2 Deoxysugars 1.3.3 Myoinositol 1.3.4 Aminosugars 1.3.5 N-acetyle muramic acid 1.3.6 Sialic acid 1.3.7 Disaccharides 1.3.8 Polysaccharide 1.4 Structural Polysaccharides 1.4.1 Cellulose and ehitin 1.5 Storage polysaccharides 1.5.1 Starch and Glycogen 1.6 Structure and biological functions of glucosaminoglycans or mucopolysaccharides 1.7 Carbohydrates of glycoprotiens and glycolipids 110 1.8 Role of sugars in biological recognition blood group substance – Ascorbic acid 1.9 Carbohydrates metabolism 1.9.1 Kreb’s cycle 1.9.2 Glycolysis 1.9.3 Glycogenesis 1.9.4 Glycogenolysis 1.9.5 Gluconeogenesis 1.9.6 Pentose Phosphate pathway. 2.0 Let us sum up 2.1 Check your progress key 2.2 Assignment/Activity 2.3 References CARBOHYDRATE 1.0 INTRODUCTION The carbohydrates, or saccharides, are most simply defined as polyhydroxy aldehydes or ketones and their derivatives. Many have the empirical formula (CH2O)n, which originally suggested they were ―hydrates‖ of carbon. Monosaccharides, also called simple sugars, consist of a single polyhydroxy aldehyde or ketone unit. The most abundant monosaccharide is the six-carbon D-glucose; it is the parent monosaccharide from which most others are derived. D-Glucose is the major fuel for most organisms and the basic building block of the 111 most abundant polysaccharides, such as starch and cellulose. Oligosaccharides (Greek oligo, ―few‖) contain from two to ten monosaccharide units joined in glycosidic linkage. Polysaccharides contain many monosaccharide units joined in long linear or branched chains. Most polysaccharides contain recurring monosaccharide units of only a single kind or two alternating kinds. Polysaccharides have two major biological functions, as a storage form of fuel and as Fig. 1 structural elements. In the biosphere there is probably more carbohydrate than all other organic matter combined, thanks largely to the abundance in the plant world of two polymers of D-glucose, starch and cellulose. Starch is the chief form of fuel storage in most plants, whereas cellulose is the main extracellular structural component of the rigid cell walls and the fibrous and woody tissues of plants. Glycogen, which resembles starch in structure, is the chief storage carbohydrate in animals. Others polysaccharides serve as major components of the cell walls of bacteria and of the soft cell coats in animal tissues. 1.1 OBJECTIVES FAMILY OF MONOSACCHARIDES Monosaccharides have the empirical formula (CH2O)n, where n=3 or some larger number. The carbon skeleton of the monosaccharides common is unbranched and each carbon 112 atom except one contains a hydroxyl group; at the remaining carbon there is a carbonyl oxygen, which, as we shall see, is often combined in an acetal or ketal linkage. If the carbonyl groups is at the end of the chain, the monosaccharide is an aldehyde derivative and called an aldose; if it is at any other position, the monosaccharides is a ketone derivative and called ketose. The simplest monosaccharides are the three carbon trioses glyceraldehydes and dihydrosyacetone. Glyceraldehyde is an aldotriose; dihydroxyacetone is a ketotriose. Also among the monosaccharides are the tetroses (four carbons), pentoses (five carbons), hexoses (six carbons), heptoses (seven carbons), and octoses (eight carbons). Each exists in two series, i.e., aldotetroses and ketotetroses, aldopentoses and ketopentoses, aldohexoses and ketohexoses, etc. The structures of D aldoses and D ketoses are shown. In both classes of monosaccharides the hexoses are by far the most abundant. However, aldopentoses are important components of nucleic acids and various polysaccharides; derivatives of trioses and heptoses are important intermediates in carbohydrate metabolism. All the simple monosaccharides are white crystalline solids that are free soluble in water but insoluble in nonpolar solvents. Most have sweet taste. 1.2 CONFIGURATION OF MONOSACCHARIDES & THEIR FAIMILY First, we must clarify two terms often confused. Configuration denotes the arrangement in space of substituent groups in stereoisomers such structures cannot be inter-converted without breaking one or more covalent bonds. Conformation refers to the spatial arrangement of substituent groups that are free to assume many different positions, without breaking bonds, because of rotation about the single bonds in the molecule. In the hydrocarbon ethane, for example, one might expect complete freedom of rotation around the C-C single bond to yield an infinite number 113 of conformations of the molecule. However, the staggered conformation is more stable than all others and thus predominates, whereas the eclipsed form is least stable. All the monosaccharides expect dihydroxyacetone contain one or more asymmetric carbon atoms and thus are chiral molecules. Glyceraldehyde contains only one asymmetric carbon atom and therefore can exist as two different stereoisomers (Figure- 4). It will be recalled that C- and L- Glyceraldehyde are the reference, or parent, compounds for designating the absolute configuration of all stereoisomeric compounds. Aldotetroses have two asymmetric carbon atoms and aldopentoses have three. The aldohexoses have four asymmetric carbon atoms and thus exist in the form of 2n = 24 = 16 Fig. 3 different stereoisomers, 8 of which are Dshown in Figure- 2. As expected, the monosaccharides with asymmetric carbon atoms are optically active. For example, the usual form of glucose found in nature in dextrorotatory ([α]20 = +52.70), and the usual form of fructose is levorotatory ([α]20 = -92.40), but both are members of the D series since their absolute configurations are related to D-glyceraldehyde. For sugars having two or more asymmetric carbon 114 atoms, the convention has been adopted that the prefixes D and L refer to the asymmetric carbon atoms farthest removed from carbonyl carbon atom. Fig4 shows the projection formulas of the D aldoses. All have the same configuration at the asymmetric carbon atom farthest from the carbonyl carbon, but because most have two or more asymmetric carbon atoms, a number of isomeric D aldoses exist, most important biologically being Dglyceraldehyde. D-ribose, D-glucose, D- mannose, and D-galactose. Figure-4 shows the projection formulas of the D ketoses; all share the same configuration at the asymmetric carbon atom farthest from the carbonyl group. Ketoses are sometimes designated by inserting ul into the name of the corresponding aldose; e.g., D-ribulose is the Fig-4 The stereoisomers of glyceraldehyde, showing projection formulas (top) and perspective formulas (bottom). ketopentose corresponding to the aldopentose D-ribose. The most important ketoses biologically and dihydroxyacetone. Dribulose, and D-fructose. Aldoses and ketoses of the L series are mirror images of their D counterparts. L sugars are found in nature, but they are not so abundant as D sugars. Among the most important are L-fucose, L-rhamnose , and L-sorbose. Two sugars differing only in the configuration around one specific carbon atom are called epimers of each other. Thus, D-glucose and D-mannose are epimers with 115 respect to carbon atom 2, and D-glucose and D-galactose are epimers with respect to carbon atom 4. 1.3 STRUCTURE AND FUNCTION OF IMPORTANT DERIVATIVES OF MONOSACCHARIDES 1.3.1 GLYCOSIDES Aldopyronoses readily react with alcohols in the presence of a mineral acid to form anomeric α- and β-glycosides. The glycosides are asymmetric mixed acetals formed by the reaction of the anomeric carbon atoms of the intramolecular hemiacetal or pyranose form of the aldohexose with a hydroxyl group furnished by an alcohol. This is called a glycosidic bond. The aromeric carbon in such glycosides is asymmetric. D- -D-glycopyranoside ([α] = 158.90), and methyl β -D-glucopyranoside ([α] = -34.20). The glycosidic linkage is also formed by the reaction of the numeric carbon of a monosaccharide with a hydrosyl group of another monosaccharide to yield a disaccharide. Oligosaccharides and polysaccharides are chains of monosaccharides joined by glycosidic linkages. The glycosidic linkage is stable to bases but is hydrolyzed by boiling with acid to yield the free monosaccharide and free alcohol. Glycosides are also hydrolyzed by enzymes called glycosidases, which differ in their specificity according to the type of glycosidic bond (α or β), the structure of the monosaccharide unit (s), and the structure of the alcohol. Whether a given glycoside exists in furanose or pyranose form can be ascertained by osicative degradation with periodic acid, which cleaves 1,2-dihydroxy compounds. Treatment of methyl α -D-gludopyranoside with periodate cleaves the pyranose ring 116 to yield a dialehyde and formic acid. (Figure 10-14). Periodate cleavage of methyl α -D-arabinofuranoside yields the same dialdehyde but no formic acid. 1.3.2 DEOXYSUGARS Several deoxy sugars are found in nature. The most abundant is 2deoxy-D-ribose, the sugar component of deoxyribonucleic acid. L- Rhamnose (6-deoxy-L-mannose) and L-fructose (6-deoxy-L-galactose) are important components of some bacterial cell walls. Fig- 5 1.3.3 SUGAR ALCOHOLS - MYOINOSITOL The carbonyl group of monosaccharides can be reduced by H2 gas in the presence metal catalysts or by sodium amalgam in water to form the corresponding sugar alcohols, D-Glucose, for example Yields the sugar alcohol D-glucitol, also formed by reduction of L-sorbose and often called L-sorbitol. D-Mannose yields D- 117 of mannitol. Such reductions can also be carried out by enzymes. Two other sugar alcohols occur in nature in some abundance. One is glycerol, an important component of some lipids. The other is the fully hydroxylated cyclohexane derivateive inositol, which can exist in Fig- 6 several stereoisomeric forms. One of the stereoisomers of inositol, myo-inositol, is found not only in the lipid phosphatidylinositol but also in phytic acid, the hexaphosphoric ester of inositol. The calcium-magnesium salt of phytic acid is called phytin; it is abundant in the extracellular supporting material in higher-plant tissues. The structures of some sugar alcohols are shown in fig.6 1.3.4 AMINO SUGARS Two amino sugars of wide distribution are Dglucosamine (25-amino-2-deoxy-D-glucose) and D-galactosamine (2-amino-2-deoxy-D- galactose), in which the hydroxyl groups at carbon atom 2 is replaced by an amino group. Dglucosamine occurs in many polysaccharides of vertebrate tissues and is also a major component of chitin, a structural polysaccharide found in the exoskeletons of insects and crustaceans. DGalactosamine is a component of glycolipids and of the major polysaccharide of cartilage,and chondroitin sulfate. Fig- 7 1.3.5 MURAMIC ACID AND NEURAMINIC ACID AND SIALIC ACID 118 These sugar derivatives are important building blocks of the structural polysaccharides found in the cell walls of bacteria and the cell coats of higher-animal cells respectively. Both are nine-carbon amino sugar derivatives; they may be visualized as consisting of a sixcarbon amino sugar linked to a three-carbon sugar acid; the amino group is usually acetylated. N-Acetylmuramic acid is a major building blocks of the polysaccharide back-bone of bacterial cell walls. It consists of N-acetyl-D-glucosamine Fig- 8 in ether linkage with the three-carbon- D-lactic acid. N-Acetylneuraminic acid is derived from N-acetyl-dmannosamine and pyruvic acid. It is an important building block of the oligosaccharide chains found in the glycoproteins and glycolipids of the cell coasts and membranes of animal tissues. N-Acyl derivatives of neuraminic acid are generically called sialic acids. The sialic acids found in human tissues contain an Nacetyl group; in some other species they contain an N-glycolyl group. 1.3.6 DISACCHARIDES Disaccharides consist of two monosaccharides joined by a glycosidic linkage. The most common disaccharides are maltose, lactose, and sucrose. Maltose, which is formed as an intermediate product of the action of amylases on starch, contains two D-glucose residues. It is a mixed acetal of the anomeric carbon atom 1 of D-glucose; one hydroxyl group is furnished intramolecularly by carbon atom 5 and the other by 119 carbon atom 4 of a second D-glucose molecule. But glucose moieties are in pyronose form, and the configuration at the anomeric carbon atom in glycosidic linkage is α. Maltose may therefore may therefore be called O- α -D-glucopyranosyl-(14)-α-Dglucopyranose. The second glucose residue of maltose had a free anomeric carbon atom capable of existing in α β forms; both the α and β forms are products of enzyme action. The first glucose residue cannot undergo oxidation, but the second residue can; it is called the reducing end. The position of the glycosidic linkage between the two glucose residues is symbolized 1 4. Exhaustive methylation of all the free hydroxyl groups, followed by hydrolysis of the glycosidic linkage, has proved that the glycosidic linkage in maltose involves carbon atom 1 of the first residue and carbon atom 4 of the second glucose unit. The resulting methylated fragments were 2,3,4,6-tetra-O-methyl-Dglucose and 2,3,6-tri-O-methylD-glucose. Two other common disaccharides that contain two D-glucose units are cellobiose and gentiobiose. Cellobiose, the repeating disaccharide unit of cellulose, has a β(14) glycosidic linkage; its full name is thus O-β-D-glucopyranosyl(14)-β-Dglucopyranose. Fig- 9 In 120 gentiobiose, the glycosidic linkage is β (16). Since both these disaccharides have a free anomeric carbon, they are reducing sugars. The disaccharide lactose [O- β -D-glucopyranosyl-(14)- β-D-glucopyranose] is found in milk but otherwise does not occur in nature. It yields D-galactose and Dglucose on hydrolysis. Since it has free anomeric carbon on the glucose residue, lactose is a reducing disaccharide. Sucrose, is a disaccharide of glucose and fructose [O- β -D-fructofuranosyl-(21)- α -D-glucopyranoside]. It is extremely abundant in the plant world and is familiar as table sugar. Unlike most disaccharides and oligosaccharides, sucrose contains no free anomeric carbon atom; the anomeric carbon atoms of the two hexoses are linked to each other. For this reason sucrose does not undergo mutarotation, does not react with phenylhydrazine to form osazones, and does not act as a reducing sugar. It is much more readily hydrolyzed than other disaccharides. 1.3.7 POLYSACCHARIDES (GLYCANS) Most of the carbohydrates found in nature occur as polysaccharides of high molecular weight. On complete hydrolysis with acid or specific enzymes, these polysaccharides yield monosaccharides or simple monosaccharide derivatives. DGlucose is the most prevalent monosaccharide unit in polysaccharides, but polysaccharides of D-mannose, D-fructose, D- and L-galactose, D-xylose, and Darabinose are also common. Monosaccharide derivatives commonly found as structural units of natural polysaccharides are D-glucosamine, D-galactosamine, Dglucuronic acid, N-acetyl-muramic acid, and N-acetylneuraminic acid. Polysaccharides, which are also called glycans, differ in the nature of their recurring monosaccharide units, in the length of their chains, and in the degree of branching. 121 They are divided into homopolysaccharides, which contains only one type of monomeric unit, and heteropolysaccharides, which contain two or more different monomeric units. Starch, which contains only D-glucose units, is a homopolysaccyharide. Hyaluronic acid consists of alternating residues of Dglucuronic acid and N-acetyl-D-glucosamine and is thus a heteropolysaccharide. Homopolysaccharides are given class names indicating the nature of their building blocks. For example, those containing D-glucose units, e.g., starch and glycogen, are called glucans and those containing mannose units are mannans. The important polysaccharides are best described in terms of their biological function. 1.4 STRUCTURAL POLYSACCHARIDES Many polysaccharides serve primarily as structural elements in cell walls and coats, intercellular spaces, and connective tissue, where they give shape, elasticity, or rigidity to plant and animal tissues as well as protection and support to unicellular organisms. Polysaccharides also are found as the major organic compounds of the exoskeletons of many invertebrates. For example, the polysaccharide chitin, a homopolymer of N-acetyl-D-glucosamine in β(14) linkage, is the major organic element in the exoskeleton of insects and crustacean. Cell walls and coats are not only important in maintaining the structure of tissues but also contain specific cell-cell recognition sites important in the morphogenesis of tissues and organs. They also contain other protective elements, such as the cellsurface antibodies of vertebrates tissues. For this reason we shall examine the 122 structural polysaccharides in the context of the molecular organization of cell walls and coats. 1.4.1 Plant Cell Walls- Cellulose & Chitin Since plant cells must be able to withstand the large osmotic pressure difference between the extracellular and intracellular fluid compartments, they require rigid cell walls to keep from sweeling. In larger plants and trees the cell walls not only must contribute physical strength or rigidity to stems, leaves, and root tissues but must also be able to sustain large weights. The most abundant cell-wall and structuaral polysaccharide in the plant world is cellulose, a linear polymer of D-glucose in β (14) linkages. Cellulose is the major component of wood and thus of paper; cotton is nearly pure cellulose. Cellulose is also found in some lower invertebrates. It is almost entirely of extracellular occurrence. On complete hydrolysis with strong acids, cellulose yields only D-glucose, but partial hydrolysis yield the reducing disaccharide cellobiose , in which the linkage between the D-glucose units is β(14). When cellulose is exhaustively methylated and then hydrolyzed, it yields only 2,3,6-tri-O-methylglucose, showing not only that all its glycosidic linkage are (14) but also that there are no branch points. The only chemical difference between starch and cellulose, both homopolysaccharides of Dglucose, is that starch α (14) linkages and cellulose β (14). Cellulose is not attacked by either α β β (14) linkages of cellulose are not secreted in the digestive tract of most mammals, and they cannot use cellulose for food. However, the ruminants, e.g., the cow, are an exception: they can utilize cellulose as food since bacteria in the rumen form the enzyme cellulase, which hydrolyzes cellulose to D-glucose. 123 The minimum molecular weight of cellulose from different sources has been estimated to vary from about 50,000 to 2,500,000 in different species, equivalent to 300 to 15,000 glucose residues. X-ray diffraction analysis indicates that cellulose molecules are organized in bundles of parallel chains to form fibrils. Although cellulose has a high affinity for water, it is completely in soluble in it. In the cell walls of plants densely packed cellulose fibrils surround the cell in regular parallel arrays, often in criss-cross layers. These fibrils are cemented together by a matrix of three other olymeric materials: hemicellulose, pectin, and extensin. Hemicelluloses are not related structurally to cellulose by are polymers of pentoses, particularly D-xylans, polymers of D-xylose in β(14) linkage with side chains of arabinose and other sugars. Pectin is a polymer of methyl D-galacturonate. Extensin, a complex glycoprotein, is attached covalently to the cellulose fibrils. Extensin resumbles its animals tissue counterpart collagen in beng rich in hydroxyproline residues; it also contains many side chains with arabinose and galactose residues. The cell walls of higher plants can be compared to cases of reinforced concrete, in which the cellulose fibrils correspond to the steel rods and the matrix materials to the concrete. These walls are capable of withstanding enormous weights and physical stress. Wood contains another polymeric substance, lignin, which makes up nearly 25 percent of its dry weight. Lignin is a polymer of aromatic alcohols. Other polysaccharides serving as cell-wall or structural components in plants include agar of seaweeds, which contains D- and L- galactose residues, some of which are esterified with sulfuric acid; alginic acid of algae and kelp, which contains Dmannuronic acid units; and gum arabic, a vegetable gum, which contains D-galactose and D-glucuronic acid residues, as well arabinose and rhamnose. CHITIN Those amino sugars which occur in nature in combination with protein and glucosamine, are called chitin. Generally, it is made up of chitibiose, a disachharide, 124 which on decomposition yield N-acytyl glucosamine when chitin is hydrolysed by acids, it yields acetic-acid and glucosamine, it occurs in shells of arthropods, lenses of eyes, lining of digestive tract, respiratory and excretory tract of insects. Bacterial Cell Walls The cell walls of bacteria are rigid, porous, boxlike structures that provide physical protection to the cell. Since bacteria have a high internal osmotic pressure, and since they are often exposed to a quite variable and sometimes hypotonic external environment, they must have a rigid cell wall to prevent swelling and rupture of the cell membrane. The structure and biosynthesis of bacterial cell walls have been intensively studied. The covalently linked framework of the cell wall actually may be regarded as a single large sacklike molecules. It is called a peptidoglycan or murein (latin murus, wall). The structure and enzymatic biosynthesis of the peotidoglycan were largely elucidated during investigations on the mechanism of action of the antibiotic penicillin by J. Strominger and his colleagues. The basic recurring unit in the peotidoglycan structure is the muropeptide. It is a disaccharides of N-acetyl-Dglucosamine and N-acetylmuramic acid in β( 4) linkage. The backbone may be regarded as a substituted chitin with D-lactic acid substituted on alternating residues. To the carboxyl group of the N-acetylmuramic acid residues of the backbone are attached tetrapeptide side chains each containing L-alanine, D-alanine, D-glutamic acid or D-glutamine, and either meso-diaminopimelic acid, L-lysine, Lhydroxyllysice, or ornithine depending on the bacterial species. The parallel polysaccharide chains of the cell wall are cross-linked through their peptide side chains. The terminal D-alanine residue of the side chain of one polysaccharide chain is joined covalently with the peptide side chain of an adjacent polysaccharide chain, either directly, as in E. coli, or through a short connecting peptide, e.g., the pentaglycine in Staphylococcus aureus. The peptidoglycan forms a 125 completely continuous, covalent structure around the cell; Gram-positive bacteria are encased by up to 20 layers of cross-linked peptidoglycan. The peptidoglycan structure of the bacterial cell wall is resistant to the action of peotide-hydrolyzing enzymes, which do not attack peptides containing D-amino acids. In addition to the peptidoglycan framework, bacterial cell walls contain a number of accessory polymers, which make up almost 50 percent of the weight of the wall. These accessory components differ from one species to the another. There are three types of accessory polymers: (1) teichoic acids, (2) polysaccharides, and (3) polypeptides or proteins. The walls of Gram-negative cells, such as E. coli, are much more complex than those of Gram-positive cells. Their accessory component consist of polypeptides, lipoproteins, and particularly, a very complex lipopolysaccharide whose structure is just beginning to be understood. It has a trisaccharide backbone repeating unit, consisting of two heptose (seven-carbon) sugars and octulosonic acid (an eight carbon sugar). To this backbone are attached oligosaccharide side chains and the fatty acid β-hydroxymyristic acid, which gives this complex structure its lipid character. The lipopolysaccharide forms an outer lipid membrane and contributes to the complex antigenic specificity of Gram-negative cells. The cell wall of Gram-positive organisms can be tough of as a rigid, brittle box, like the 126 shell of a crustacean, whereas the cell wall of Gram-negative organisms has an outer lipid-rick skin, with the rigid peptidoglycan skeleton buried underneath. 1.5 STORAGE POLYSACCHARIDE These polysaccharides, of which starch is the most abundant in plants and glycogen in animals, are usually deposited in the form of large granules in the cytoplasm of cells. Glycogen or starch granules can be isolated from cell extracts by differential centrifugation. In times of glucose surplus glucose units are stored by undergoing enzymatic linkage to the ends of starch of glycogen chains; in times of metabolic need they are released enzymatically for use as fuel. 1.5.1 STARCH Starch occurs in two forms, α -amylose and amylopectin. α -Amylose consists of long unbranched chains in which all the D-glucose units are bound in α (14) linkages. The chains are polydisperse and vary in molecular weight from a few thousand to 500,000. Amylose is not truly soluble in water but forms hydrated micelles, which give a blue color with iodine. In such micelles, the polysaccharide chain is twisted into a helical coil. Amylopectin is highly branched; the average length of the branches is from 24 to 30 glucose residues, depending on the species. The backbone glycosidic linkage is α (14), but the branch points are α (16) linkages. Amylopectine yields colloidal or micellar solutions, which give a red-violet color with iodine. Its molecular weight may be a high as 100 million. The major components of starch can be enzymatically hydrolyzed in two different ways. Amylose can be hydrolyzed by α α(14)]-glucan 4- glucanohydrolase], which is present in saliva and pancreatic juice and participates in the digestion of starch in the gastrointestinal tract. It hydrolyzes α(14) linkages at random to yield a mixture of glucose and free maltose; the latter is not attacked. 127 Amylose can also hydrolyzed by β α(14)-glucan maltohydrolase]. This enzyme, which occurs in malt, cleaves away successive maltose units beginning from the nonreducing end to yield maltose quantitatively. The α- and β- amylases also attact amylopectine. The polysaccharides of intermediate chain length that are formed from starch components by the action of amylases are called dextrins. Neither α- nor β-amylases can hydrolyze the α (16) linkages at the branch points of amylopectine. The end product of exhaustive β -amylase action on amylopectin is a large, highly branched core, or limit dextrin, so called because it represents the limit of dextrin, so called because it represents the limit of the attack of β -amylase. A debranching enzyme [α (16)-glucan 6-glucanohydrolase, also called α (16)glucosidase] can hydrolyze the α (16) linkages at the branch points. The combined action of a β -amylase and an α(16)-glucosidase can therefore completely degrade amylopectin to maltose and glucose. 1.5.2 GLYCOGEN Glycogen is the main storage polysaccharide of animal cells, the counterpart of starch in plant cells. Glycogen is especially abundant in the liver, where it may attain up to 10 percent of the wet weight. It is also present to about 1 to 2 percent in skeletal muscle. In liver cells the glycogen is found in large granules, which are themselves clusters of smaller granules composed of single, highly branched molecules with a molecular weight of several million. Like amylopectin, glycogen is a polysaccharide of D-glucose in α (14) linkage. However, it is a more highly branched and more compact molecule than amylopectine; the branches occur about ever 8 to 12 glucose residues. The branch linkages are α (16). Glycogen can be isolated from animal tissues by digesting 128 them with hot KOH solutions, in which the nonreducing α (14) and α (16) linkages are stable. Glycogen is readily hydrolyzed by α - and β -amylases to yield glucose and maltose, respectively; the action of β -amylase also yields a limit dextrin. Glycogen gives a red-violet color with iodine. 1.5.3 OTHER STORAGE POLYSACCHARIDES Dextrans, too, are branched polysaccharides of D-glucose, but they differ from glycogen and starch in having backbone linkages other than α (14). Found as storage polysaccharides in yeasts and bacteria, they vary in their branch points, which may be 12, 13, 14, or 16 in different species. Dextrans form highly viscous, slimy solutions. Fractans (also called levans) are homopolysaccharides composed of D-fructose units; they are found in many plants, Inulin, found in the artichoke, consists of D-fructose residues in β (21) linkage. Mannans are mannose homopolysaccharides found in bacteria, yeasts, molds, and higher plants. Similarly, xylans and arabinanas are homopolysaccharides found in plant tissues. 1.6 STRUCTURE AND FUNCTION OF MUCOPOLYSACCHARIDES OR GLUSOSAMINOGLYCANS The acid mucopolysaccharides are a group of related heteropolysaccharides usually containing two types of alternating monosaccharide units of which at least one has an acidic group, either a carboxyl or sulfuric group. When they occur as complexes with specific proteins, they are called mucins or mucoproteins; in this class of glycoproteins the polysaccharide makes up most of the weight. Mucoproteins are jellylike, sticky, or slipery substances; some provide lubircation, and some function as a flexible intercellular cement. 129 Table 10-2 Acid mucopolysaccharides Polysaccharides Constituents Occurrence Hyaluronic acid Glucuronic acid, N-acetyl-D-glucosamine Synovial fluid Chondroitin Chondroitin Glucuronic acid, N-acetyl-D-glucosamine Cornea 4- Glucuronic acid, N-acetyl-D-glucosamine 4- Cartilage sulfate sulfate Dermatan sulfate Iduronic acid, N-acetyle-D-galactosamine 4- Skin sulfate Keratan sulfate Galactose, galactose 6-sulfate, N-acetyl-D- Cornea glucosamine 6-sulfate Heparin Glucosamine 6-sulfate, glucuronic acid 2- Lung sulfate, iduronic acid The most abundant acid mucopolysaccharides is hyaluronic acid, present in cell coats and in the extracellular ground substance of the connective tissues of vertebrates; it also occurs in the synovial fluid in joints and in the vitreous humor of the eye. The repeating unit of hyaluronic acid is a disaccharide composed of Dglucuronic acid and N-acetyl-D-glucosamine in β(3) linkage. Since each dissacharide unit is attached to the next by β (4) linkages, hyaluronic acid contains alternating β (3) and β (4) linkages. Hyaluronic acid is a linear polymer. Because its carboxyl groups are completely ionized and thus negatively charged at pH 7.0, hyaluronic acid is soluble in water, in which it forms highly viscous 130 solutions. The enzyme hyaluronidase catalyzes hydrolysis of the β (4) linkages of hyaluronic acid; this hydrolysis of accompanied by a decrease in viscosity. Another acid mucopolysaccharide is chondroitin, which is nearly identical in structure to hyaluronic acid; the only difference is that it contains N-acetyl-Dgalactosamine instead of N-acetyl-D-glucosamine residues. Chondroitin itself is only a minor component of extracellular material, but its sulfuric acid derivatives, chondroitin 4-sulfate (chondroitin A) and chondroitin 6-sulfate (chondroitin C), are major structural components of cell coats, cartilage, bone, cornea, and other connective-tissue structures in vertebrates. Dermatan sulfate and keratan sulfate are acid mucopolysaccharides found in skin, cornea, and bony tissues. A related acid mucopolysaccharide is heparin, which prevents coagulation of blood, It is found in the lungs and in the walls of arteries. Recently it has been discovered that some acid mucopolysaccharides contain the element silicon, which is essential in the nutrition of rats and chicks. The silicon is bound to the polysaccharides in covalent form. 1.7 CARBOHYDRATES OF GLYCOPROTEINS AND GLYCOLIPIDS 1.7.1 GLYCOPROTEINS Among the several different classes of conjugated proteins, the glycoproteins, which contains carbohydrate groups attached covalently to the polypeptide chain, represent a large groups of wide distribution and considerable biological significance. In fact, on closer study many proteins once thought to be simple proteins, i.e., containing only amino acid residues, have been found to contain carbohydrate groups. The percent by weight of carbohydrate groups in different glycopoteins may vary from less than 1 percent in ovalbumin to as high as 80 percent in the mucoproteins. Glycoproteins having a very high content of carbohydrate are called proteoglycans. 131 Glycoproteins are found in all forms of life. In vertebrates most but not all of the glycoproteins are extracellular in occurrence and function or are secreted from cells; it has accordingly been suggested that one pupose of the attached sugar residues is to label the protein for export from the cell. Among the glycoproteins having extracellular location or function are the cell-coat glycoproteins, the blood glycoproteins, the cirulating forms of some protein hormones, the antibodies, various digestive enzyems secreted into the intestine, the mucoproteins of mucous secretions, and the glycoproteins of extracellular basement membranes. Many different monosaccharides and monosaccharide derivatives have been found in glycoproteins. The linear or branched side chains of glycorproteins may contain from two to dozens of monosaccharide residues, usually of two or more kinds. Often the terminal monosaccharide unit is a negatively charged of N-acetylneuraminic acid, a sialic acid. The oligosaccharide groups of most glycoproteins are convalently attached to the R groups of specific amino acid residues in the polypeptide chain. Three different types of linkages have been found. In some glycoproteins, e.g., ovalbumin and the immunoglobulins, the oligosaccharide is attached via a glycosylamine linkage between N-acetyl-D-lucosamine of the bligosaccharide to the amide nitrogen of an asparagine residue in the polypeptide chain. In a second class of glycoproteins, including the submaxillary mucoprotein, there is a glycosidic bond between Nacetyl-D-galactosamine of the side-chain oligosaccharide and the hydroxyl group of a serine or threonine residue. The submaxillary mucins have recurring units of about 28 amino acid residues; each redcurring unit contains theree oligosaccharide side chains. In the third class of glycoproteins, respresented by collagen, the oligosaccharide side chains are attached to the hydroxyl groups of hydroxylysine 132 residues. The precise sequences of residues in the oligosaccharide side chains are known in only a few cases. The antifreeze proteins found in the blood plasma of Antarctic fishes are particularly interesting. Their backbones consist of the recurring amino acid sequence Ala-AlaThr; the disaccharide galactosy-N-acetyl-galactosamine is attached to every threonine residue. The molecular weights of these proteins vary from 10,000 to 23,000. The antifreeze proteins have a flexible, expanded structure in water which presumably interferes with the formation of the crystal lattice of ice. The human blood-group proteins contain oligosacchride side chins with residues of L-fructose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine; these side chains determine the glood-group specificity. Table - Some glycoproteins, grouped according to biological occurrence. Note that most glycoproteins are extracellular. Blood plasma Fetuin a1-Acid glycoprotein fibrinogen Immune Globulins Thyroxine-binding protein Blood-group proteins Urine Urinary glycoprotein Hormones Chorionic gonadotrophin Follicle-stimulating hormone Thyroid-stimulating hormone 133 Enzymes Ribonuclease B b-Glucuronidase Pepsin Serum cholinesterase Egg white Ovalbumin Avidin Ovomucoid Mucus secretions Submaxillary glycoproteins Gastric glycoproteins Connective tissue Collagen Cell membranes Glycophorin of erythrocyte membrane Extracellular Membranes Basement-membrane glycoprotein Lens-capsule glycoprotein 1.7.2 GLYCOLIPIDS These are compounds containing a fatty acid, a carbohydrates, a complex alcohol, and nitrogen, but no phosphorus. Glycolipids, galactolipids or cerebrosides occur in considerable amounts in the white matter of the 134 brain and of all nervous tissue. They usually occur in the amorphous state, but are also known as liquid crystals. They are insoluble in ether but soluble in hot alcohol. On hydrolysis, they give a fatty acid, sphingosinol, and a sugar, usally glactose. There are four individual memebers of this group which differ only in the nature of their fatty acids, The general structural formula for galactolipids is as below. The sphingosin-fatty acid moieiy is called a ceramide. The nature of the fatty acid radical (R.CO) in the four galactolipids is given in the following table. Name of the lipid Fatty acid radical (R.CO -) Name of the fatty acid Cerasin CH (CH2)22 COOH Lignoceric acid Phrenosin CH3 (CH2)22 CHOH.CHOH A (cerebron) Nervone Hydroxylignoceric (phrenosinic) acid CH3 Nervonic acid (CH2)7.CH=CH.(CH2)12.COOH Hydroxy nervone C24H46O3 Hydroxynervonic acid In addition to the above compound lipids there are globosides, hematosides and gangliosides. There are structurally similar to glycosides with the only difference that in the former the sugar residues are acetylated amino sugars, e.g. Dgalactosamine, in the middle one they are sialic acid while in the latter acetylated amino sugars and sialic acid both are present. 1.8 ROLE OF SUGARS IN BIOLOGICAL RECOGNITION BLOOD GROUP SUBSTANCES- VITAMIN C (ASCORBIC ACID) Ascorbic acid is a derivative of carbohydrates. It possesses an asymmetric carbon atom marked by *) and is therefore optically active. It exists in the following two forms. 135 L-Ascorbic acid and L-dehydroascorbic acid are the only known naturally occurring biologically active substances, the corresponding D-forms are generally inactive. It is a white crystalline solid freely soluble in water, its most important chemical property is its powerful reducing activity during which it gives up its two hydrogen atoms and is oxidised to dehydroascorbic acid. This oxidation of ascorbic acid in the body is reversible and affected by the – SH group of glutathione. Howerver, ascorbic acid may be irreversibly oxidised beyond the stage of dehydroascorbic acid to oxalic acid via deketagulonic acid. The stability of ascorbic acid is affected by the pH, temperature,, contact with oxygen and traces of metals especially copper. It is rapidly oxidised in the presence of light, oxygen and traces of coppeer at pH above4, expecially if the solution is warmed. Thus ascorbic acid is liable to be destroyed during preparation and cooking of many foods. Moreover, fruits and vegetables contain an enzyme, ascorbic acid oxidase, which accelerates the oxidation of ascorbic acid in the presence of oxygen. The vitamin is distributed both in plant and animal kingdoms. In plant community the important sources are leaves and flowers (e.g., rose hips, pine needles), fruit (e.g., lemons oranges, black currants, grapefruit, guava, gooseberries, strawberries, apples, bananas, etc) and green vegetables (e.g. cauliflowers, cabbage, green peas, beans, tomatoes, etc.) Because of the chemical instability of ascorbic acid, much of it is 136 destroyed in the preparation of food. In animal, the vitamin occurs in tissue and various glands or organs, e.g. adrenal gland, thymus, pituitary, corpus luteum, liver, lung and heart muscle. Milk, potatoes and blood also contain small quantity of ascorbic acid. In plants, mostly, the vitamine occurs as such but in animsl and some plants it is found in equilibrium with dehydroascorbic acid. Besides these two forms ascorbic acid is also found in the combined form (ascorbigen). Ascorbic acid is absorbed readily from the small intestice, peritoneum, and subcutaneous tissue. Although the human body has reserve stores of the vitamin, there is no evidence to show that any particular organ or tissue serves this function. Under normal dietary conditions, about 25-50 % of the ingested vitamin is excreted in the urine; the rest being degraded to diketogulonic acid and oxalic which are excreted in the urine. It is also secreted in the milk. Function: The biological activity of this vitamin is due to its reversible oxidation. It has been seen that there are special enzymes or compounds which help in the oxidation and reduction of ascorbic acid; e.g., glutathione reduces the oxidised form of vitamin C; whereas some purines (xanthine, uric acid, theophyline etc.) protect the vitamin against oxidation. On the other hand, the enzyme ascorbic acid oxidase brings about the oxidation of ascorbic acid. On the basis of the above behavious or ascorbic acid, Szent-Gyorgyl suggested that the vitamin takes part in the respiratory system according to the follwing reactions. Ascorbic acid + O2 Flavone + H2O Dehydro-ascorbic acid + H2O ..i Oxidised flavone + H2O Oxidized flavone + Ascorbic acid ..ii Dehydroascorbic acid + flavone ..iii Dehydroascorbic acid + glutathione Ascorbic acid + Oxidized glutathione … iv 137 Oxidized glutathione + Glucose phosphate Glutathione + H2O2 + H2O …v In the absence of the compounds such as flovones the reactions (ii) and (iii) are replaced by H2O2 H2O + ½ O2 Ascorbic acid is also found to be linked with the amino acid metabolism. Check your progress – 1 1. Note : - Write your answer in the space given 2. Compare your answer with the one given at the end of the unit. 1. What are carbohydrates? What is their functions significance. 2. Write one function of : 3. Deoxy sugars, N-acitylmeramic acid, sialic acid & cellulose, Glycogen. 4. What are muco polysaccharides. 1.9 CARBOHYDRATE METABOLISM ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.AN OVERVIEW OF RESPIRATION. --------------------------------------------------------------------------------------------------------- 138 a) You must bear a clear understanding in mind that both photosynthesis and respiration involves gaseous exchange but light reaction of photosynthesis requires sunlight whereas respiration occurs all the time. O2 is utilized in the process &Co2 is released. b) The sites of respiration are cytoplasms and mitochondria. The organic compounds are broken down inside the cells by oxidation process, known as cellular respiration. The energy released is stored in pyrophosphate bonds of ATP. ATP(ADP˜P) ADP + H3PO4 Energy stored in ATP is utilized for carying out different cellular and biological activites because of this, energy is called energy currency of the cell. c) The overall reaction is as follows: C6H12O6 + 6O2 + 38ADP+38iP 6CO2 + 6H2O + 38ATP The main features of respiration are: Oxidation of organic compounds occurs in under aerobic conditions Complete oxidation occurs End products are CO2 & H2O Higher amount of (673 Kcal )energy is liberated out Process occurs in cytoplasm and mitochondria Various respiratory substance are: glucose, fructose, fats, protein, etc. The ratio of volume of CO2 released to the volume of O2 absorbed during respiration is called respiratory ratio or R.Q. Volume of CO2 released R.Q. = 139 Volume of O2 absorbed To develop a clear understanding of the process let us understand the mechanism of respiration MECHANISM OF RESPIRATION Cellular respiration is a complicated process which is completed in many steps. for every step, a particular enzyme is required which works in a sequential manner one after the another. it is completed in 3 steps: d) Glycolysis / EMP pathway e) Oxidation of pyruvic acid f) ETC & oxidative phosphorylation 1.9.1 GLYCOLYSIS/ EMP PATHWAY Greek, glucose – sugar, lysis – dissolution. If I say that glycolysis is a fermentive pathway would you agree? Reasons to support my statement are: d) It does not involves O2 intake e) ATP generated is through substrate level phosphorylation. f) Organic compound donates electrons and organic compound accepts it. This process was discovered by three German scientists Embden, Meyernhof and Parnas. On their name the pathway is also called EMP pathway. All the reactions of glycolysis take place in the cytoplasm and through the glycolysis glucose is oxidized into pyruvic acid in presence of many 140 enzymes present in the cytoplasm. Thus the process of sequential oxidation of glucose into pyruvic acid is known as glycolysis. Energy production during glycolysis: During glycolysis process two molecules of ATP are utilized to convert glucose into glucose-6-PO4 & fructose -1, 6 diphosphate wherea 4 molecules of ATP and 2 molecules of NADH2 are produced during following steps. 141 (One molecule of NADH2 gives three molecules of ATP by ETC) Total production of ATP in glycolysis cycle Reaction number No. of ATP molecule produced (vii) 1,3 –DPG-Ald 1,3-DPGA (viii)1,3 – DPGA 3-PGA (xi) PEPA Pyruvic Acid 2NADH2(2*3) = 6ATP 2ATP = 2ATP 2ATP = 2 ATP 10ATP As 2 molecules of ATP are utilized during glycolysis, thus net gain of ATP molecules during this process is 8 molecules of ATP 10 ATP – 2 ATP Net gain of ATP = 8 ATP SIGNIFICANCE OF GLYCOLYSIS: a) Generates ATP b) Precursor metabolic generation c) Generates reducing power Main enzymes are: 1) phosphofructokinase 2) pyruvate kinase 3) pyruvate enol carboxylase 142 General patter of metabolism leading to synchronization in Ecoli cells Role of ATP : Adenosine - P ~ P ~ P + H2O adenosine - P~P + P 4° = -7.8Kcal Adenosine - P ~ P + H2O adenosine ~P + P 4° = 7.3Kcal Adenosine ~ P + H2O adenosine + P 4° =3.4Kcal High energy compounds other than ATP Compound cause action in Priosyn.of: GTP Protein(ribosome function) CTP Phospholipids UTP Peptidoglycan layer of bacterial wall Dcoxythymidine~ P~P~P lipopolysaccarid layer of bacterial wall dTTTP Acyl~SCoA Fatty acids OXIDATION OF PYRUVIC ACID The fat of pyruvic acid produced during glycolysis depends on whether oxygen is available or not A) In case of anaerobic condition it is used as hydrogen acceptor for the two molecules of NADH generated during glycolysis and is converted into lactic acid. Alcoholic fermentation of pyruvic acid in plants: in yeast cells anaerobic oxidation of pyruvic acid takes place as follows: Decarboxylation of pyruvic acid in presence of pyruvic decarboxylase enxyme to produced acetaldehyde. CH3COOH Pyruvic Decarboxylase CH3CHO + O2 143 1) In presence of alcohol dehydrogenase enzyme acetaldehyde reacts with NADH2 to produce ethyl alcohol and NAD. 2CH3.CHO+2NADH2 A.dehydrogenase Acetaaldehyde CH3.CH2.OH + 2NAD Ethylalcohol In animal cells lactic acid is formed B) Aerobic oxidation of pyruvic acid according to Wood et.al.(1942) and H.A.Kreb‘s (1943) in the presence of O2 oxidation of pyruvic acid takes place through Kreb‘s cycle or T.C.A cycle. Before entering Kreb‘s cycle pyruvic acid gets decarboxylated to produce acetylCoA which enters the Kreb‘s cycle and oxidize to produce CO2 . H2O and ATP. Pyruvic Decarboxylase Pyruvic acid + Coenzyme A + NAD Acetyl-CoA +NADH2 C) Fate of pyruvic acid to alanine during amino acid synthesis pyruvic acid react with glutamic acid alanine. Alanine +α-Keto glutaric acid Pyruvic acid + Glutamic acid 1.9.2 T.C.A. CYCLE/KREB’S CYCLE: This cycle was described for the first time by H.A.Kreb in 1943. It is also known as T.C.A. cycle because it produces tricarboxylic acids the process completes in mitochondrial crests. 144 Diag ram of Mitochondria All the chemical reaction of Kreb‘s cycle can be summarized in following steps: 145 10.Aerobic oxidation of P.A 11.Condensation of Acetyl-CoA with oxalo-acetic acid 12.Isomerisation of citric acid into isocitric acid ,{(a) dehydration and (b) hydration)} 13.Oxidative decarboxylation of isocitric acid (a) dehydration and (b) decarboxylation) 14.Oxidative decarboxylation of α-Keto glutaric acid. 15.Conversion of succinyl CoA into succinic acid. 16.Dehydrogenation of succinic acid into fumaric acid 17.Hydration of fumaric acid into malic acid 18.Dehydrogenation of malic acid in OAA. Overall reaction of respiration is: Glycolysis + Kreb‘s cycle = Glucose + 4ADP + 4H3PO4+ 8NAD++ NADP+ +2FAD 6CO2 + 4 ATP + 8NADH + 10H+ +2NADPH + 2FADH2 Thus as a result of oxidation of pyruvic acid, one molecule of CO 2 in oxidative decarboxylation and two molecules of CO2 in Kreb‘s cycle are liberated. The total number of CO2 evolved becomes 3 which indicates that 3 carbon pyruvic acid has been completely oxidized in glycolysis. Because two molecules of P.A. which are formed by one molecule of glucose in glycolysis, enter into Kreb‘s cycle for oxidation, a total of 6CO 2 molecule will be evolved. 2PA * 3CO2 = 6CO2 All the NADH2 and FADH2 are oxidized to NAD and FAD through a chain of reaction c/a etc.in this process ATP molecules are released (1NADH2 = 3ATP, 1FADH2 = 2ATP). In the process of Kreb‘s cycle 8 molecules of NADH2 =24ATP , 2FADH2 = 4 ATP and two molecules of ATP are synthesized from 2GTP. ELECTRON TRANSPORT SYSTEM AND OXIDATIVE PHOSPHORYLATION 146 Electron Transport System (ETC) During repiration simple carbohydrates and intermediate compounds like phosphoglyceraldehyde, pyruvic acid, isocitric acid, α ketoglutaric acid, succinec acid and malie acid are oxidized. Each oxidative step involves release of a pair of hydrogen atoms which dissociates into two protons and two electrons. 2H 2H+ + 2e- These protons and electrons are accepted by various hydrogen acceptors like NAD,NADP, FAD etc. After accepting hydrogen atoms these acceptors get reduced to produce NADH2, NADPH2 and FADH2. The pairs of hydrogen atoms released a series of coenzymes and cytochromes which form electron transport system, before reacting with O2 to form H2O. ½ O + 2H+ + 2e2NADH + O2 + 2H+ H2O 2NAD++ 2H2O As you know that H ions and electrons removed from the respiratory substrate during oxidation do not directly react with oxygen. Instead they reduce acceptor molecules NAD and FAD to NADH2 and FADH2. These molecules then transfertheir electron to a system of electron acceptors and transfer molecules. The proteins of the inner mitochondrial membrane act as electron transporting enzymes. They are arranged in an ordered manner in the membrane and function in a specific sequence. This assembly of electron transport enzymes is known as mitochondrial respiratory chain or the electron transport chain. Specific enzymes of this chain receive electrons from reduced prosthetic groups, NADH2 or FADH2 produced by glycolysis and the TCA cycle. The electrons are then transported successively from enzyme to enzyme, 147 down a descending ‗stairway‘ of energy yielding reactions. This process takes place in mitochondrial cristae which contain all the components of E,T.S. Components of electron transport system: the electron transport system is made up of following enzymes and proteins: 10.Nicotinamide adenine dinucleotide (NAD). 11.Flavoproteins (FAD and FMN). 12.Fe-S protein complex. 13.Co-enzyme Q or ubiquinone. 14.Cytochome-b 15.Cytochrome-c1. 16.Cytochrome-c 17.Cytochrome-a 18.Cytochrome-a3. All the above enzymes are found in F1 particles of mitochondria. Mechanism of action of electron transport system: During respiration electron pairs liberated from respiratory compounds are accepted by coenzymes like NAD or NADP and FMN etc. The transfer of electrons in all compounds except succinic acid takes place first in NAD+ or NADP+ and later on in FAD. The transfer of electgrons from succinic acid takes place diretly to the FAD and not through NAD+ or NADP+. Due to this reason only two molecules of ATP are formed in the formation of fumaric acid from succinic acid whereas in case of other compounds 3 ATP molecules are produced because these cases the electrons are first picked up by NAD. Different Steps of E.T.S. are as follows: 148 3. Hydrogen pairs released from different substrates of Krebs cycle except succinic acid reacts with NAD+. The electrons and proton are transferred to NAD causing its reduction and one proton is released in the medium. 2H 2H+ + 2e- (protons) (electrons) NAD + 2H+ + 2e- NADH + H+ (reduced) (ion pool) 4. Now, 2e- and one H+ are transferred from NADH to FAD causing oxidation of NADH to NAD and reduction of FMN into FMNH2. One H+ is picked up from hydrogen ion pool to complete this reaction. The free energy released at this step is stored during oxidative phophorylation and one molecule of ATP is generated fronm ADP and inorganic phosphate. The hydrogen pair from succinic acid is first transferred to FAD to form FADH2. The FADH2 transfers electrons to coenzyme Q throught Fe-S and CoQ. The electrons pass to cytochromes Cyt-b, Cyt-c1, Cyt-c, Cyt-a, Cyt-a3 and then to oxygen atoms. Oxygen atom accepts those electrons and reacts with hydrogen ions of the matrix to form water. O2 + 4e- 2(O--) 2(O--) + 4e+ 2H2O Oxygen is thus the terminal electron acceptor of the mitochondrial respiratory chain. At each step of electron acceptor has a higher electron affinity than the electron donor from which it receives the electron. The energy from such electron transport is utilized in transporting protons from the matrix across the inner membrane to its outer side. This creates a higher proton concentration outside the inner membrane 149 than in the matrix. The difference in proton concentration across the inner membrane is called proton gradient. The reduction of various cytochromes requires only electrons and no protons. Each cytochromes possesses an iron elements in the centre which functions for accepting (Fe3+ Fe2+) or donating (Fe2+ Fe3+) When a cytochrome accepts electrons, it is reduced and if it donates electrons, it is oxidized. Oxidative Phosphorylation In all living beings ATP generated during oxidative breakdown of complex food products. This process of synthesis of ATP molecules from ADP and inorganic phosphate by electron transport system of aerobic respiration called as oxidative phosphorylation. ADP + iP O 2 ATP E.T. Chain The process of oxidative phosphorylation takes place in mitochondrial crests through electron transport chain. Due to high proton concentration outside the inner membrane, protons return to the matrix down the proton gradient. Just as a flow of water from a higher to lower level can be utilized to turn a water-wheel or a hydroelectric turbine, the energy released by the flow of protons down the gradient is utilized in synthesizing ATP. The return of proton occurs through the inner membrane particles. In the F0-F1 complex the F1 head piece functions as ATP synthetase. The latter synthesizes ATP from ADP and inorganic phosphate using the energy from the proton gradient. Transport of two electrons from NADH2 by the electron transport chain simultaneously transfers three pairs of protons to the outer compartment. One high energy ATP bond is produced per pair of protons returning to the matrix through the 150 inner membrane particles. Therefore, oxidative phosphorylation produces three ATP molecules per molecules of NADH2 oxidized. Since FADH2 donates its electrons further down the chain. Its oxidation can only produce two ATP molecules. During oxidative phosphorylation ATP molecules are produced during following steps: IV. When NADH2 is oxidized to NAD by reacting with FAD. V. When the electron transfer from cytochrome-b to cytochrome-c1. VI. When the electron transfer from cytochrome-a to cytochrome-a3. Now it is clear that oxidation of one molecule of reduced NADH2 or NADPH2 results in the formation of 3 molecules of ATP while oxidation of FADH2 leads to the formation of 2 molecules of ATP. 1.9.3 PENTOSE PHOSPHATE PATHWAY : AN ALTERNATIVE PATHWAY FOR GLUCOSE BREAKDOWN. 151 Warberg et al (1935) & Dicken‘s suggested an alternative oxidative pathway for glucose oxidation. It is named as Pentose Phosphate Pathway or Hexose Monophosphate shunt. Out of 6 only molecule of glucose monophosphatic is oxidized into CO2 in each cycle of P.P.P & 5 molecules of fructose monophosphatic & glucose monophosphatic are formed. Total 12 NADPH2 molecules are formed in each cycle which are oxidized by cytochrome cycle into 12 molecules of NADP. Total 36 ATP molecules are produced during whole process. Significance of P.P.P. 1. It is substitute for glycolysis and Kreb‘s cycle. 2. 5-carbon compounds produced are used in the synthesis of nucleic acids. 152 3. This pathway can supply required quantity of the energy to the cell, when glycolysis & Kreb‘s cycle do not occur due to some reason. 153 CHECK YOUR PROGRESS 2 1. Note : - Write your answer in the space given 2. Compare your answer with the one given at the end of the unit. 1. What is EMP Pathway? How many ATP are generated through it 2. Name of end product of a. Glycolysis b. Kreb‘s Cycle c. Glycogenesis ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------- 154 1.9.4 GLYCOGEN SYNTHESIS - STORAGE OF CARBOHYDRATESGlycogen synthesis and breakdown. In the absence of urgent physiological demands for oxidative energy or conversion to special products. Excess of glucose is stored in the form of glycogen in the liver and other tissues: the process of biosynthesis of glycogen from glucose (or other sugars) is known as glycogenesis. Glycogen is synthesized in practically all the tissues of the body but the major sites are liver and muscles. The storage of glucose in the form of glycogen, which is converted back to glucose, as the time of requirement, is very important because in the absence of this the tissues would be flooded with excess of glucose immediately after mean and starved of it at all other times. The carbohydrate reserve in the adult of it at all other times. The carbohydrate reserve in the adult human liver (1.8 kg) is about 100 gm and since accumulation of such a large amount of the smaller molecule like glucose will give concentration of glucose inside the liver cells will nearly be doubled leading to disastrous results. It is, therefore, advantageous to the organism to store its glucose in the form of a polymer, glycogen, which has a high molecular weight and correspondingly low osmotic pressure. In case carbohydrate rich diet is taken, the liver tissue may immediately store glycogen about 5-6% of its weight. The liver glycogen may be exhausted after a fast of 12-18 hours. Other important store of glycogen is muscles which may contain about 0.7-1.0% glycogen and since an adult human has about 35 kg. of muscles. Muscle glycogen is utilised in case of severe body exercise or when the liver glycogen is completely exhausted. Now since the amount of glycogen which can be stored in the body is limited, excess quantities of glucose are converted to fatty acids and stored as triglycerides (fat). Glycogenesis Pathway 155 Although synthesis of glycogen from glucose can occur in most of the tissues of the body; liver and muscles are the most important sites. Further, liver is the only viscera which can synthesize glycogen from monosaccharides other than glucose. However, the biochemical reactions for the polymerization of glucose to glycogen in liver and muscles are found to be similar. The various steps involved in glycogenesis are described here. 1. Phosphorylation of glucose. First of all glucose1 is phophorylated (activated) by A.T.P. in presence of the enzyme hyxokinase or more specifically glucokinase and Mg++ (activator) to glucose-6-phosphate. Both of these enzymes are found to be present in the high mammals. Glucose + ATP Glucose-6-phosphate + ADP The enzyme glucokinase (hexokinase) is inhibited by adrenal cortical hormones (gluco-corticoids) and this inhibition is facilitate by anterior pituitary hormones. However, the inhibition is removed by insulin. It is important to note that formation of glucose-6-phosphate acts as a locking mechanism to keep the glucose within the cell since it is not permeable to cell membrane while glucose is readily permeable. 2. Conversition of glucose-6-phosphate to glucose-1-phosphate. Glucose-6phosphate is reversibly transferred to glucose-1-phosphate in presence of phosphoglucomutase; this reaction requires the presence of glucose-1, 6diphosphate as coenzyme. Glucose –6-P Glucose-1-P 3. Conversion of glucose-1-phosphate to UDPG. The glucose 1-phosphate now combines with uridine triphosphate (UTP) in presence of uridine diphosphate glucose pyurophosphorylase (UDPG pyrophosphorylase) to form uridine diphosphoglucose (UDPG) with the elimination of pyrophosphate2 (P~P). 156 Glucose-1-P + UTP UDPG + Pyrophosphate (P~P) (converted into H3PO4) 4. Conversion of UDPG to glycogen. The uridine diphosphoglucose molecule now transfers its glucose molecules to the non-reducing outer end of an existing glycogen chain under the influence of the glycogen-UDP glucosyl transferas (commonly called glycogen synthetase); one glucose unit is thus added to a primer glycogen via 1-4 linkage. This reaction is driven forward when excess of glucose –6-phosphate is present. The UDP formed as a byproduct is converted to UTP is presence of ATP. UDPG + Glycogen primer UDP + ATP (1, 4-Glucosylunit)x + UDP UDP + ADP Glycogen synthesis is found to exist in two forms namely synthesis-D (dependent) which is active only in presence of glucose-6-phosphate and synthetase –I (independent) which is independent of the concentration of glucose –6-phosphate and is the active enzyme for all practical purposes. Insulin favours the conversion of synthetase-D to synthetase-I* and hence accelerates glycogen synthesis while epinephrine and glucagons favours the conversion of synthetase-I to synthetase-D, by their stimulating action of the production of cyclic AMP, and hence inhibit glycogen synthesis. α β 5. Branching of glycogen. As soon as the straight chain polysaccharide attains a length of eight glucose units, the enzyme amylo-1, 41, 6-trans-glucosidase (branching enzyme) cleaves it into two fragments which then re-attach by means of an α -1, 6-linkage. (1, 4-Glucosyl unit)x (1, 4 Glucosyl and 1,6-glucosyl units)x Glycogen The existing branches of the glycogen molecule are extended by the addition of new glucose molecules from UDPG under the influence of glycogen 157 synthetase. Thus under the combined action of glycogen synthetase and branching enzyme, the glycogen molecule grows like a tree. The molecular weight of glycogen thus synthesized may vary from 1 to 4 millions or more. Inborn error of glycogen anabolism. A form of glycogen storage deficiency resulting from inherited lack of glycogen synthetase has been described. Such individuals are not being able to form proper amount of glycogen. This inborn error of metabolism is characterized by fasting hypoglycemia (less than normal blood glucose levels) with convulsions and mental retardation. Normal individuals store approximately 100 gm. of glycogen in the liver and about 250 gm. in muscles. 1.9.5 GLYCOGENOLYSIS. The process of breakdown of glycogen to glucose (as in liver and kidney) or glucose 6-phosphate (as in the muscles) is known as glycogenolysis. The process involves the following steps 1. Cleavage of α -1, 4-linkage. The breakdown of glycogen is catalysed by the enzyme phosphorylase in presence of inorganic phosphate. The reaction, being known as phosphorolysis, splits up the terminal glucose as glucose 1phosphate. In this way a glycogen chain can be shortened by one glucose unit at a time. It is important to note that the enzyme phosphorylase attacks only the α -1, 4-linkages in glycogen, it can neither attack the α -1, 6linkages at the branching points nor can by-pass them to attack the next α 1, 4-linkages. Thus if glycogen is subjected to the action of phosphorylase alone, a shorter glycogen, known as limit dextrin is formed. 2. Cleavage of α -1, 6-linkages. The α -1, 6-linkages are cleaved by another enzyme known as debranching enzyme (α -1, 6-glucosidase) which removes the glucose unit linked by α -1, 6-linkage to free glucose and thus allows the phosphorylase to continue its attack on the chain. Thus a 158 glycogen molecule can be completely broken down by the combined of phosphorylase and debranching enzyme. 3. Conversion of glucose-1-phosphate to glucose-6-phosphate. Glucose-1phosphate obtained under the influence of phosphorylase is isomerised to glucose 6-phosphate by the action of phosphoglucomutase. 4. Hydrolysis of glucose6-phosphate to glucose. The glucose phosphate is 6then hydroiysed under the influence of glucose-6-phosphatase (present only in liver and kidney) to free glucose. Thus glycogen is hydrolysed finally to free glucose in liver and and kidney and to glucose 6-phosphate in muscles. 1.9.6 GLUCONEOGENESIS A continuous supply of glucose is necessary as a source of energy especially for the nervous system and the erythrocytes. For example, man‘s brain uses about 110 gm glucose per day and his blood converts about 24 gm. of glucose to lactate per day. On the basis the minimum requirement of glucose in a normal adult man receiving no dietary 159 carbohydrate is more than 130 gm./day during rest and further increases during exercise. In addition to this, glucose plays the following important roles. i. It is required in adipose tissue as a source of glyceride-glycerol. ii. It helps in maintaining the levels of intermediates of the citric cycle in many tissues. iii. Glucose is always required at a basal rate as a source of energy even under conditions where fat supplies most of the caloric requirement of the organism. Moreover, glucose is the only fuel that supplies energy to skeletal muscles under anaerobic conditions. iv. It is the precursor of lactose (milk sugar) in the mammary gland and it is taken up actively by the fetus. Thus it is not surprising to note that certain mechanisms have been studied in certain specialized tissues for the conversion of non-carbohydrates to glucose. The process of glucose (carbohydrate) synthesis from onocarbohydrate substances is known as gluconeogenesis (reversal of glycolysis) and such non-carbohydrates substances are called as gluconeogenic substances. In mammals, this process occurs mostly in the liver and kidney which show low glycolytic activity during gluconeogenesis. This process usually occurs at a basal rate but becomes very active when diet is not able to meet the carbohydrate requirement of the body at the required rate. The most important gluconeogenic substances are lactic acid (continuously produced in muscles and blood) and glycerol (from fats) followed by propionic acid, certain amimo acids (derived from the dietary and tissue proteins), certain α -keto acids such as pyruvic acid, α -ketoglutaric acid and oxaloacetic acid. In general all the gluconeogenic susbstances at some stage of their metabolism are linked with glycolytic or citric acid cycle 160 reactions and thus are converted into glucose of glycose or glycogen by the reversal of these reactions. The process of gluconeogenesis from lactate, amino-acids, propionic acid and α -keto acids-takes place via the formation of oxaloacetic acid. The whole of the process may be sketched as in Fig. 6.21, while the details of glucose synthesis from the amino acids (the so called glucogenic or antiketogenic amino acids) are discussed in the metabolism of individual amino acids. Physiological functions of gluconeogenesis : The process of gluconeogenesis performs the folowing physiological functions. i. During starvation (i.e. when aletary carbohydrates are not supplied) the stored glycogen in the tissues of well-nourised man is exhausted after only a few hours. At such critical times the tissues or dietary proteins break down to yield glucogenic amino acids which may be converted into glucose of glycogen and thus the process of gluconeogenesis helps in maintaining the nomal blood sugar level at the times when dietary carbohydrates are insufficient to meet the body carbohydrate requirement. The process is especially important for nervous tissues which can derive energy only from carbohydrates (not from fats 1) and are irreparably damaged if their supply of blucose is insufficient. ii. The proces of gluconeogenesis brings about proper disposal of lactic acid produced by the muscles during and after exercise and that of glycerol produced in the adipose tissues. iii. It also helps in establishing a dynamic equillibrium among carbohydrates, fats and proteins and thus at the time of emergency one can be converted into another. 161 2.0 LET US SUM UP. Carbohydrates: 1. Carbohydrates are polyhydroxy aldehydes or ketones and their derivatives. Their general formula is (CH2O)n. Originally were called as hydrates of carbon. Monosaccharides are simple sugars e.g. glucose. It is the basic building block of polysaccharides starch cellulose. Oligasaccharides have mo to ten monosaccharides units joined together. Polysaccharides units joined together in linear or branched chain. Eg. Cellulose 2. Carbohydrates Monosaccharides Oligosaccharides Plysaccharides - Eg. derivatives eg. 1Cellulose, Chitin- Glycosides structural Polysaccharides Deoxysugars; 2 Starch & Glycogen Myoinositol, Storage Polysaccharides. Aminosugars, N-acetyl muramic acid Sialic acid. 3. Glycoproteins are a class of conjugated proteins which contain carbohydrate group attacched covalently to the poly peptide chain. They are found in all forms of life. They occur as cell-cbats, in blood, and in some proteins harmones in vertabrates. 4. Glycolipids. 5. Carbohydrate metabolism At the time of break down of carbohydrates, cell utilizes the enzymes of the cytoplasm and mitochondria. The process involves a number of steps. The 162 steps that occur in cytoplasm comprises glycolysis, & kreb‘s cycle in mitochondria The Various steps in which oxidation of glucose into CO2 & H2O 1. Glycolysis or EMP pathway. 2. Kreb‘s cycle or citric acid cycle. 3. ETC 4. Oxidative phosphorylation 5. Biosynthesis of Ribose, a pentose sugar occurs through pentose phosphate pathway. 6. The process of formation of D-glucose from non-carbohydrates precursors is called gluconeogenesis. Important precursors are lactate, pyruvic acid, glycerol, certain amino acids. 2.1 CHECK YOUR PROGRESS 1 THE KEY: 1. See sum up. Point 1. 2. A. occurs in DNA 3. Content of peptidoglycan. 4. In human tissue. 5. Structural component of plant cell. 6. Storage product of animal cell. 1. They are a group of related heteropolysaccharides containing two types of alternating monosaccharides units with one acidic gr, either a carboxyl or sulfuric gr. CHECK YOUR PROGRESS 2: THE KEY: 1. It is a fermentative pathway that occurs in cytoplasm for glucose break down. Net yield is – 8 ATP 163 2. a. Pyruvic acid a. CO2 & H2O & utric acid is regenerated b. Glycogen 2.2 ASSIGNMENT/ACTIVITY 1. Draw flow chart of Glycolysis, Kreb‘s Cycle, PPP, gluconeogenesis, 2. Write notes on structural and storage polysaccharides 3. List functions of carbohydrate 2.3 REFERENCES. 1. Agarwal‘s Textbook of Biochemistry (Physiological Chemistry) Goel Publishing house meerut. 2. Lehninger, Biochemistry, Kalyani Publication. 3. Jain J.L., Biochemistry, S. Chand Publication. 4. Stryer Lubert, Biochemistry 5. Verma S.K. & Verma Mohit, A text book of Plant Physiology, Biochemistry & Biotechnology, S.chand Publication 164 UNIT-III LIPIDS 3.1 INTRODUCTION 3.2 OBJECTIVES 3.3 FATTY ACIDS 3.4 ESSENTIAL FATTY ACIDS 3.5 STRUCTURE AND FUNCTION OF TRIACYLGLYCEROLS 3.6 GLYCEROPHOSPHOLIPIDS 3.7 SPHINGOLIPIDS 3.8 CHOLESTEROL 3.9 BILE ACID 3.10 PROSTAGLANDINS 3.11 LIPOPROTEINS COMPOSITION AND FUNCTION ANTHEROSCLEROSIS 3.12 PROPERTIES OF LIPID AGGREGATES ROLE IN 3.12.1 MICELLES, BILAYERS, LIPOSOMES & THEIR POSIBBLE BILOGICAL FUNCTIONS. 3.12.2 BIO-LOGICAL MEMBRANE 3.12.3 FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE. 3.13 LIPID METABOLISM -OXIDATION OF FATTY ACIDS. 3.14 LET US SUM UP 3.15 CHECK YOUR PROGRESS: THE KEY 3.16 ASSIGNMENT / ACTIVITY 3.17 REFERENCES 3.1 INTRODUCTION Lipids are esters of higher fatty acids. Lipids are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents, e.g., choloroform, ether, or benzene. There are several different families or classes of lipids but all derive their distinctive properties from the hydrocarbon nature of a major portion of their structure. (1) as structural components of membranes, (2) as storage and trasport forms of metabolic fuel, (3) as a protective coating on the surface of many organisms, and (4) as cell-surface components concerned in cell recognition, species specificity, and tissue immunity. Some substances classified among the lipids have intense biological activity; they include some of the vitamins and hormones. 165 Although lipids are a distinct class of biomolecules, we shall see that they often occur combined, either covalently or through weak bonds, with members of other classes of bio-molecules to yield hybrid molecules such as glycolipids, lipoproteins, which contain both lipids and proteins. In such biomolecules the distinctive chemical and physical properties of their components are blended to fill specialized biological functions. 3.2 OBJECTIVES In this unit you are expected to 1. is removed Learn the general properties and occurrence of lipids in cells. 2. There structure and possible biological functions. 3. Membrance Structure and metabolism of lipids. 3.3 FATTY ACIDS Although fatty acids occur in very large amounts as building block components of the saponifiable lipids, only traces occur in free (unesterified) form in cells and tissues. Over 100 different kinds of fatty acids have been isolated from various lipids of animals, plants, and microorganisms. All possess a long hydrocarbon chain and a terminal carboxyl group. The hydrocarbon chain may be saturated, as a in palmitic acid, or it may have one or more double bonds, as in oleic acid; a few fatty acids contain triple bonds. Fatty acids differ from each other primarily in chain length and in the number and position of their saturated bonds. They are often symbolized by a shorthand notation that designates the length 166 of the carbon chain and the numbers, posistion, and configuration of the double bonds. Thus palmitic acid (16 carbons, saturated) is symbolized 16:0 and oleic acid [18 carbons and one double bond (cis) at carbons 9 and 10] is symbolized 18:1 9. it is understood that the double bonds are cis unless indicated otherwise. Some generalizations can be made on the different fatty acids of higher plants and animals. 6. Even numbered straight chain fatty acids are found distributed both in plants and animals, they are between 14 and 22 carbon atoms long, but those with 16 or 18 carbons predominate.In animal fats most abundantly found fatty acids are palmitic acid (C16) and stearic acid (C18). 7. Unsaturated fatty acids predominate over the saturated ones, particularly in higher plants and in animals living at low temperatures. 8. Unsaturated fatty acids have lower melting points than saturated fatty acids of the same chain length 9. In most monounsaturated (monoenoic) fatty acids of higher organisms there is a double bond between carbon atoms 9 and 10. In most polyunsaturated (polyenoic) fatty acids one double bond is between carbon atoms9 and 10; the additional double bonds usually occur between the 9, 10 double bond and the methy-terminal end of the chain. 10.In most types of polyunsaturated fatty acids the double bonds are seperated by one methylene group, for example, -CH=CH-CH2-CH=CH-; only in a few types of plant fatty acids are the double bonds in conjugation, that is, CH=CH-CH=CH-. 3.4 ESSENTIAL FATTY ACID 167 When weanling or immature rats are placed on a fat-free diet, they grow poorly, develop a scaly skin, lose hair, and ultimately die with many pathological signs. When linoleic acid is present in the diet, these conditions do not develop. Linolenic acid and arachidonic acid also prevent these symptoms. Saturated and monounsaturated fatty acids are inactive. It has been concluded that mammals can synthesize saturated and monounsaturated fatty acids from other precursors but are unable to make linoleic and γ-linoleic acids. Fatty acids required in the diet of mammals are called essential fatty acids. The most abundant essential fatty acid in mammals is lionoleic acid, which makes up from 10 to 20 percent of the total fatty acids of their triacylglycerols and phosphoglycerides. Linoleic and γ -linolenic acids cannot be synthesized by mammals but must be obtained from plant sources, in which they are very abundant. Linoleic acid is a necessary precursor in mammals for the biosynthesis of arachidonic acid, which is not found in plants. Although the specific functions of essential fatty acids in mammals were a mystery for many years, one function has been discovered. Essential fatty acids are necessary precursors in the biosynthesis of a group of fatty acid derivatives called prostaglandins, hormone like compounds which in trace amounts have profound effects on a number of important physiological activities. 3.5 STRUCTURE AND FUNCTION OF TRIACYLGLYCEROLS (TRIGLYCERIDES) Fatty acid esters of the alcohol glycerol are called acylglycerols or glycerides; they are sometimes referred 168 to as "neutral fats," a term that has become archaic. When all three hydroxyl groups of glycerol are esterified with fatty acids, the structure is called a triacylglycerol. Triacyglycerols are the most abundant family of lipids and the major components of depot or storage lipids in plant and animal cells. Triacyglycerols that are solid at room temperature are often referred to as "fats" and those which are liquid as "oils" Diacylglycerols (also called diglycerides) and monoacylglycerols (or monoglycerides) are also found in nature, but in much samller amounts. Triacylglycerols occur in many different types, according to the identity and position of the three fatty acid components esterified to glycerol. 3. Those with a single kind of fatty acid in all three positions, called simple triacylglycerols, are named after the fatty acids they contain. Examples are tristearoylglycerol, tripalmitoylglycerol, and trialeoyglycerol; the trivial and more commonly used names are tristearin, tripalmitin, and triolein, respectively. 169 4. Mixed triacyglycerols contain two or more different fatty acids. 3.6 PHOSPHOGLYCERIDES (GLYCEROPHOSPHOLIPIDS) The second large class of complex lipids consists of the phosphoglycerides, also called glycerol phosphatides. They are characteristic major components of cell membranes; only very small amounts of phosphoglycerides occur elsewhere in cells. Phosphoglycerides are also loosely referred to as phospholipids or phosphatides, but it should be noted that not all phosphorus-containing lipids are phosphoglycerides; e.g., sphingomyelin is a phospholipid because it contains phosphorus, but it is better classified as a sphingolipid because of the nature of the backbone structure to which the fatty acid is attached. In phosphoglycerides one of the primary hydroxyl groups of glycerol is esterifed to phosphoric acid; the other hydroxyl groups are esterfied to fatty acids. The parent compound of the series is thus the phosphoric ester of glycerol. This compound has an asymmetric carbon atom and can be designated as either D-glycerol 1-phosphate or L-glycerol 3phosphate. 170 Phosphoglycerides possess a polar head in addition to their nonpolar hydrocarbon tails, they are called amphiphatic or polar lipids.The different types of phosphoglycerides differ in the size, shape, and electric charge of their polar head groups. Each type of phosphoglyceride can exist in many different chemical species differing in their fatty acid substituents. Usually there is one saturated and one unsaturated fatty acid, the latter in the 2 position of glycerol. The parent compound of the phosphoglycerides is phosphatidic acid, which contains no polar alcohol head group. It occurs is only very small amounts in cells, but it is an importnat intermediate in the biosynthesis of the phosphoglycerides. The most abundant phosphoglycerides in higher plants and animals are phosphatidylethanolamine and phosphatidylcholine, which contain as head groups the amino alcohols ethanolamine and choline, respectively. Plasmologens differ from all the other phosphoglycerides. 3.7 SPHINGOLIPIDS Sphingolipids, complex lipids containing as their backbone sphingosine or a related base, are important membrane compoenents in both plant and animal cells. They are present in especially large amounts in brain and nerve tissue. Only trace amounts of sphingolipids are found in depot fats. All sphingolipids contain three characteristic building-block components: one molecule of a fatty acid, one molecule of sphingosine or one of its derivatives, and a polar head group, which in some sphingolipids is very large and complex. Sphingosine is one of 30 or more different long-chain amino alcohols found in sphingolipids of various species. In mammals sphingosin and dihydrosphingosine are the major bases of sphingolipids, in higher plants and yeast phytosphingosine is the major base, and in marine invertebrates doubly unsaturated bases such as 4, 8sphingadiene are common. The sphingosine base is connected at its amino group by an amide linkage to a long saturated or monounsaturated fatty acid of 18 to 26 171 carbond atoms. the resulting compound, which has two nonpolar tails and is called a ceramide, is the characteristic parent structure of all sphingolipids. Different polar head groups are attached to the hydroxyl group at the 1 position of the sphingosine base. Sphingomyeline The most abundant sphingolipids in the tissues of higher animals are sphingomyelins, which contain phosphorylethanolamine or phosphorylcholine as their polar head groups, esterifed to the 1-hydroxyl group of ceramide. Sphingomyelines have physical properties very similar to those of phosphatidylethanolamine and phosphatidylcholine; they are zwitterions at pH 7.0. Neutral Glycosphingolipids A second class of sphingolipids contains one or more neutral sugar residues as their polar head groups and thus has no electric charge; they are called neutral glycosphingolipids. The simplest of these are the cerebrosides, which contain as their polar head group a monosaccharide bound in β -glycosidic linkage to the hydroxyl group of ceramid. The cerebrosides of the brain and nervous system contain Dgalactose and are therefore called galactocerabrosides. Neutral glycosphingolipids with disaccharides as their polar head groups are called dihexosides. Also known as trihexosides and tetra hexosides. 172 Acidic Glycosphingolipids (Gangliosides) The third and most complex group of glycosphingolipids are the gangliosides; they contain in their oligosaccharide head groups one or more residues of a sialic acid, which gives the polar head of the gangliosides a net negative charge at pH 7.0 Waxes Waxes are water-insoluble, solid esters of higher fatty acids with long-chain monohydroxylic fatty alcohols or with sterols. They are soft and pliable when warm but hard when cold. Waxes are fond as protective coatings on skin, fur, and feathers, on leaves and fruits of higher plants, and on the exoskeleton of many insects. The major components of beeswax are palmitic acid esters of long-chain fatty alcohols with 26 to 34 carbon atoms. Lanolin, or wool fat, is a mixture of fatty acid esters of the sterols lonosterol and agnosterol. Simple (Nonsaponifiable) Lipids The lipids discussed up to this point contain fatty acids are building blocks, which can be released on alkaline hydrolysis. The simple lipids contain no fatty acids. They occur in smaller amounts in cells and tissues than the complex lipids, but they include many substance having profound biological activity- vitamins, hormones, and other highly specialized fat-soluble biomolecules. 3.8 STEROIDS (CHOLESTEROL) Steroids are derivatives of the saturated tetracylic hydrocarbon opentanophenanthrene. perhydrocylA great many different steroids, each with a distinctive 173 function of activity, have been isolated from natural sources. Steroids differ in the number and position of double bonds, in the type, location, and number of substituent functinal groups, in the configuration (α or β) of the bonds between the substituent groups and the nucleus, and in the configuration of the rings in relation to each other, since the parent hydrocarbon has six centers of asymmetry. The main points of substitution are carbon 3 of ring A, carbon 11 of ring C, and carbon 17 of ring D. All steroids originate from the linear triterpene squalene, which cyclizes readily. The first important steroid product of this cyclization is lanosterol, which in animal tissues is the precursor of cholestrol, the most abundant steroid in animal tissues. Cholesterol and lanosterol are members of a large subgroup of steroieds called the sterols. Cholestrol melts at 1500 C and is insoluble in water but readily extracted from tissues with chloroform, ether, benzene, or hot alcohol. Cholestrol occurs in the plasma membranes of many animal cells and in the lipoproteins of blood plasma. Cholestrol occurs only rarely in higher plants, which contain other types of sterols known collectively as phytosterols. Among these are stigmasterol and sitosterol. Fungi and yeasts contain still other types of sterols, the mycosterols. Cholesterol is the precursor of many other steroids in animal tissues, including the bile acids, detergentlike compounds that aid in amulsification and absorption of lipids in the intestine; the androgens, or male sex hormones; the estrogens, or female sex hormones; the progestational hormone progesterone; and the adrenocortical hormones. 174 3.9 BILE ACIDS These are also steroid compounds and are formed from cholesterol in the body.In the bile of higher animals ,cholic deoxycholic and lithocholic acids are found.They are conjugated with glycinr and taurine forming peptide bonds at the COOH group,resulting glychocolic and taurocholic acids.They may be considered as derivatives of cholanic acid. 3.0 PROSTALGLANDINS Prostaglandins are a family of fatty acid derivatives which have a variety of potent biological activities of a hormonal or regulatory nature. The name prostaglandin was first given in the 1930s by the Swedish physiologist U.S. von Euler to a lipid-soluble 175 acidic substance found in the seminal plasma, the prostate gland, and the seminal vesicles. At least 14 prostaglandins occur in human seminal plasma, and many others have been found in other tissues or prepared synthetically in the laboratory. The structure of prostaglandins was established by S. Bergstrom and his colleagues in Sweeden. All the natural prostaglandins are biologically derived by cyclization of 20 carbon unsaturated fatty acids, such as arachidonic acid, which is formed from the essential fatty acid linoleic acid. Five of the carbon atoms of the fatty acid back-bone are looped to form a five-membered ring. The prostaglandins are named according to their ring substituents and the number of additional side-chain double bonds, which have the cis configuration. The best known are prostaglandins E1, F1α, and F2α, abbreviated as PGE1, PGF1α and PGF2α, respectively. These in turn are the parent compounds of further biologically active prostaglandins. The prostaglandins differ from each other with respect to their bilogical activity, although all show at least some activity in lowering blood pressure and inducing smooth muscle to contract. Some, like PGE1, antagonize the action of certain hormones. PGE2 and PGE2α may find clinical use in inducing labor and bringing about therapeutic abortion. CHECK YOUR PROGRESS – 1 Note: Write your answer in the space given below. Check your answer with the one at the end of the unit. Fill in the blanks. 10.Lipids are ________ of higher fatty acids. 11.Fluid mosaic model of membrane was proposed by ________ 12.Glycerol phosphatides is another name of ________. 13.________ is most abundant in the tissues of higher animals. 176 14.Steroids are ________. 3.1 LIPOPROTEIN COMPOSITION AND FUNCTION, ROLE IN ANTHEROSCLEROSIS Certain lipids associate with specific proteins to form lipoprotein systems in which the specific physical properties of these two classes of biomolecules are blended. There are two major types, transport lipoproteins and membrane systems. In these systems the lipids and proteins are not covalently joined but are held together largely by hydrophobic interactions between the nonpolar portions of the lipid and the protein components. Antherosclerosis (Greek – adhere-mush) is a complex disesase charaterized by hardening of artries due to accumlation of lipids. Hyper cholesterolemia is associated with anthero scholerosis and coronary heart disease. Antherosclerosis is characterized by deposition of cholesteryl esters and other lipids in the intima of the arterial walls often leading to hardening of coronary artries and arteral blood vessels LDL – cholesterol is positively related, where as HDL-cholesterol is negatively co-related with cardio vascular diseases (LDL stands for lethaly dangerous lipoprotein & HDL is highly desirable lipoprotein) CAUSE OF ANTHERROSCLEROSIS & CHD :Antherosis deve & CHD related to plasma cholesterol & LDL Plasma HDL is inversly related to LHD. DISORDERS THAT MAY CAUSE ANTHEROSCLEROSIS: Certain diseases are associated with it like dieabetes mellitus, hyper lipoproteins, nephrotic syndrome, hypthyroidism etc. obesity, smoking, high consumption of fat, lack of physical exercise, stress etc. are probable cause of anthersclerosis. 177 Antioxidants in general decreases the oxidation of LDL studies suggest that taking of antioxidants reduces the risk of antherosclerosis. CONTROL OF HYPER CHOLESTEREMIA Various measures to lower plasma cholesterol are – 1- Consumption of PUFA :- dietary entake of polyunsaturated F.A. reduces cholesterol level. PUFA rich oils are soyabean oil. Cornoil, fish oil –etc 2- Dietary cholesterol :- avoidance of cholesterol rich food i.e. animal food is recommended. 3- Dietary fibers : - fibers present in vegetable decreases cholesterol absorption from intestine. 4- Avoidance of high carbohydrate diet:- diets rich in carbohydrates if avoided controls hyper cholestrolemia. 5- Use of drugs :- Drugs such as lovastatin which inhibit HMG CoA reductase & decrease cholesterol sym. Transport Lipoproteins of Blood Plasma The plasma lipoproteins are complexes in which the lipids and proteins occur in a relatively fixed ratio. They carry water-insoluble lipids between various organs via the blood, in a form with a relatively samll and constant particle diameter and weight. Human plasma lipoproteins occur in four major classes that differ in density as well as particle size. 3.2 PROPERTIES OF LIPID AGGREGATES 3.2.1 LIPID MICELLES AND BILAYERS When a polar lipid, like a phosphoglyceride, is added to water, only a small fraction dissolves to form a true molecular solution. Above the 178 critical micelle concentration the polar lipids associate into various types of aggregates resembling the micelles formed from soaps. In such structures the hydrocarbon tails are hidden from the aqueous environment and form an internal hydrophobic phase whereas the hydrophilic heads are exposed on the surface. Triacylglycerols do not form such aggregates since they have no polar heads.Phosphoglycerides also form monolayers on air-water interfaces as well as bilayers separating two aqueous compartments. Liposomes are completely closed, vasicular bilayer structures formed by exposing phosphoglyceride-water suspensions to sonic oscillation. Bilayer systems of this sort have been extensively studied as models of natural membranes, which appear to contain polar phospholipid bilayers as their continuous phase. 3.2.2 BIOLOGICAL MEMBRANE STRUCTURE The most 179 satisfactory model of membrane structure to date appears to be the fluid-mosaic model, postulated by S.J. Sanger and G.L. Nicolson in 1972. This model postulated that the phospholipids of membranes are arranged in a bilayer to form a fluid, liquidcrystalline matrix. The fluid-mosaic model postulates that the membrane proteins are globular. Some of the proteins are partially embedded in the membrane, penetrating into the lipid phase from either side, and others completely span the membrane .It views membranes as a fluid mosaic in which proteins are inserted into a lipid bilayer. While phospholipids provide the basic structural organization of membranes, membrane proteins carry out the specific functions of the different membranes of the cell. These proteins are divided into two general classes, based on the nature of their association with the membrane. Integral membrane proteins are embedded directly within the lipid bilayer. Peripheral membrane proteins are not inserted into the lipid bilayer but are associated with the membrane indirectly, generally by interactions with integral membrane proteins.The fluid-mosaic model accounts satisfactorily for many features and properties of bilogical membranes. 1. It provides for membranes with widely different protein content, depending on the number of different protein molecules per unit area of membrane. 2. It provides for the varying thickness of different types of membranes. 3. It can account for the asymmetry of natural memebranes, since it permits proteins of different types to be arranged on the two surfaces of the lipid bilayer. 4. It accounts for the electrical properties and permeability of membranes. 5. It also accounts for the observation that some protein components of cell membranes move in the plane of the membrane at a rather high rate. 180 a. LIPID METABOLISM b. As you know metabolism involves both anabolism and catabolism, here we will learn anabolism/biosynthesis of fatty acid first and then their breakdown. FATTY ACID BIOSYNTHESIS Since, two types of fatty acids Viz , saturated and unsaturated fatty acid occurs in nature different pathways are followed for synthesis of different F.A 1.Biosynthesis of saturated F.A. (malonyl CoA pathway) 2.Enzymatic biosynthesis of unsaturated F.A. (i) Biosynthesis of saturated Fatty Acids: Naturally occurring fatty acids may be saturated and main pathway of biosynthesis of fatty acid in plants, animals and bacteria is common and takes through malonyl CoA pathway. In fatty acids(participating in fat synthesis) the number of carbon atoms varies form 16 to 18.Complete biosynthesis of fatty acid takes place in cytosol. Overall reaction is catalysed by the complex of 7 proteins-the fatty acid synthetase complex. Ultimate source of carbon atoms of fatty acid is acetyl-CoA which is produced from carbohydrates and aminoacids. Acetyl-CoA is regenerated with the help of citrate cleaving enzymes as follows: Citrate Cleaving Citric Acid + CoA +ATP Acetyl-CoA + ADP+Pi + OAA Enzymes 181 Acetyl-CoA acts as a primer. Molecules of malonyl-CoA are successively attached to the primer molecules of Acetyl-CoA accompanied by decarboxylation. Before starting of fatty acid biosynthesis an important preparatory reaction, the formation of malonyl-CoA takes place in cytosol. According to Green (1960). Two enzymes complexes and five cofactors-ATP, Mn++ , biotin, NADPH and CO2 are essential for the synthesis of fatty acids. The long chain compounds of fatty acids are synthesized from two carbon compounds Acetyl-coenzyme A (Acetyl-CoA) which is highly reactive compound and is produced as an intermediate in respiration of sugar and fats. The synthesis of fats takes place in stepwise reaction taking place again and again. In each step, 2-carbons atoms of acetyl-CoA are added in the chain. In presence of biotin-acetyl-CoA carboxylase enzyme, acetyl-CoA react with CO2 and ATP to produce malonyl-CoA,ADP and inorganic phosphate. Mn++ acts as cofactor in this reaction. 182 Carboxylase CH3 – CO – SCOA+CO2+ATP Biotin – Acetyl - CoA (Acetyl – CoA)(2C) Mn++ H__ H | C__ O || C __ S.CoA + ADP +IP | O == C__ OH Malonyl-CoA(3C) Malony I-CoA react with acetyl-CoA to produce acetomalonyI-CoA (5C compound). H O | || H__ C __ C __ S.CoA+CH3___CO___ S.CoA | O==C__ OH Malonyl-CoA(3C) Acetyl-Coa(2C)3 H O H O | || | || __ __ __ __ __ H C C C C S.CoA | | _ H O = C OH Acetomalonyl-CoA(5C) In presence of specific enzyme and coenzyme NADPH, acetomalonyl-CoA is con verted into 4 carbon compound butyryl-CoA. CO2 is released during this reaction and water (H2O) and NADP are formed. H | C O || __ C H O | || __ __ __ H C C __ S.CoA+ 4NADPH | | H O == C__ OH Acetomalony-CoA(5C) H | __ H C __ | H || C | H | C __ O || __ C __ S.CoA+CO2 +4NADP=H2O | 183 H H H Butyryl-cOa(4C) The butyryl-CoA then reacts with another molecule of malonyl-CoA and cycle is repeated and 6-carbon compounds is produced. This will again react with still another molecule of malonyl-CoA to produce 8-carbon compounds. When the chain length reaches 16 or 18-carbons, the fatty acid is released. (ii) Biosynthesis of unsaturated fatty acid: Biosynthesis of unsaturated fatty acid has not been completely studied in higher plants. During their synthesis, double bonds are introduced into previously formed saturated fatty acid by the enzyme desaturase. Enzymes acyl-CoA desaturase and stearyl-CoA desaturase have been isolated from yeast and Euglena respectively. OXIDATION OF LIPIDS OR DEGRADATION OF FATS. Lipid is a storage material and can be used as energy rich fuel by cells during seed germination. Degradation or oxidation of fats involves following three processes: 1) Hydrolysis of fat into glycerol and fatty acids 2) Metabolism of glycerol 3) Oxidation of fatty acids. I) Hydrolysis of Fat into Glycerol and Fatty Acids Degradation of fatty acids starts with their hydrolysis. During seed germination, the enzyme lipase catalyses this reaction. During this process triglycerides react with water to produce fatty acids and glycerol. The whole process is completed in three steps.The fats first split to produce diglycerides,part of these are then split to monoglycerides.Finally part of the monoglycerides split to yield fattyacid and glycerol.This reaction occurs at alkaline pH. 2) Metabolism of Glycerol : 184 Glycerol is produced during hydrolysis of triglycerides, enters the carbohydrates metabolism and metabolized into CO2 and H2O through various steps. According to Stumpf (1955) and Beevers (1956), the metabolism of glycerol takes place according to following diagram(Figure 13.9). After complete metabolism, glycerol is converted into acetyl-CoA, which may be oxidized into Krebs cycle to CO2 and H2O . 3) Oxidation of Fatty Acids: The oxidation of fats depend upon α- or β-carbon atom. On the basis of α- and β-carbon atom, the oxidation of fatty acids is of the following kinds: (i) α- oxidation , (ii) β- oxidation. α- OXIDATION OF FATTY ACIDS α- oxidation of fatty acids is discovered by Newcomb and stumpf in 1952. this type of oxidation is found only in the fats in which number of carbon atoms is limited from 13 to 18 carbon. In this oxidation only one carbon atom in every step. It is called α-oxidation because in this process oxidation of α-carbon atom takes place. * α-oxidation of fatty acids takes place only in cotyledons and young leaves. Mechanism of α-oxidation The α-oxidation is completed in following two steps: a. Decarboxylation: First of all in the presence of fatty acid peroxidase enzyme, peroxidative decarboxylation of fatty acids takes place. In this process fatty acids react with hydrogen peroxide(H2O2) to yield an aldehyde (one carbon atom shorter than fatty acid) CO2 and H2O. Peroxidase R-CH2- CH2-COOH + H2O2 R-CH2-CHO + CO2 + H2O α-carbon fatty acids Hydrogen Peroxide 185 Aldehyde b. Dehydrogenation : the enzyme aldehyde dehydrogenase now catalyses the oxidation of aldehyde(formed by first enzyme)to yield the corresponding acid. Dehydrogenase R-CH2-CHO + H2O R-CH2-COOH Aldehyde Acid + + NAD NADH + H This resulted acid is again utilized by the enzyme fatty acid peroxidase as substrate for another turn round the two stage of α-oxidation spiral. The enzyme of αoxidation are specific for long chain saturated fatty acids. The acids with more than C12(usually C14,C16,and C18)are utilized as substrate in α-oxidation. *The fatty acids formed after dehydrogenation may also be oxidized through βoxidation. β-OXIDATION OF FATTY ACID According to Knoop, degradation of fatty acids takes place by successive removal of C2 units after oxidation of the β-carbon atoms. β-oxidation is the chief process of fatty acids degradation in plants. β-oxidation takes place in mitochondrial matrix (and also in glyoxyzones) and involves sequential removal of 2-C in the form of acetyl-CoA molecules from the carboxyl and of fatty acids. This is called β-oxidation because β-carbon (i.e.C-3) of the fatty acid is oxidized during this process. Requirements of β-oxidation β-oxidation requires the following substance: a) A fatty acid 186 b) An energy source- ATP c) Coenzyme-A d) A carrier molecule – carnitine e) five enzymes: 1) Acetyl-CoA synthetase 2) Acetyl-CoA dehydrogenase 3) Enoyl-CoA hydrase 4) β-hyrdoxy acyl CoA dehydrogenase 5) Thiolase Mechanism of β-oxidation : Fatty acids are formed in cytosol. Now the question is, how fatty acids enter mitochondria for their degradation: These are the following steps by which fatty acid enters mitochondria: a. Activation and entry of fatty acid into mitochondria i. Activation of fatty acids into mitochondria. ii. Transfer to carnitine. iii. Transfer to intramitochondrial membrane. 187 b. OXIDATION OR DEGRADATION OF FATTY ACID 1) Activation and entry of fatty acids into mitochondria: It is completed in following three steps: (a) Activation of fatty acids: the first step involves the activation of fatty acid in the presence of ATP and enzyme thiokinase. CoASH is consumed and CoA derivative of fatty acid is produced. In this reaction esterification of fatty acid takes place. Thiokinase,Mn++ R.CH2.CH2COOH+CoASH+ATP ============== 188 RCH2CH2C.SCoA || + AMP+PPi O Fatty acyl-CoA Pyrophosphate (b) transfer to carnitine: the acyl group of fatty acid CoA is transferred to carnitine. Carnitine s a carrier protein which is found in inner mitochondrial membrane which transport fatty acyl CoA through inner mitochondrial membrane to the actual site of degradation. CH3 SCoA+H R__ CH2.CH2 C || O Fatty acyl-CoA O__ CH__CH2__N+ | CH2COO_ Carnitine CH3 CH3 Acyl-CoAcarnitine transferase CH3 R__ CH2.CH2 C__O__ CH__CH2__N+ || O CH3 + COASH | CH3 CH2COO_ Fatty acyl-CoA Carnitine (C) Transfer to intramitochondrial membrane: Dergadation of fatty acid takes place in mitochondrial matrix, which requires Acyl-CoA as substrate. In this step acyl group of fatty acyl carnitine is transferred to intramitochondrial CoaA. 189 CH3 R__ CH2.CH2 C__O__ CH__CH2__N+ || CH3 + COASH | CH3 O CH2COO_ Fatty acyl-CoA Carnitine Acyl-CoAcarnitine transferase CH3 R__ CH2.CH2 C__S__ CoA+OH__CH__ CH2__ N+ || | CH3 CH2__COO_ O Fatty acyl S.CoA 2) CH3 Carnitine Oxidation or degradation of fatty acid : The oxidation of fatty acid involves in the following fur steps: (a) First dehydrogenation : During oxidation, first of all two hydrogen atoms are reomoved from α- and β-carbon atoms of fatty acyl-CoA and trans – α, β-unsaturated fatty acyl-CoA is formed. This reaction is catalysed by FAD containing enzyme acyl-CoA dehydrogenase. β α Acyl CoA dehydrogenase R – CH2-CH2-CO-S-CoA + FAD 190 β α R-CH=CH-CO-S-CoA+ FADH2 Trans α,β-unsaturated fatty acid b) Hydration: In the second step the addition of water molecule takes place to form ,β-hydroxyacyl-CoA in the presence of Enoyl hydrase. Enoyl Hydrase R-CH=CH-CO-S-CoA +HOH R-CH(OH)-CH2-CO-S-CoA β -Hydroxy fatty acyl- CoA c) Second dehydrogenation: Now β-hydroxy acyl-CoA is dehydrogenated in the presence of NAD specific β- hydorxy acyl-CoA dehydrogenase. Two hydrogen atoms are removed from β-C atom (βoxidation), which now bears a carboxyl function and β-keto fatty acylCoA is formed. β- hydorxy acyl-CoA R-CH(OH)-CH2-CO-S-CoA + NAD+ Dehydrogenase R-CO-CH2-CO-S-CoA + NADH +H+ β-keto fatty acyl-CoA 191 d) Thiolysis : β- fatty acyl-CoA is unstable and it releases 2-C fragment as actyl –CoA by the process of thiolysis. The thioclastic cleavage of βketo fatty acyl-CoA takes place in the presence of enzyme β-keto acylthiolase and results in the formation of an active 2-C unit actyl-CoA and β-keto fatty acyl-CoA molecule which is shorter by 2-C atoms then when it entered the β-oxidation spiral. *Acetyl CoA is used in TCA (=Krebs cycle) and Keto acyl-CoA reenters into β-oxidation for its complete oxidation and in every step two carbon atoms are released. The sequence continues until whole molecule is degraded. e.g.palmitic acid enters seven times in the β-oxidation pathway for their complete oxidation. Energetics of β-oxidation For complete oxidation of palmitic acid, it is passed through β-oxidation pathway seven times and get completely oxidized to form CO2 and H2O. C16H32O2 + 23O2 16CO2 + 16H2O Palmitic acid *Each turn of β-oxidation pathway produces 5ATP molecules, however the first turn shows a net gain of only 4 ATP, one ATP molecule is utilized in activating the fatty acid molecule. * Each acetyl CoA molecule after complete oxidation through TCA cycle produces 12 ATP molecules. Thus , the total number of ATP molecules produced by a fatty acid depends upon the number of carbon atoms present in that fatty acid molecule. For e.g.: one molecule of palmitic acid (C16) after complete oxidation to CO2 and H2O produces 130 molecules of ATP as follows: 192 1. During activation 1 ATP is udes and 8 energy rich acetyl-CoA are formed. 2. FADH2 and 1 NADH2 are formed in each cycle. Their reoxidation takes place through ETS Step 1 : 8 actyl –CoA +14 electron pairs Palmitic acid 7 pairs of electrons via FAD+ 7*2 = 14 ATP 7 pairs of electrons via NAD+ 7*3 = 21 ATP 35 ATP 1 ATP used in activation of fatty acid – 1 ATP Net 34 ATP Step 2 : Now Acetyl-CoA enters the T.C.A cycle and oxidized. As we know that 3 ATP molecules are formed from each O2 atom during oxidative phosphorylation. Here 32 atoms of O2 are used, hence, T.C.A. cycle 8 Acetyl-CoA + 16 O2 16 CO2 + 8H2O + 8CoA Therfore 32*3 = 96 ATP formed. Thus total ATP formed will be 34 + 96 = 130 ATP molecules gained. The efficiency can be calculated 130*8000*100 = = 49% 2340,000 The remaining energy is lost in the form of heat. 193 194 CHECK YOUR PROGRESS – 2 Note: Write your answer in the space given below. Compare your answer with the one given at the end of the unit. Q.1 Write Short notes on. a. Fluid mosaic model of membrane. b. -oxidation of F-A Q.2 What are liposomes. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 3.4 LET US SUM UP. Lipids are esters of higher fatty acids. There are several different families or classes of lipids but all derive their distinctive properties from the hydrocarbon nature of a major portion of their structure Lipids are the important constitutnets of cell membranes. Fluid mosaic model of the membrane is the most accepted model of membrane structure. 3.5 CHECK YOUR PROGRESS 1 : THE KEY 1. Esters 2. Sanger and Nicholson in 1972 3. Phosphoglycerides 4. Sphingomylines 195 5. Derivatives of the Saturated tetracyclic hydrocarbon perhydrocyclopentano phenanthrene. CHECK YOUR PROGRESS 2 : THE KEY Hint Q.a 1 Draw diagram of fluid mosaic model of membrane. 2 Write about structure and function of protein molecules 3 Explain structue of phospholipids. b 2 Draw pathway & mention names of enzymes also. Liposomes are membrane bound molecules used for target directed drug delivery. 3.6 ASSIGNMENT/ACTIVITY 1. Prepare a model of cell membrane. 2. Draw a chart showing lipid classification. or 3. Draw a chart showing β-oxidation of lipids. 3.7 REFERENCES. 6. Agarwal‘s Textbook of Biochemistry (Physiological Chemistry) Goel Publishing house meerut. 7. Lehninger, Biochemistry, Kalyani Publication. 8. Jain J.L., Biochemistry, S. Chand Publication. 9. Stryer Lubert, Biochemistry 10.Verma S.K. & Verma Mohit, a Text Book of Plant Physiology, Biochemistry & Biotechnology, S. Chand Publication 196 UNIVERSITY MADHYA PRADESH BHOJ OPEN UNIVERSITY - BHOPAL (M.P.) PROGRAMME - PAPER TITLE OF PAPER - BKOCK NO . UNIT WRITER M.Sc.P.chemistry - v (A-II) BIOLOGY FOR CHEMIST III UNIT - I Smt. Shikha Mandloi Asst. Prof. Microbiology Sri Sathya Sai College for Women UNIT – II Smt. Shikha Mandloi Asst. Prof. Microbiology Sri Sathya Sai College for Women EDITOR - Dr.(Smt.) Renu Mishra, HOD, Botany & Microbiology, Sri Sathya Sai College for Women, Bhopal COORDINATION COMMITTEE - Dr. Abha Swarup, Director, Printing & Translation Major Pradeep Khare, Consultant, Printing & Translation 197 POST GRADUATE PROGRAMME M.Sc.P. CHEMISTRY DISTANCE EDUCATION SELF INSTRUCTIONAL MATERIAL Paper-v(A-II) BIOLOGY FOR CHEMISTS MADHYA PRADESH BHOJ OPEN UNIVERSITY BHOPAL (M.P.) 198 INTRODUCTION Proteins are the most abundant molecules in cells, consisting 50 percent or more of their dry weight. They are found in every part of every cell, since they are fundamental in all aspects of cell structure and function. There are many different kinds of proteins, each specialized for a different biological function. Moreover, most of the genetic information is expressed by proteins. Proteins consist of long chains, in which amino acids occur in specific linear sequences. Yet we know that in each type of protein the polypeptide chain is folded into a specific three-dimensional conformation, which is required for its specific biological function and activity. In this unit, we examine various aspects of the primary structure of proteins, which we have defined as the covalent backbone structure of polypeptide chains, including the sequence of amino acid residues. The three major aspects covered are: (1) the determination of amino acid sequence in polypeptide chains, (2) the significance of variations in the amino acid sequences of different proteins in different species, and (3) the laboratory synthesis of polypeptide chains.(4) structure of proteins The nucleic acids are of considerable importance in biological systems.Two types of nucleic acids are found in the cells of all living organisms. These are: 1. Deoxyribonucleic acid DNA 2. Ribonucleic acid RNA The name nucleic acid was given to it after knowing its acidic property. They are of two types; (1) Ribose nucleic acid, and(2) Deoxyribose nucleic acid . The basic chemical subunits of the nucleic acids are nucleotides. The nucleotides are made up of three components: (i) A heterocyclic ring containing nitrogen, known as a nitrogenous base, (ii) a five carbon pentose sugar, and (iii) A phosphate group. The bases found in nucleic acid are of two kinds- purines and pyrimidines.Adenine and guanine are purine and cytosine, uracil and thymine are pyrimidine bases. The nucleotides found in 199 nucleic acids are much fewer in number than the α-amino acids. DNA is found in almost all the cells as a major component of chromosomes of the nucleus. Certain viruses, including many of the bacterial viruses or bacteriophages, are DNA-protein particles. Mostly the plant viruses are RNAprotein particles. Ribose nucleic acid (RNA) is also of common occurrence in plants as well as animals. It is of three types- (i)ribosomal RNA (r-RNA); (ii) soluble RNA or transfer RNA (t-RNA) and (iii) messenger RNA (m-RNA). Ribosomal-RNA is found in small sub-cellular particles, the ribosomes. RNAs with sendimentation Coefficient value, 5S, 16S and 23S have been reported from 70S ribosomes, while 18S, 28S, 5.8S and 5S r-RNAs have been reported from 80S ribosomes. T-RNA is found in free from in the cytoplasm. M-RNA is found in small quantities in association with ribosomes. Unit IV AMINO ACIDS, PEPTIDES & PROTEINS 1.1 Introduction 1.2 Objectives 1.3 Chemical & enzymatic hydrolysis of proteins to peptides & amino acid sequencing 1.4 Structure of proteins 1.4:1 Forces responsible for holding secondary structure 1.4:2 α-helix, β-sheets, super-secondary structure 1.4:3 Structure of collagen 1.4:4 Tertiary structure- folding & domain structure 1.4:5 Quaternary structure 1.5 Amino acid metabolism degradation & Biosynthesis of amino acid sequence determination 200 1.6 Chemistry of oxytocin & tryptophan releasing hormone. 1.7 Let us sum up 1.8 Check your progress – The Key 1.9 Assignment / Activity 2.0 References 1.1 INTRODUCTION Proteins are the most abundant molecules in cells, consisting 50 percent or more of their dry weight. They are found in every part of every cell, since they are fundamental in all aspects of cell structure and function. There are many different kinds of proteins, each specialized for a different biological function. Moreover, most of the genetic information is expressed by proteins. The structure of protein molecules and its relationship to their biological function and activity are central problems in biochemistry today. Proteins consist of long chains, in which amino acids occur in specific linear sequences. Yet we know that in each type of protein the polypeptide chain is folded into a specific three-dimensional conformation, which is required for its specific biological function and activity. How is the linear, or one-dimensional, information inherent in the amino acid sequence of polypeptide chains 201 translated into the three-dimensional conformation of native protein molecules? The answer to this question comes from some of the most significant advances in modern biological research. These discoveries, made possible by the application of physical-chemical measurements to pure proteins, have illuminated the function and comparative biology of proteins. In this chapter, we examine various aspects of the primary structure of proteins, which we have defined as the covalent backbone structure of polypeptide chains, including the sequence of amino acid residues. We begin by considering the properties of simple peptides. Then we examine three major aspects: (1) the determination of amino acid sequence in polypeptide chains, (2) the significance of variations in the amino acid sequences of different proteins in different species, and (3) the laboratory synthesis of polypeptide chains.(4) structure of proteins 1.2 OBJECTIVES :- This unit emphasizes on developing a 1. Basic understanding of the structure of amino acids and proteins. 2. Make you understand the various aspects and forces involved in formation of primary, secondary tertiary and quarternary structure of proteins. 3. Information about general occurrence and distribution of proteins in living system. 202 1.3 CHEMICAL & ENZYMATIC HYDROLYSIS OF PROTEINS INTO PEPTIDES & AMINO ACID SEQUENCY The structure of peptide Simple peptides containing two, three, four, or more amino acid residues, i.e., dipeptides, tripeptides, tetrapeptides, etc., joined covalently through peptide bonds, are formed on partial hydrolysis of much longer polypeptide chains of proteins. Many hundreds of different peptides have been isolated from such hydrolyzates or synthesized by chemical procedures. Peptides are also formed in the gastrointestinal tract during the digestion of proteins by proteases, enzymes that hydrolyze peptide bonds. Peptides are named from their component amino acid residues in the sequence beginning with the amino-terminal ( abbreviated N-terminal) residue. Much evidence supports the conclusion that the peptide bond is the sole covalent linkage between amino acid in the linear backbone structure of proteins. This evidence comes not only from chemical and enzymatic degradation studies, but also from various physical measurements. For example, proteins have absorption bands in the far ultraviolet (180 to 220 nm) and infrared regions that are similar to those given by authentic peptides. Furthermore, x-ray diffraction analysis directly shows the presence of peptide bonds in native proteins. There is only one other major covalent linkage between amino acids: the disulfide bond of cystine serves in some proteins as a cross-linkage between two separate polypeptide chains ( interchain disulfide bond) or between loops of a single chain( intrachain disulfide bond ). Peptides may be regarded as substituted amides. Like the amide group, the peptide bond shows a high degree of resonance stabilization. The C―N single bond in the peptide linkage has about 40 percent double-bond 203 character and the C=O double-bond about 40 percent single-bond character. This fact has two important consequences: (1) The imino (―NH―) group of the peptide linkage has no significant tendency to ionize or protonate in the pH range 0 to 14.(2) The C―N bond of the peptide linkage is relatively rigid and cannot rotate freely, a property of supreme importance with respect to the three-dimensional conformation of polypeptide chains. Chemical Properties of Peptides The free N-terminal amino groups of peptides undergo the same kinds of chemical reactions as those given by the α-amino groups of free amino acids, such as acylation and carbamoylation. The N-terminal amino acid residue of peptides also reacts quantitatively with ninhydrin to form colored derivatives; the ninhydrin reaction is widely used for detection and quantitative estimation of peptides in electrophoretic and chromatographic procedures. Similarly, the C-terminal carboxyl group of a peptide may be esterified or reduced. Moreover, the various R groups of the different amino acid residues found in peptides usually yield the same characteristic reactions as free amino acids. One widely employed color reaction of peptides and proteins that is not given by free amino acids is the burette reaction. Treatment of a peptide or protein with Cu 2+ and alkali yields a purple Cu 2+ - peptide complex, which can be measured quantitatively in a spectrophotometer. 1.3:1 Steps in the Determination of Amino Acid Sequence. With this information on the properties of simple peptides as background, we can examine the general strategy used to determine the amino acid 204 sequence of peptides and proteins devised by Frederick Sanger in 1953 in his epoch-making determination of the amino acid sequence of the polypeptide chains of insulin, the first protein for which the complete covalent structure became known. Although each protein offers special problems, the following sequence of steps are generally used: 1. If the protein contains more than one polypeptide chain, the individual chains are first separated and purified. 2. All the disulfide groups are reduced and the resulting sulfhydryl groups are alkylated. 3. A sample of each polypeptide chain is subjected to total hydrolysis, and its amino acids composition is determined. 4. On another sample of the polypeptide chain the N-terminal and Cterminal residues is identified. 5. The intact polypeptide chain is cleaved into a series of smaller peptides by enzymatic or chemical hydrolysis. 6. The peptide fragments resulting from step 5 is separated, and their amino acid composition and sequence are determined. 7. Another sample of the original polypeptide chain is partially hydrolyzed by a second procedure to fragment the chain at points other than those cleaved by the first partial hydrolysis. The peptide fragments are separated and their amino acid composition and sequence are determined. 8. By comparing the amino acid sequences of the two sets of peptide fragments, particularly where the fragments from the first partial hydrolysis overlap the cleavage points in the second, the peptide fragments can be placed in the proper order to yield the complete amino acid sequence. 205 9. The positions of the disulfide bonds and the amide groups in the original polypeptide chain are determined. Cleavage of disulfide bonds and separation of polypeptide chains Before analyzing the amino acid sequence of a protein, the investigator must determine whether the protein contains more than one polypeptide chain. The number of chains is usually deduced from the number of N-terminal amino acid residues per molecule of protein, by methods to be described below. Clearly, the number of polypeptide chains will be equal to the number of N-terminal amino acid residues per molecule of protein. If the polypeptide chains have no covalent cross-linkages, they can be separated by treating the protein with acid, base, or high concentrations of salt or a denaturing agent. If the polypeptide chains are covalently cross-linked by one or more disulfide bonds between half residues of cystine, these cross-linkages must be cleaved by appropriate chemical reactions. The commonest procedure is to reduced the disulfide bond to sulfhydryl groups with an excess of mercaptoethanol. An alkylating agent like iodoacetate is then used to alkylate the sulfhydryl group of the cysteine residues to yield their S-carboxymethyl derivatives. When the polypeptide chain is subsequently hydrolyzed, these residues appear as S-carboxymethylcysteine, which is easily identified by the chromatographic procedures used for amino acid analysis. Alkylation of cysteine residues is desirable because of sulfhydryl group of cysteine is relatively unstable and tends to undergo oxidation. Other reagents such as iodoacetamide and ethyleneimine are also employed for alkylation of sulfhydryl groups. An older and less common method for cleaving disulfide cross-linkages, first developed by Sanger, is to oxidize the disulfide group to yield cysteic acid residues from the half-cysteines. Once the interchain 206 disulfide bonds have been cleaved, the individual polypeptide chains are separated, usually by eletrophoresis. Even if the protein to be examined contains a single polypeptide chain, its intrachain disulfide bonds, if any, must be cleaved and all cysteine residues alkylated to the more stable S-carboxymethyl derivatives. Fig - 1 Complete hydrolysis of polypeptide chains and determination of amino acid composition Once the polypeptide chain to be examined has been obtained in homogeneous form, with no remaining disulfide cross-links or free sulfhydryl groups, it is completely hydrolyzed and its amino acid composition determined. Peptide bonds are readily hydrolyzed by heating with either acid 207 or base. Heating polypeptides with excess 6N hydrochloric acid at 100 to 120˚C for 10 to 24 h, usually in an evacuated, sealed tube, is the usual procedure for complete hydrolysis. Little or no recemization of the amino acids takes place under these conditions. However, not all the amino acids are recovered quantitatively following acid hydrolysis; tryptophan is usually destroyed by this treatment. Moreover, the amide groups of asparagines and glutamine undergo complete hydrolysis in acid, to yield free aspartic and glutamic acids, respectively, plus free ammonium ions. Fig - 2 Polypeptides can also be hydrolyzed by boiling with strong sodium hydroxide solutions, but alkaline hydrolysis causes destruction of cysteine, serine, and threonine and recemization of all the amino acids. Alkaline hydrolysis is normally used only for the separate estimation of tryptophan, which is unstable to acid but stable to base. 208 The amino acid composition of hydrolyzates of polypeptides and proteins is determined by automated ion-exchange chromatography in an amino acid analyzer. The first pure protein for which the complete amino acid composition was deduced was β-lactoglobulin of milk. This analysis, which required several years of work by older methods, was completed in 1947. Today, the amino acid analyzer determines the complete amino acid composition of a protein hydrolyzates within 2 to 4 hrs. for which very small samples are required. At this point it is instructive to consider the amino acid composition of representative pure proteins. Some generalizations may be made from these and other available data: 1. Not all proteins contain all the 20 amino acids normally found in proteins; e.g., ribonuclease lacks tryptophan. Fibrous proteins, e.g., silk fibroin and collagen, lack several amino acids. 2. Some amino acids occur much less frequently in proteins than others. For example, in most proteins there are relatively few histidine, tryptophan, and methionine residues. 3. In most proteins 30 to 40 percent of the residues are amino acids with nonpolar R groups. Membrane proteins tend to have a somewhat higher content. Over 90 percent of the amino acid residues of the insoluble fibrous protein elastin, are nonpolar. 4. In some proteins, such as lysozyme, cytochrome c, and the histones, the positively charged R groups predominant (at pH 7.0); such proteins are basic. In others, the negatively charged R groups of glutamic or aspartic acid predominate, as in pepsin, which is highly acidic. Identification of the N-terminal residue of a peptide 209 Very important in the procedure for establishing amino acid sequence are methods for identifying the terminal amino acid residues. The first useful method for the N-terminal residue of polypeptides was described by Sanger, who found that the free unprotonated α-amino group of peptides reacts with 2,4-dinitrofluorobenzene (DNFB) to form yellow 2,4dinitrophenyl derivatives. When such a derivative of a peptide, regardless of its length, is subjected to hydrolysis with 6 N dinitrophenyl derivative HCl, all the peptide bonds are hydrolyzed, but the bond between the 2,4dinitrophenyl group and the α-amino group of the N-terminal amino acid is relatively stable to acid hydrolysis. Consequently, the hydrolyzates of such a dinitrophenyl peptide contains all the amino acid residues of the peptide chain as free amino acids except the N-terminal one, which appears as the yellow 2,4-dinitrophenyl derivative. This labeled residue can easily be separated from the unsubstituted amino acids and identified by chromatographic comparison with known dinitrophenyl derivatives of the different amino acids. 210 Fig.- 3 Sanger's method has been largely supplanted by more sensitive and efficient procedures. One employs dimethylaminonaphthalene-5-sulfonyl the chloride labeling ( reagent abbreviated 1- dansyl chloride ), as shown in figure 5-7. Since the dansyl group is highly fluorescent, dansyl derivatives of the N-terminal amino acid 211 Can be detected and measured in munite amounts by fluorimetric methods. The dansyl procedure is 100 times more sensitive than the Sanger method. The most important and most widely used labeling reaction for the Nterminal residue is that designed by P.Edman. In the Edman procedure phenylisothiocyanate reacts quantitatively with the free amino group of a peptide to yield the corresponding phenylthiohydantoin derivatives, which can be separated and identified, usually by gas-liquid chromatography. Alternatively, the N-terminal residue removed as the phenylthiocarbamoyl derivative can be identified simply br determining the amino acid composition of the peptide before and after removal of the N-terminal residue; this is called subtractive Edman method. The great advantage of the Edman method is that the rest of the peptide chain after removal of the N-terminal amino acid is left infact for further cycles of this procedures; thus the Edman method can be used in a sequential fashion to identify several or even many consecutive amino acid residues starting from the N-terminal end. This great advantage has been further exploited by Edman and G. Begg, who have perfected an automated degradation amino of acid peptides "sequenator" by the for carrying out sequential phenylisothiocyanate procedure. Automated amino acid sequencers, now widely used, permit very rapid determination of the amino acid sequence of peptides up to 20 residues. 212 Fig - 4 In some native proteins the N-terminal residue is buried deep within the tightly folded molecule and is inaccessible to the labeling reagent; in such cases denaturation of the protein can render it accessible. In other proteins, e.g., the tobacco mosaic virus coat protein, the α-amino group of the N-terminal amino acid is acetylated and hence not reactive to labeling reagents. Some natural peptides have no free N-terminal α-amino group because they are cyclic; e.g., the antibiotic tyrocidin A has 10 amino acid residues in a circular arrangement (figure 5-2). However, there is no evidence that circular polypeptide chains occur in proteins. 213 Identification of the C-terminal residues of peptides The C-terminal amino acid of peptides can be reduced with lithium borohydride to the corresponding α-amino alcohol. If the peptide chain is then completely hydrolyzed, the hydrolyzates will contain one molecule of an α-amino alcohol corresponding to the original C-terminal amino acid. This can be easily identified by chromatographic methods; all the other residues will be found as free amino acids. 214 Fig – 5 Another important procedure is hydrazinolysis (figure 5-9), which cleaves all the peptide bonds by converting all except the C-terminal amino acid residues into hydrazides. The C-terminal residue appears as a free amino acid, which can be readily identified chromatographically. 215 Fig. - 6 C-terminal amino acid of a peptide can be also be selectively removed by action of the enzyme carboxypeptidase, which specifically attacks Cterminal peptide bonds. A drawback is that the enzyme, after removal of the terminal residue, proceeds to attack the new C-terminal peptide bond. It is therefore necessary to measure the rate of liberation of different amino acids from the peptide by carboxypeptidase in order to identify the C-terminal residue unequivocally. 1.4 STRUCTURE OF PROTEINS 1.4:1 Forces responsible for structure of proteins 216 The following types of bonds play an important role in the formation of proteins. 1.Peptide bond and peptides The peptide bonds helps in the formation of primary structure of proteins. A single peptide bond is formed when two amino acids are involved in a reaction and the carboxyl group(-COOH) of one amino acid reacts with the amino group (NH2) of another amino acid, with the elimination of one molecule of water. The bond between the two amino acids (-CO-NH-)is called peptide bond and the compound formed by the condensation of amino acids is known as a dipeptides. H H | | R―C― CO OH + H NH―C―R‟ | | NH2 COOH H H | | → R― C― CO―NH ― C ―R‟ ―H2O | peptide bond | NH2 COOH Dipeptides Formation of peptide linkage Depending upon the number of amino acids involved in a reaction, the compound is known as a dipeptides (two amino acids with one peptide bond), a tripeptides (three amino acids with two peptide bonds), a tetrapeptide (four amino acids with three peptide bonds) or a polypeptide [many (n….) amino acids with n-1 peptide bonds] where n=number of amino acids. When a polypeptide chain is formed, one free amino and one free amino and one free carboxyl group is left at the two different ends. The end having free amino group is referred as amino terminal or N-terminal, while the end having free carboxyl group is called carboxy terminal or C-terminal. 217 2. Disulphide bond Disulphide bond is also characteristic of the primary structure of proteins. It is a covalent bond and is generally established between cysteine residues as in insulin and in ribonucleases.The disulphide bond may also be established in other sulphur containing amino acids like Cystine and methionine. When the thiol groups of two cysteine molecules are reversibly oxidized, they form the disulphide compound, cysteine, and the linkage established between them is known as disulphide (―S―S―) linkage. The disulphide bond may be formed either within the single polypeptide chain (intramolecular) or between the two polypeptide chains (intermolecular).Disulphide bond helps in stiffening the folded polypeptide chain and also in joining two or more polypeptide chains together. HS― CH2―CH―COOH | NH2 HS― CH2―CH―COOH | NH2 2 molecules of cysteine S ―CH2―CH―COOH | NH2 S ―CH2―CH―COOH | NH disulphide bond 2 cysteine Formation of disulphide bond 3.Hydrogen bonds Hydrogen bonds are commonly found among the proteins. They are electrostatic in origin and reflect the interaction of the incompletely shielded nucleus of the hydrogen atom―which is a portion of unit positive charge, with the electronic system of another atom. The hydrogen bonds are formed by only electronegative atoms. This bond results by sharing of electrons between hydrogen atom and other electronegative atoms like oxygen. 1.Hydrophobic bond 218 Hydrophobic bonds arise from the mutual cohesion of non-polar hydrocarbon side chains. In biological systems there are a number of amino acids having side-chains which are of hydrocarbon nature. These are hydrophobic groups in that they do not form hydrogen bonds with water molecules. On the other hand, water molecules have a strong tendency to form hydrogen bonds among themselves. As a result of the hydrogen bonding among water molecules, hydrocarbons are forced out of any water phase in which they may be placed. Similarly, hydrocarbon side-chains of the various amino acids tend to be forced together as a result of the hydrogen bonding among water molecules. The hydrophobic bonds are believed to contribute most of the structural stabilization energy for the majority of proteins. Primary, Secondary, Tertiary & Quarternary Structure of Proteins The sequential arrangement of the amino acids in a protein molecule is known as the primary structure. When an interaction between polypeptide takes place, it gives rise to a helical type structure known as secondary structure. Further folding and coiling gives rise to the highly specific and complex tertiary and quarternary structures Primary Structure The sequential arrangement of the various amino acids in a protein ( Polypeptide chain ) through the peptide bonds is known as the primary structure. Each protein molecule consists of one or more polypeptide chains in which the amino acids are linked by peptide linkages. Myoglobin, a protein, consists of only single polypeptide chain, whereas hemoglobin molecule consists of four polypeptide chains. The covalent bonds and disulphide (―S―S―) bonds are again characteristics of the primary structure of proteins. The disulphide bond is generally established between cysteine residues, as in insulin and ribonucleases.If the protein has only one 219 polypeptide chain, it can have only one free α-amino group(-NH2 terminal) and one free carboxyl (-C-terminal) group. In the determination of primary structure of protein, it is essential to know what amino acids are a N-terminal and C-terminal ends. The free α-amino group can react with the reagents like dinitrofluorobenzenes to give a dinitrophenyl (DNP) derivative which on hydrolysis yields the yellow coloured DNP –amino acids. These can be isolated chromatographically. Secondary Structure of Protein The covalent backbone of a polypeptide chain is formally single-bonded. We would therefore expect the backbone of a polypeptide chain to have an infinite number of possible conformations and the conformation of any given polypeptide to undergo constant change because of thermal motion. However, it is now known that the polypeptide chain of a protein has only one conformation (or a very few) under normal biological conditions of temperature and pH. This native conformation, which confers biological activity, is sufficiently stable so that the protein can be isolated and retained in its native state. This fact therefore impiles that the single bonds in the backbone of native proteins cannot rotate freely. When the long polypeptide chains in a protein undergo folding, they form the secondary structure or helical structure. The secondary structure is determined by hydrogen bonding between the components of the peptide chain itself. The hydrogen bonds can occur either within one polypeptide chain or between different polypeptide chains of the protein molecule.Thus, the secondary structure of proteins is represented by helical structures which are ultimately formed by the hydrogen bonding between the chains or chain. Fine Structure of Proteins 220 α-Structure: Fine structure of proteins was discovered by Pauling and Corey (1951) of California Institute of Technology and were awarded Nobel Prize in chemistry for this discovery. They proposed the α-helical conformation of protein based on theoretical grounds. β-Structure : Asbury and Street (1933) proposed β-Structure of proteins which was later modified by Pauling and Corey. The β-structure is represented by parallel zig-zag polypeptide chain which form a pleated sheet-like structure. The hydrogen bonds are formed between NH and C=O groups on the neighboring chains which stabilize the β-structure of proteins. The side chain attached to the amino acid residues lies above and below the hydrogen bonded sheets. This structure is a stable arrangement where side chains are small and do not cause distortion of the pleated structure. In fibroin, the chains run anti-parallel to each other, i.e the free amino (or carboxyl) groups are at opposite ends of neighbouring chains. β-Structure is found in milk and keratin. Fibrous Proteins We shall consider the conformation of fibrous proteins first. Not only are they very abundant, particularly in higher animals, but they also have simpler conformations than the globular proteins, since their polypeptide chains are usually arranged or coiled along a single dimension, often in parallel bundles. As a result the conformation of the polypeptide chains in some fibrous proteins has been easier to examine experimentally; actually, the fibrous proteins gave the first important clues to the constraints on the freedom of rotation of the single bonds in the polypeptide-chain backbone of proteins.Two major classes of fibrous proteins, the keratins and collagens, will be considered here. Study of the keratins has been especially important 221 in revealing the most prevalent conformations of the polypeptide chains in native proteins, namely, the α-helix and β conformation. The Keratins The Keratins are fibrous, insoluble proteins of animals derived from ectodermal (skin) cells. They include the structural protein elements of skin ( leather is almost pure keratin) as well as the biological derivatives of ectoderm, such as hair, wool, scales, feathers, quills, nails, hoofs, horns, and silk. There are two classes of keratins. The α-keratins are relatively rich in cystine residues and thus contain many disulfide cross bridges; in addition, they contain most of the common amino acids. The α-keratins include the hard, brittle proteins of horns and nails, which have a very high content of cysteine (up to 22 percent), as well as the softer, more flexible keratins of skin, hair, and wool, which contain about 10 to 14 percent cystine. The β– keratins, on the other hand, contain no cysteine or cysteine but are rich in amino acids with small side chains, particularly glycine, alanine, and serine. The β–keratins are found in the fibers spun by spiders and silkworms and in the scales, claws, and beaks of reptiles and birds. Another important difference is, that the α-keratins stretch when heated; hair, for example, stretches to almost double its length when exposed to moist heat but contracts to its normal length on cooling. The β–keratins do not stretch under these conditions. Electron microscopy has revealed that hair and wool fibers contain bundles of macrofibrils, each made up of thinner fibrils consisting in turn of parallel bundles of protein filaments arranged along a single axis. This structural feature allows them to be examined readily by x-ray diffraction analysis. The α-Helix and the Structure of α-Keratins 222 Pauling and Corey used precisely constructed models to study all the possible ways of twisting or coiling the backbone of the polypeptide chain along one axis, in view of the constraint imposed by the planar peptide bonds, to account for the observed repeat units of 0.50 to 0.55 nm in αkeratins. The simplest arrangement they found is the helical structure shown in the figure 6-5. In this structure, the α-helix, the backbone is arranged in a helical coil having about 3.6 amino acid residues per turn. The R groups of the amino acids extend outward from the rather tight helix formed by the backbone. In such a structure the repeat unit, consisting of a single complete turn of the helix, extends about 0.54 nm,(5.4 Å) along the long axis, corresponding closely to the major periodicity of 0.50 to 0.55nm deduced from the x-ray pattern of natural α-keratins. The rise per residue is about 0.15 nm, corresponding to the minor periodicity of 0.15 nm also observed in the diffraction patterns. Such an α-helix permits the formation of intrachain hydrogen bonds between successive coils of the helix, parallel to the long axis of the helix and extending between the hydrogen atom attached to the electronegative nitrogen of one peptide bond and the carbonyl oxygen of the third amino acid beyond it. The electrical vectors of these hydrogen bonds are so oriented that they give nearly maximal bond strength. But especially significant is that the α-helical arrangement allows every peptide bond of the chain to participate in intrachain hydrogen bonding. Although other kinds of helical coils of polypeptide chains can be formed, such as a π helix (4.4 residues per turn), they cannot account for the characteristic spacing of the repeat units in the α-keratins family of proteins, nor would they be as stable as the α-helix. An α-helix may form with either L-or D-amino acids, but a helix cannot form from a polypeptide chain containing a mixture of L and D residues. 223 Furthermore, starting from the naturally occurring L-amino acids, either righthanded or left-handed helical coils can be built; however, the right-handed helix is significantly more stable. In all native proteins examined to date, the α-helix is right-handed. From these structural considerations Pauling and Corey proposed that the α-keratins consist of polypeptide chains in righthanded α helical coils. In the α-keratins of hair and wool, three or seven such α helixes may be coiled around each other to form three-stranded or sevenstranded ropes (figure 6-6), held together by disulfide cross-linkages. The αhelix represents the secondary structure of α-keratins, i.e., the regular, coiled conformation of their polypeptide chains around and along their long axis. β -keratins: The β Conformation and the Pleated Sheet We have seen that x-ray study of α-keratins led to our present knowledge of the α-helix; in a similar way, x-ray studies of β–keratins have revealed important clues to the β conformation of the polypeptide chain. We recall that when fibers of α-keratins are subjected to moist heat, they can be stretched to almost double their original length. In this stretched condition they yield x-ray diffraction patterns resembling that of silk fibroin, an example of a β–keratin. Pauling and Corey concluded that the transition from α-keratin to β–keratin structure when hair or wool is steamed is caused by the thermal breakage of the intrachain hydrogen bonds that normally stabilize the α-helix and the consequent stretching of the relatively tight α-helix into a more extended, zigzag conformation of the polypeptide chain, characteristic of β– keratins generally, which they designated the β conformation. Side-by-side polypeptide chains in the β conformation are arranged in pleated sheets, which are cross-linked by interchain hydrogen bonds. All the peptide linkages participate in this cross-linking and thus lend the structure great stability; the R groups lie above or below the zigzagging planes of the pleated sheet. This 224 is the type of secondary structure found in fibroin secreated by the silkworm Bombyx mori. In most types of fibroin every other amino acid is glycine, so that all the R groups on one side of the pleated sheet are hydrogen atoms. Since alanine makes up most of the rest of the amino acids of fibroin, most of the R groups on other side of the sheet are methyl groups. Fibroin and other β–keratins are rich in amino acids having relatively small R groups, particularly glycine and alanine. If the R groups are bulky or have like charges, the pleated sheet cannot exist because of R group interactions. This is why the stretched form of α-keratins is unstable and reverts spontaneously to the α-helical form; the R groups of α-keratins are bulkier and more highly charged than those of silk fibroin. There are two other differences between α-keratins and native β–keratins. In the α forms all the polypeptide chains are parallel, i.e., run in the same Nterminal to C-terminal direction, whereas in fibroin, the adjacent polypeptide chains are antiparallel, i.e., run in opposite directions. Also, α-keratin contains many cystine residues so arranged as to provide interchain ―S―S― cross-linkages between adjacent polypeptide chains. In contrast, the β–keratins, such as fibroin, have no ―S―S― cross-linkages. Collagen Another major type of fibrous protein in higher animals, the collagen of connective tissues, is the most abundant of all proteins in higher vertebrates, making up one-third or more of the total body protein. The larger and heavier the animal, the greater the fraction of its total proteins contributed by collagen. It has been aptly said, for example, that a cow is largely held together by the collagen fibrils in its hide, tendons, bones, and other connective tissues. Collagen fibrils are arranged in different ways, depending on the biological function of the , particular type of connective tissue. In 225 tendons collagen fibers are arranged in parallel bundles to yield structures of great strength but little or no capacity to stretch. In the hide of the cow the collagen fibrils form an interlacing network laid down in sheets. The organic material of the cornea of the eye is almost pure collagen. Whatever the arrangement of collagen fibrils in connective tissue, the fibrils always show a characteristic cross-striated appearance under the electron microscope, in which the repeat distance is between 60 and 70 nm, depending on the type of collagen and spices of organism. Boiling in water converts collagen into gelatin, a mixture of polypeptides. Collagens also have a distinctive x-ray diffraction pattern, different from those of α- and β-keratins. From comparisons of the x-ray patterns of collagen and of polyproline it has been deduced that the secondary structure of collagens is that of a triple helix of polypeptide chains. Each of the chains is a left-handed three-residue helix; the chains are held together by hydrogen bonds. The frequent praline residues determine the distinctive type of helical arrangement of the chain, whereas the smaller R groups of the glycine residues, which occur in every third position, allow the chains to intertwine. The complete amino acid sequence of the collagen chains is not yet known, but –Gly-X-Pro-. –Gly- Pro-X-, and –Gly-X-Hyp- are frequently occurring sequences, in which X may be any amino acid. No proteins other than the collagens appear to contain similar triple-helical chains. Collagens is built of recurring subunit structure, triple–standard tropocollagen molecules, having distinctive"heads". These subunits are arranged heads to tail in many parallel bundles, but the heads are staggered, thus accounting for the characteristic 60-to 70-nm spacing of the repeat units in collagen fibrils from different species. The polypeptide chains of tropocollagen are covalently cross-linked by dehydrosinonorleucine residues, formed by an 226 enzymatic reaction between two lysine residues of adjacent tropocollagen subunits. The secondary structure of the polypeptidechains in other fibrous proteins is not yet known. Studies are under way on elastin of the elastic connective tissue of ligaments and on sclerotin, the structural protein of the light, rigid exoskeleton of insects. Elastin is especially interesting since its polypeptide chains are covalently connected to form a stretchy, two-dimensional sheet resembling a trampoline net. The polypeptide chains are joined through covalent attachment to residues of demosine and isodemosine . Another structural protein of great interest, resilin, found in the wing hinges of some insects, is remarkable for its perfectly reversible elastic properties. Tertiary Structure of Globular Proteins Very few protein molecules exist as a simple α-helix. Further degrees of folding or coiling of polypeptide chains in α-helix give a complex threedimensional structure (tertiary structure) which often contains helical and non-helical regions Folding of the α-helix occurs where the amino acid proline has an imino group instead unstability in the α-helix by of an amino group which causes producing hindrance in the regular internal hydrogen bonding. Three main types of bonds, ionic, hydrogen and hydrophobic, are responsible for the formation of the tertiary structure of a protein. Dipole-dipole interaction and disulphide linkages are also responsible for the formation of tertiary structure. Tertiary structure of proteins is probably thermodynamically the most stable and is of most stable and is of much importance, because the enzymatic properties of a protein depend on it. 227 Types of interaction which may contribute to the stabilization of protein structure. Electrostatic bonds (b) hydrogen bonds, (C and D) Hydrophobic bonds, (e) disulphide linkage. (a) Fig. - 7 We now turn from the fibrous proteins, which have relatively simple structures, to the far more complex globular proteins, which have polypeptide chains tightly folded into compact three-dimensional structures with many different kinds of specialized biological activities. Until x-ray analysis of crystalline globular proteins became feasible, next to nothing could be learned about how their polypeptide chains are folded in three dimensions. In fact , only the barest outlines of the shape of the globular proteins can be deduced from other physical methods, e.g., measurement of viscosity, sedimentation, and diffusion, which allow calculation of the axial ratio of protein molecules but can give no information on their internal structure. Interpretation of the x-ray diffraction patterns is far more difficult for globular than for fibrous proteins because the polypeptide chains of globular proteins are not arranged along one axis but are irregularly and compactly folded into nearly spherical shapes. However, the introduction of intensely diffracting, 228 electron-dense heavy-metal atoms into the molecules of globular proteins to provide reference points for the mathematical interpretation of the diffraction patterns has made it possible to determine the three-dimensional structures of a number of globular proteins to a resolution of 0.6 nm, and in cases 0.2 nm. Among the globular proteins whose tertiary structures are now well known are trypsin, carboxypeptidase A, cytochrome c, lactate dehydrogenase, and subtilisin, a proteolytic enzyme from a bacterium. Although the conformations of only a few proteins are known in detail, the results have already yielded some important generalizations that are probably applicable to many globular proteins. The Stabilization of Tertiary Structure of Globular Proteins Once the native tertiary structure of a globular protein has formed, four mojor types of week interactions or bonds cooperate in stabilizing it: (1) hydrogen bonds between peptide groups, as in α-helical or β–pleated sheets; (2) hydrogen bonds between R groups; (3) hydrophobic interactions between nonpolar R groups; and (4)ionic bonds between positive charged and negatively charged groups, such as the―COO- of aspartate or glutamate R groups and the ―NH3+ of lysine R groups. From studies on the relative contribution of each of these four types of weak bond to the total conformational stability of native protein molecules it is now clear that hydrophobic interactions between the nonpolar R groups are by far the most important. Most proteins contain from 30 to 50 percent of amino acids with nonpolar R groups; x-ray analysis shows that nearly all these R groups in the interior of native globular proteins, shielded from exposure to water. To fully understand the important role of hydrophobic interactions in stabilizing protein structure, we must ask a fundamental question: Why does a denatured, randomly coiled polypeptide chain tend to fold spontaneously 229 into a highly ordered, biologically active conformation, a process that apparently decreases the entropy of the polypeptide chain? Is protein folding a violation of the second law of thermodynamics, which states that all processes proceed in that direction which maximizes entropy, or randomness? The answer of this dilemma is found in a balance of forces. One force is the tendency of the polypeptide chain to seek its own conformation of maximum randomness or entropy. The opposing force is the tendency of the surrounding water molecules to seek their position of maximum randomness or entropy. The critical factor in this balance of forces is represented by the nonpolar R groups. When nonpolar groups are inserted into water, a new interface is created, which requires the adjacent water molecules to assume a more ordered arrangement than they would have in pure liquid water; thus input of energy is required to force a nonpolar R group into water. A random polypeptide chain,with its nonpolar R groups exposed, will thus tend to assume a conformation in which the nonpolar R groups are shielded from exposure to water. It is the tendency of the surrounding water molecules to relax into their maximum-entropy state that brings about the transition of the plypeptide chain from a random unfolded state to a highly ordered tertiary conformation. At equilibrium, when the random chain is fully folded, the increase in the entropy of the surrounding water molecules is greater than the decrease in the entropy of the now correctly coiled polypeptide chain. The second law has not been violated because the combination of the system (the polypeptide) and the surroundings (the water) has undergone a net increase in entropy. However, much evidence suggests that the folded, native conformation is more stable than the unfolded, or denatured, conformation by only a relatively small margin. The stability of a native globular protein is thus the result of a 230 delicate balance between two relatively massive and opposing forces:(1) the tendency of the polypeptide chain to unfold into a more random arrangement and(2) the tendency of the surrounding water molecules to seek their most random state.We have assumed in this discussion that the native conformation of a globular protein is more stable. i.e., has less free energy, than the random-coil from under biological conditions, but this assumption may not be true for all proteins; it is the subject of much debate and study. Proteins that spontaneously refold into their native from may indeed be more stable than their denatured forms under specific conditions of pH, ionic strength, and temperature. On the other hand, the unfolded form of some proteins may have less free energy than the native form. In such cases the transition from the native to the random state may have a very high activation-energy barrier, thus locking the polypeptide chain into its native conformation. In this case, once the native form is unfolded, the polypeptide chain will not spontaneously refold into the native conformation. The Quaternary Structure of Oligomeric Proteins This defines the degree of polymerization of a protein unit. The quarternary structure is exhibited by haemoglobin molecule which was determined by Perutz and coworkers (1960). They showed that this protein undergoes further organization, being made up of 4-polypeptide chains. This further organization is known as the quarternary structure. The chains undergo secondary folding, two of the structures consisting of α-chain and other two of β-chain. All 4-chains fit together in a compact tetrahedral arrangement to form a complete haemoglobin molecule. The forces maintaining the quarternary structure are similar to those involved in tertiary structure, but the association of the sub-units, in general is more flexible. 231 We have seen that some globular proteins are oligomeric, i.e., contain two or more separate polypeptide chains or subunits. The quaternary structure designates the characteristic manner in which the indivisual, folded polypeptide chains fit each other in the native conformation of an oligomeric protein. Among the simplest oligomeric proteins is hemoglobin, which has four polypeptide chains. Because oligomeric protein have relatively high molecular weights, and because they contain multiple chains each of which may have a characteristic conformation, their three-dimensional conformation is far more difficult to analyze by x-ray methods than that of single-chain proteins. Haemoglobin Haemoglobin was the first oligomeric protein for which the complete tertiary and quaternary structure became known from x-ray analysis. This achievement, accomplished by M.F.Perutz and his colleagues in England, culminated some 25 years of detailed study of the structure of this important protein. Because of the similarity of function and the homology of amino acid sequence of the polypeptide chains of myoglobin and hemoglobin, a number of extremely important relationships have developed from concurrent investigations of the structure of these two proteins, which were carried out in the same laboratory. Haemoglobin contains two α chains and two β chains, to each of which is bound a heme residue in noncovalent linkage. The molecule was examined in its oxygenated form, which has a compact spheroidal structure of dimensions 6.4 by 5.0 nm. Figure 6-19 shows the low-resolution outlines of the haemoglobin chains, and figure 6-20 shows how the chains fit together in an approximately tetrahedral arrangement. Each chain has an irregularly folded conformation, in which lengths of pure α-helical regions are separated 232 by bends. Both the α and β chains have about 70 percent α-helical chapter, as is true for myoglobin. The α and β chains are very similar to each other in their tertiary structure, which consists of similar lengths of α helix with bends of about the same angles and directions. But most remarkable is that the tertiary structure of the α and β chains is very similar to that of the single chain of myoglobin, consonant with the similar biological function of these two proteins, namely, their capacity to bind oxygen reversibly, myoglobin in muscle and haemoglobin in blood. In haemoglobin there is very little contact between the two α chains and between the two β chains, but there are numerous R-group contacts between the pairs of unlike chains. Of special interest is the location of the four heme groups, one in each subunit, that bind the four molecules of oxygen. These heme groups, flat molecules in which the iron atoms forms square-planar co-ordination complexes, are quite far apart from each other and are situated at different angles from each other. Each is partially buried in a pocket lined with nonpolar R groups. The fifth coordination bond of each iron atom is to an imidazole nitrogen of a histidine residue; the sixth position is available for co-ordination with an oxygen molecule, lined with polar R groups. The amino acid sequences of hemoglobin chains of many species have been compared. Although only nine of the residues in each chain are absolutely invariant, the amino acid replacements in many other positions suggest that the polypeptide chain subunits of the haemoglobins from nearly all species have the same tertiary structure. Moreover, in nearly all haemoglobins a histidine R group co-ordinates with the iron atom of the heme group. 233 The quaternary conformation of other oligomeric proteins has now been established, in particular the enzyme lactate dehydrogenase, which also has four polypeptide chains. Check your progress- 1 Note:1. Write your answer in the space given below. 2. Check your answer with the one at the end of the unit. Q 1. Write short notes on a) α helix b) β-conformation c) Structure of haemoglobin Q.2. Write a note on amino acid sequencing. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 234 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 235 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1.5 AMINO ACID METABOLISM 1.5:1 Biosynthesis of amino acids. The lower animals are capable of synthesizing all the amino acids present in proteins from the amphibolic intermediates. On the other hand, the higher animals can‟t synthesize certain amino acids in adequate quantities and therefore such amino acids must be taken in the diet. These are the nutritionally essentials amino acids and thus those which can be synthesized from amphibolic intermediates are known as nutritionally non-essentials amino acids. Biosynthesis of non-essential amino acids. 1. L-Glutamate α-ketoglutarate; the reaction is catalyzed by L-glutamate dehydrogenase. Although the reaction is reversible, the equilibrium constant favours glutamate formation. α-ketoglutarate + NH2+ NAD(P)H + H+←→ L-Glutamate + NAD+(P)+ 2. Glutamine. It is synthesized from glutamate and ammonia molecule in presence of glutamine synthetase, a mitochondrial enzyme ( present in highest amounts in renal tissue) which requires ATP and Mg++ ions. NH2+ L-Glutamate+ ATP Glutamine synthetase L-Glutamine + ADP+ Pi Mg++ 3. Proline. It is also synthesized from glutamate by the reversal of the catabolic route of praline to glutamate. Hydroxyproline is derived from praline by an oxygenase reaction; the substract being a prolylcontaining polypeptide and oxygen is supplied air. 4. Alanine and aspartate. These are formed by the transamination reactions of pyruvate and oxaloacetate respectively. Like glutamine, 236 asparagine is obtained from aspartate and ammonia in a reaction catalysed by asparagines synthetase. 5. Tyrosine. It is obtained by the hydroxylation of phenylalanine ( an essential amino acid) in presence of phenylalanine hydroxylase. 6. Cysteine. As discussed earlier, it is synthesized from methionine (essential) and serine(non-essential) ; the former supplies sulphur by transulfuration, and the latter supplies the carbon skeleton. 7. Serine. It has been shown to be obtained from D-3 phosphoglycerate (an intermediate in glycolysis) by two different routes. Of the two routes one involves the phosphorylated intermediates and the other involves non-phosphorylates. The three important reactions in both the routes are same, viz. oxidation, transmination and hydrolysis although with specific sequence which is clear from the following figure no. 237 Fig. - 8 It is probable that the route involving phosphorylated intermediates accounts for most of the serine synthesized by mammalian tissues. 8. Glycine. In mammalian tissues, it can be synthesized by three different routes. 238 a. From glyoxalic acid: The cytosol of liver tissue contains active glycine transaminase which catalyses the transamination of glyoxalate with glutamate to glycine. CHO | + COOH Glyoxalate COOH COOH | | CH.NH2 CH2 NH2 CO Glycine | transaminase | + | (CH2)2 COOH (CH2)2 | | COOH COOH Glutamate α-ketoglutarate Glycine b. From serine: Serine, ammonium ions, bicarbonate, tetrahydrofolate (FH4), pyridoxal phosphate (PLP), and a source of reducing power (NADH) condense in presence of an enzyme system found in the liver mitochondria of mammals to form two moles of glycine. CH2OH | CH.NH2 + CO2 + NH2 + 2H | COOH serine FH4 PLP CH2. NH2 2 | + H2O COOH Glycine c. From choline: Choline (derived from serine) may also be converted into glycine in the following way (see also catabolism of serine). + CH2OH.CH2.N.(CH3)2 choline oxidase + COOH. CH2.N.(CH3)2 ( -2H) Choline Betaine aldehyde NDA+ (-NADH,-H+) COOH. CH2.N.(CH3)2 Demethylation (-CH3) Dimethylglycine Dimethylglycine oxidase (- CH2O) Sarcosine oxidase COOH.CH2.NH.CH3 239 Betaine aldehyde dehydrogenase + COOH. CH2.N.(CH3)2 Betaine COOH.CH2.NH2 Sarcosine (- CH2O) Glycine d. In clostridia, not in mammals, L-threonine or L-allothreonine undergoes aldol type cleavage to form glycine and acetaldehyde. COOH | H2N― C ―H | H―C―OH | CH3 L-Threonine COOH | Or H― C ― NH2 | O―C―H | CH3 L-Allothreonine COOH → | + CH3CHO CH2. NH2 Glycine 9. 5-Hydroxylysine. It is formed by the hydroxylation of lysine. The mechanism of hydroxylation has been extensively studied in developing chick embryo. The probable mechanism is as below. CH2. NH2 | CH2 | CH2 | CH2 | CH. NH2 | COOH Lysine O2[H] ―H2O CH2. NH2 | CHOH | CH2 | CH2 | CH. NH2 | COOH 5-Hydroxylysine 5-Hydroxylysine is found to be present in collagen and collagen products such as gelatin. 1.5:2 GENERAL CATABOLISM OF AMINO ACIDS. Although several amino acids pursue individual catabolic pathways, a few general reactions are found to be common in the catabolism of nearly all the amino acids. With few exceptions, the catabolic pathway of amino acids begins with their conversion to α-keto acids. The amino group, eliminated in the form of ammonia, joins the ammonia pool either as such or in the combined form. The majority of α-keto acids produced from amino acids join 240 the carbohydrate metabolism, while a minority is more closely related to the ketone bodies and fatty acids. However, a few amino acids do not undergo catabolism in this way but they behave in different individualistic way, they will be discussed separately. Conversion of α-amino acids to α-keto acids There are two important ways by which amino acids are converted into their corresponding α-keto acids and thus the amino group is removed from the carbon skeleton in the form of ammonia. 1.Oxidative deamination: In oxidative deamination, the amino acids lose hydrogen atoms to produce an imino acid which is then hydrolysed to ammonia and a keto acid. R | CH.NH2 | COOH Amino acid R | C=NH | COOH Imino acid R | CO + NH3 | COOH Keto acid +2H2O The reaction is catalysed by the amino acid oxidase and the coenzyme (FAD or FMN) which takes up hydrogen. Amino acid oxidase are of two types depending upon the nature of the substrate on which they act. One of these is the L-amino acid oxidase which attacks most of the L-amino acid. The Lamino acid oxidase contains FMN as hydrogen acceptor and is found mainly in liver and kidney. But even in these organs its concentration is too less to be of any significant importance. The other type of amino acid oxidases are specific for D-amino acids and hence known as D-amino acid oxidases. These contain FAD as the hydrogen acceptor and are of wide occurrence in animal tissues. Owing to their high concentration, they are quite active but their action is limited owing to non-availability of D-amino acids in the organisms. 241 Oxidative deamination of glutamic acid may also be catalysed by an important enzyme, the L-glutamate dehydrogenase. This enzyme is highly active and found abundantly in many tissues. This enzyme is not a flavoprotein dependent enzyme; but requires NAD as coenzyme. In this case the deamination is reversible (difference from the deamination by L-amino acid oxidases). It brings the oxidation of glutamic acid to α-keto-glutaric acid (α-oxoglutaric acid). COOH | CH2 | + NAD+ CH2 | CH.NH2 | COOH Glutamic acid COOH | CH2 | CH2 | C=NH | COOH Imino acid COOH gluamate dehydrogenase | CH2 | CH2 | C=NH | COOH Imino acid + NADH + H+ H2O oxidative chain + NAD+ COOH | H2O CH2 | + NH2 CH2 | CO | COOH α-ketoglutaric acid A number of agents dissociate the enzyme into sub-units (probably four) and inhibit its activity as a glutamate dehydrogenase. Agents acting in this way include (in the presence of NADH) ATP and GTP, oestrogens, androgens, progesterone, and thyroxine; ADP, NAD+ and NADP+ act in the opposite direction. Since the above reaction is reversible, it functions both in amino acid catabolism and biosynthesis. The latter (biosynthetic) function is of particular importance in plants and bacteria, which can synthesise large amounts of amino acids from glucose (source of α-keto acid) and ammonia. 242 Glycine is another amino acid which is acted upon by a specific enzyme glycine oxidase. glycine oxidase H2N.CH2.COOH + 1/2O2 Glycine CHO.COOH + NH3 glyoxalic acid Lastly since the amino acids serine and threonine have one hydroxyl group in their molecules, they are deaminated non-oxidatively by enzymes known as dehydrases CH2OH | CH.NH2 | COOH Serine serine dehydrase CH2 | C=NH | COOH Imino acid H2O CH2 | C=O | COOH Pyruvic acid + NH3 2.Transamination : This is the most important mechanism for the conversion of an amino acid to a keto acid and involves the transference of an amino group from a donor amino acid to a recipient keto acid under the influence of a transaminase or aminotransferase. R1 | CH.NH2 | COOH I + R2 | CO | COOH II R1 | CO | COOH III + R2 | CH.NH2 | COOH IV The donor amino acid thus becomes a keto acid and recipient keto acid becomes an amino acid, the coenzyme required for this reaction is pyridoxal phosphate. Pyridoxal phosphate reacts with the amino acid to form a Schiff‟s base type complex which then yields the keto acid and pyridoxalamine phosphate. The latter now reacts with a second keto acid to form a Schiff‟s base complex which again decomposes to produce an amino acid and pyridoxal phosphate. 243 However, there are certain limitations to the general transamination reaction. Although most of the amino acids may act as donor (I), the reciepient keto acid (II) may only be either α-keto-glutaric acid or oxaloacetic acid or Pyruvic acid. The amino acid (IV) formed from these three keto acids are glutamic acid, aspartic acid and alanine, respectively. Thus on the whole there are mainly three types of transaminases; out of which that involving α-ketoglutaric acid as the recipient keto acid (II) is the most important type. COOH | CH2 | CH.NH2 | COOH + Aspartic acid COOH | CH2 | CH2 | CO | COOH α-ketoglutaric acid COOH | CH2 | CO | COOH + Oxaloacetic acid COOH | CH2 | CH2 | CH.NH2 | COOH Glutamic acid The reaction is catalysed by the aspartate aminotransferase also known as glutamate-oxaloacetate transaminase (GOT). The normal value of this transaminase in the blood serum (SGOT) is 30-40 units/100ml. When there is much damage to the cardiac and hepatic tissues, of course, with high concentration in heart and liver tissues. Most of the amino acids (but not all) undergo transamination except lysine, threonine and the cyclic amino acids, proline and Hydroxyproline. Moreover, transaminations involving the β , γ , or δ-amino acids, aldehydo-acids, and even D-amino acids (in bacteria) are also known. 244 CH.NH2 | CH2 | CH2 + | CH.NH2 | COOH L-ornithine (a δ-amino acid) COOH | CH2 | CH2 | CO | COOH α-ketoglutaric acid CHO | CH2 | CH2 + | CH.NH2 | COOH L-glutamate γ-semialdehyde COOH | CH2 | CH2 | CH.NH2 | COOH glutamic acid Since L-glutamate is the only amino acid in mammalian tissues which can undergo oxidative deamination owing to the presence of highly active and widely abundant L-glutamate dehydrogenase, all other amino acids are converted to glutamic acid by transamination with α-ketoglutaric acid. The glutamic acid then undergoes oxidative deamination to form α-ketoglutaric acid and ammonia. The process of amino acid catabolism by the combined action of an aminotransferase (transaminase) and glutamate dehydrogenase may now be summarized as below. NH2 | R―CH―COOH Amino acid α-ketoglutaric acid Transaminase R―CO―COOH keto acid NH3 glutamate dehydrogenase Glutamic acid overall catabolism (transdeamination) of amino acids This process takes place mainly in the liver but occurs also in the kidneys. In the liver, ammonia is converted into urea. 245 3.Transamidation: In this reaction, the amide group of glutamine is transferred to a keto group. If the amide group is transferred to an α-keto acid, an amino acid is formed ; while if the amide group is transferred to the keto group of fructose, glucosamine is formed. Transamination and transamidation serve in the interconversion of amino acids and synthesis of the non-essential amino acids. Liver is the most active site for both of these reactions. Disposal of the Nitrogen. Since ammonia, constantly produced in the tissues by the processes described above, is very toxic compound, it must rapidly be removed from the circulation (detoxication). This function is performed by the liver which removes ammonia rapidly from the circulation by converting it to glutamate, glutamine, or urea. Liver performs this function so rapidly that only traces of ammonia (10-20μg/100ml.) are present in the blood. In the event of failure of hepatic function, concentration of ammonia ion in blood increases and different tissues including brain are poisoned. Other disadvantages of increased ammonia concentration is that it converts α-keto acids to amino acids nd thus hinders Kreb‟s cycle ( a major source of energy in the brain) reactions. The various paths for he fixation of ammonia, obtained from amino acids, acids are : (i) synthetic pathways, (ii) glutamine pathways, (iii) formation of urea, (iv) direct excreation, and (v) formation of creatine and creatinine. Let us discuss them one by one. 1.Synthetic pathways. Ammonia may be used in the reductive amination of α-keto acids, (derived from carbohydrate) to form new amino acids (reversal of transdeamination reaction). O || R.C.COOH NH2 | R.CH.COOH + NH3 246 Ammonia may also be used for the synthesis of purines, pyrimidines and porphyrins, although in these compounds ammonia is generally introduced in the form of a carrier, such as glutaminate, aspartate, carbamyl phosphate, and glycine, rather than in the free state. 2.Glutamine pathways (detoxication) : Free ammonia is a toxic substance in cells and may lead to coma unless removed or detoxified. However, in the extrarenal tissues it is converted into glutamine. The metabolic reaction of ammonia leading to the formation of glutamine is catalysed by glutamine synthetase, a mitochondrial enzyme present primarily in the brain and liver. This reaction resembles somewhat with the synthesis of a peptide linkage, and similarly requires a source of energy, i.e. ATP. COOH | CH2 | CH2 + NH4 + ATP | CH.NH2 | COOH L-Glutamic acid glutamine synthetase, Mg++ CO NH2 | CH2 | CH2 +ADP + H3PO4 | CH.NH2 | COOH L-Glutamine The mechanism of this reaction presumably involves the intermediary formation of γ-glutamyl phosphate and the subsequent exchange of the phosphate group for the ― NH2 group. Glutamine acts as NH2 donor in general metabolism, e.g. in the synthesis of purines and of glucosamine. Glutamine is an important form for transporting ammonia in the organism because in this form ammonia is no longer toxic. Actually the glutamine travels from the various tissues through the blood to the kidneys, where it is hydrolysed by glutaminase to glutamic acid and ammonia. CONH2 | CH2 | COOH | CH2 | glutaminase 247 CH2 | CH.NH2 | COOH L-Glutamine CH2 + | CH.NH2 | COOH L-Glutamic acid (+ H2O) NH2 The ammonia which is thus liberated accounts for about 60% of the urine ammonia. An analogous reaction is the formation and hydrolysis of another acid amide (asparagine) catalysed respectively by the enzymes asparagines synthetase and asparaginase. Glutaminase and asparaginase have been employed as anti-tumour agents since certain tumours exhibit abnormally high requirements for glutamine and asparagines. 3.Direct excretion: usually deamination of amino acids occurs in ectrarenal tissues where the ammonia is immediately channeled into certain metabolic pathways which bind it. In case the removal of amino group from the amino acid (deamination) occurs in kidney in the absence of immediate physiological requirements for synthetic purposes, the liberated ammonia may be excreted directly into the urine. This source of urinary ammonia accounts to about 40% of the total urinary ammonia (60% of urinary ammonia is derived by the hydrolysis of glutamine in kidney). It is important to note in this respect that the direct excreation of ammonia as ammonium salts is very less (although it occurs in states of metabolic acidosis), the vast majority of ammonia is excreted as urea. 4. Formation of Urea: The urinary area constitutes about 95% of the excreted nitrogen. On an average, about 30 gm. of urea is excreted per 24 hours. It has conclusively been proved by experiments in animals that the formation of urea occurs only in the liver. From liver it is released into the blood, and then cleared by the kidney .The conversion of ammonia to urea in the liver is not a simple combination of ammonia with carbon dioxide and 248 water to form ammonium carbonate for direct transformation to urea. Most probably it occurs by way of the ornithine cycle, proposed by kreb. The ornithine cycle is a cyclic process which, like the tricarboxylic acid cycle, can be considered to start with a carrier molecule, the ornithine (amino acid). Before the actual arnithine cycle take place, ammonia (derived by deamination of amino acids) and carbon dioxide (derived from kreb cycle) combine with the aid of ATP to form carbamyl phosphate (amidophosphate). Since two molecules of ATP are required, it has been suggested that the reaction occurs in two steps. In the first step carbon dioxide is activated with the consumption of 1 mole of ATP in presence of Mg++ and N-acetyl glutamate as a cofactor; the ―COOH group is bound presumably to the N atom. In the second step active carbon dioxide unites with an ammonium ion, with the aid of another ATP to form carbamyl phosphate. This step is catalysed by the enzyme carbamyl phosphate synthetase present in liver mitochondria. O Mg++ CO2 + ATP C + ADP OP Activated carbon Dioxide Mg++ O C +NH3 + ATP O H2N―C OP + ADP + Pi OP In bacteria, glutamine rather than ammonia serves as a substrate for carbamyl phosphate synthesis. The reaction is catalysed by the enzyme carbamate kinase. After the formation of carbamyl phosphate, the proper ornithine cycle begins in which the former compound reacts with the δ-amino 249 group of orthinine to form citrulline in the presence of ornithine carbamyl transferase. Citrulline then condenses with the amino group of aspartate to form arginosuccinate. The reaction requires ATP and is catalysed by the arginosuccinate synthetase. Arginosuccinate is cleaved reversibly to fumarate and arginine by the enzyme argnosuccinase. Finally, arginine is cleaved by the well-known enzyme arginase into urea and ornithine. This completes the cycle; and orthinine molecule accepts another molecule of carbomyl phosphate to repeat the cycle. In summary, two moles of ammonia (from glutamate and aspartate) join with one mole of CO2 to give one mole of urea. In the process, 3 moles of ATP are consumed and thus the formation of urea, from the energy view point, is a luxury in which the cell apparently indulges in order to escape the deleterious effect of high concentration of free ammonia. The fact that urea is synthesised via the ornithine-arginine cycle is proved with the aid of isotopes. With the help of labeled carbon it has been proved that the carbon of urea is derived from cabon dioxide. Similarly, when ammonia or amino acids containing labeled nitrogen (N15) are fed to animals, the labeled nitrogen is found in the urea of the urine and in the arginine of the tissue proteins. Of the four nitrogen atoms in arginine only the two in the amidine group are involved in urea formation, while the other two remain in the amino acids of the cycle. Urea is a highly diffusible substance and is found in almost all the fluids of the body. The concentration of urea in blood is usually between 20-35 mg./100ml. and actually changes with the nature of diet. It is high in individuals taking high protein diets and vice versa. Blood urea level may 250 increase in cases of early and terminal nephritis, in renal damages, renal disfunctioning, such as inmercury poisoning and double polycystic kidney, in cardiac failure. On the other hand, lower concentration of urea in blood is found in hepatic damage and nonhemorrhagic nephritis with edema. Urea is mainly excreted by the kidney. 5.Creatine and creatinine: Creatinine (the anhydride of creatine) derived from creatine, is a significant excreatory from of amino acid nitrogen. Aconstant amount (related to muscle mass) of creatinine is excreated daily. Formation of these compounds is discussed elsewhere because these are formed only from three (glycine, arginine, and methionine) rather than the entire group of amino acids. (Glycine, Arginine, Thionine) ATP ADP CH3 ―N―CH2―COOH | C=NH | NH2 CH3 ―N―CH2―COOH | C=NH | NH ~PO3H2 CREATINE PHOSPHOCREATINE CH3 ―N―CH2 H3PO4 H2O C=O HN= C ―NH CREATININE Disposal of carbon skeleton: As we have already seen, amino acids yield keto acids by remova of the amino group as ammonia. The fate of these keto acids may be either of the following. 251 1.Synthetic pathway: Like the disposal of nitrogen, the α-keto acids (resulting from deamination) also may be reductively aminated by reversal of the transdeamination mechanism, thus reforming the original amino acids. Like the deamination, this process is also continuous and the net change is determined by physiological requirements. Certain fragments of the carbon skeletons are also used for special synthesis which are described in the metabolism of individual amino acids. 2.Glucogenic pathways: The carbon skeleton of most of the amino acids are convertible to carbohydrates (gluconeogenesis from protein). Such amino acids are known as glucogenic or antiketogenic amino acids. A few amino acids which are involved in carbohydrate metabolism directly, are shown below, Glucose Pyruvate Oxaloacetate Alanine Aspartate α- Ketoglutarate Glutamate The various glucogenic amino acids are given in the table given below. Table of amino acids according to the fate of their carbon skeleton. Glycogen forming Fat (Glycogenic) amino acids forming Both glycogen and fat (Ketogenic) amino forming ( glycogenic as acids well as Ketogenic) amino acids Alanine, Hydroxyproline, Leucine Isoleucine Arginine, Methionine, Lysine Aspartic acid, Proline Phenylalanine Cystine, Cysteine, Serine, Tyrosine Glutamic acid, Threonine Tryptophan 252 Glycine, Valine, Histidine The keto acids obtained from these amino acids may also directly enter the tricarboxylic acid cycle and thus oxidized ultimately to CO2 and H2O. For example, Pyruvic acid obtained by the deamination of alanine is oxidized to CO2 and H2O via TCA cycle. 3. Ketogenic Pathway: The α-keto acid (isovaleryl formic acid) obtained from the deamination of leucine, on its way of oxidation to CO2 and H2O passes through the stage of acetoacetic acid and thus forms ketone bodies instead of glucose. Such amino acids are known as ketogenic amino acids. 4. Glucogenic as well as ketogenic pathways: The keto acids obtained from certain amino acids may enter the above mentioned glucogenic as well as ketogenic pathways, i.e., they can give rise to both glucose and ketone bodies. Such amino acids include isoleucine, lysine, phenylalanine, tyrosine and tryptophan. Thus on the whole, each amino acid in the form of its keto acid is convertible either to carbohydrate (13 amino acids), fat (1 amino acid), or both (5 amino acids);see the above table. This fact supports the concept of the interconvertibility of fat, carbohydrate, and protein carbons which is also confirmed by isotopically labeled amino acids. 5. Other pathways : Certain amino acids traverse metabolic pathways which do not correspond with either of the above pathways. These routes are highly individual, and are discissed in the appropriate sections. Disposal of Sulphur: Sulphur is an important constituent of the body. It is derived largely from proteins having cystine and methionine as amino acids. In the body, the essential amino acid methionine may be converted into nonessential amino acid cystine but not vice versa. Most of the Sulphur of these 253 amino acids is eventually oxidised to sulphate, which is excreted in the urine mainly as inorganic sulphate with a small fraction as organic or ethereal sulphate. Sulphur present in these sulphates is known as oxidized Sulphur. The amount of oxidized Sulphur in the urine varies with the protein intake. However, all of the Sulphur is not oxidized in the body. A constant amount, known as neutral Sulphur, is always found in the urine in the unoxidized form. Compounds containing neutral Sulphur of the urine in the unoxidized form. Compounds containing neutral Sulphur of the urine are cystine, methyl mercaptan, ethyl sulphide, thiocyanates, and taurine derivatives. In a certain inborn error of metabolism, the ability of kidney to reabsorb cystine is decreased and large amounts of cystine may be excreted in the urine (cistinuria). Under certain conditions cystine may form deposits in the kidney with serious consequences. Energetics of Amino acid Oxidation: Due to the diverse catabolic pathways and the multiplicity of the amino acids, it is difficult to form broad generalization about the energetics of the process. However, if it is assumed that most of the nitrogen derived from amino acids is excreted in the form of urea, and that most of the keto acids are oxidized to CO2 ( HCO3 – at physiological pH) and water through the kreb cycle, then it is possible to arrive at a definite conclusion about the energetics of oxidation of a typical amino acid. Let us discuss the energetics of glutamate oxidation since its catabolic pathway is in accord with the foregoing assumptions. The overall oxidation of one mole of glutamate yields bicarbonate, water and half a mole of urea with the liberation of 490 kcal of free energy. The extent of conservation of this energy in the from of ATP can be calculated as below. 1. Transdeamination of glutamate to α-ketoglutarate and ammonia yields a reduced DPN or TPN which corresponds with 3 moles of ATP. 254 2. Since concersion of ammonia to urea is an endergonic process, and requires the expenditure of 3 moles of ATP per mole of urea, the half mole of urea formed during glutamate catabolism utilizes 1.5 moles of ATP. 3. Since the path of ketoglutarate to CO2 and water through kreb cycle involves the oxidation of ketoglutarate to succinate (yielding 4 moles of ATP), succinate to fumarate (yielding 2 moles of ATP), and malate to oxaloacetate (yielding 3 moles of ATP), a total of 9 moles of ATP are produced from this part of the cycle. The pyruvate, obtained by decarboxylation of the oxaloacetate, then undergoes its usual oxidation to CO2 and H2O and yields 15 moles of ATP. Thus on the whole 9+15=24 moles of ATP are produced from the complete oxidation of the carbon skeleton. By combining the above three steps it is clear that complete catabolism of glutamate molecule yields a net amount of 3+24-1.5=25.5 moles of ATP. Assuming an average energy of one ATP mole as 7.5 kcal; 7.5 x 25.5 = 191 kcal ( or 191x100/490 = 39%) of free energy is conserved in the complete oxidation of glutamate. 1.6 CHEMISTRY OF OXYTOCIN HORMONE & TRYPTOPHANE HARMONE. 1.6:1 Introduction: A hormone is commonly defined as a chemical substance which is produced in one part of the body, enters the circulation and is carried through the blood stream to distant organs or tissues (except local hormones which function at the site of their production) to modify their structure and function. These distant organs or tissues on which hormones act are called as target cells or target organs. Like enzymes they act as 255 catalysts and are required only in very small amounts However, they differ from enzymes in the following respects. I. They are formed in one organ and perform functions in other organ. II. Before being utilized, these are always secreted into the blood. III. Chemically, they may be proteins, polypeptides, single amino acids, and steroids. Although most of the hormones are produced by specialized cells known as endocrine glands, ductless glands or glands of internal secretion, some are also secreted by other organs, viz., secretion and cholecystokinin from GIT, noradrenaline and acetylcholine from nerve-endings and erythropoietin and rennin from kidney. Moreover, it is important to note that although the term٬٬hormone‟‟ implies an ٬٬exiting‟‟ influence, certain hormones are now known to exert a depressing effect on certain of their target tissues. Mode of Action of Harmones: Hormone are found to act on three sites, namely cell membrane, intracellular enzyme systems and the cell nucleus. Chemical Nature of Hormones: Chemically, hormones are amino acid derivatives, polypeptides or proteins, and steroids in nature. Thus chemically there are three distinct types of hormones. 1.Steroid hormones: They contain a stetiod nucleus (cyclopentenophenanthrene) in their molecules. Male sex hormones (androgens), Female sex hormones (estrogens and gestogens) and hormones of the adrenal cortex (adrenocorticoids) belong to this group. 2. Amino acid derivatives: A few of the hormones are amino acid derivatives. Thyroxine, adrenaline and noradrenaline fall in this category. 3.Polypeptide and protein hormones: Hormones of this group are proteinous in nature. This major group includes insulin, glucagons, oxytocin, 256 vasopressin, parathormone, hormones from the anterior lobe of the pituitary body, and hormones from the GIT. Posterior Lobe (Neurohypophyseal) Hormones: The posterior lobe of the pituitary secretes mainly two hormones: vasopressin (pitressin) having the pressor and antidiuretic effects, and oxytocin (pitocin) having the oxytoxic effect. Although these two hormones differ greatly in physiological action, they are very similar in chemical structure. Both of them are octapeptides with one disulphide bridge. Oxytocin contains isoleucine in position 3, in position 8 oxytocin contains leucine. 1 2 H.Cys------Tyr | | 3 S Ile | | 4 S Glu (NH2) |6 5| Cys――Asp(NH2) |7 8| Pro――Leu―Gly―NH2 Structure of Oxytocin Harmone In the non-mammalian vertebrates (e.g. frog, other amphibians, reptiles,and fishes), These hormones and replaced by vasotocin, which has relatively weak vasopressor and oxytoxic activity. Oxytocin acts on the smooth muscles of the uterus and enhances contraction. It undoubtedly plays a major role during parturition. It also causes contraction of the smooth muscles of the lactating mammary gland, causing ejection of milk. Oxytocin secretion is increased by suckling; it has been suggested that it stimulates release of prolactin. It also stimulates contraction of the gallbladder, intestine, urinary bladder and ureters. The secretion of oxytocin is controlled by hypothalamus. 257 1.6:2 Tryptophan. (α-Amino-β-indolepropionic acid). It is the only amino acid containing the indole ring. It is an essential amino acid. Tryptophan is required in the dite for growth and for the maintenance of nitrogen balance in the adult human. In rats the appetite fails and the serum albumin and globulin fall markedly, if the tryptophan is ommited from the diet. Haemoglobin also diminishes and the animals develop cataracts. In addition to its importance as a constituent of proteins, this amino acid is also a source of several compounds of importance to cellular metabolism. Tryptophan replaces nictotinic acid as a dietary constituent for growth maintenance since it is converted to nicotinic acid via pathway of kinurenine. It is also a precursor of serotonin (5-HT), a substance which is present in a number of tissues e.g. blood,GIT, GHS. Serotonin is a powerful vasoconstrictor, smooth muscles stimulator and probably a neurotransmitter. The plant growth hormone indoleacetic acid (auxin) also originates from tryptophan. The D- and L-isomers of tryptophan both can maintain nutrition and growth equally well, indicating that the unnatural D-isomers is converted to L-isomer via the common indole pyruvic acid. Hence indole pyruvic acid, corresponding keto acid of tryptophan, hydroxyl acid (indole lactic acid), etc. can replace the tryptophan in the diet Metbolic breakdown. It is metabolized through several pathways which vary somewhat in different species of animals. The important pathways is discussed below one by one. (A) Kynurenine or Nicotinamide Pathway. This is the main pathway for the metabolic breakdown of tryptophan. By this pathway, tryptophan is converted into nicotinic acid (a vitamin), NAD+ and NDA+P.It appears contradictory to the statement that a vitamin is formed by an organism, but nicotinamide deficiency can indeed be demonstrated only with a tryptophan-poor diet and 258 the concurrent deficiency of vitamin B6. Tryptophan largely replaces the vitamin nicotinamide even in man. The catabolism of tryptophan seems to have many steps in common with the biosynthesis of nicotinamide. Initially the tryptophan molecule is attacked by the enzyme tryptophan pyrrolase (oxygenase) which opens up the indole ring. As in other cases of oxidative ring opening, the double bond is split and each new end receives one oxygen atom to form N-formylkynurenine. The latter compound is then hydrolyzed to formate and kynurenine; the hydolysis is catalysed by kynurenine formylase of mammalian liver. Kynurenine may be deaminated by transamination of the amino group of the side chain to the diketo derivative whioch undergoes spontaneous cyclisation by losing water to form kynurenic acid. Kynurenic acid is a by-product of kynuresine; it is not formed in the main pathway of tryptophan breakdown. Kynurenine is oxidized by atmospheric oxygen to yield 3-hydrxy kynurenine (an NADPH-catalysed reaction). 3-hydrxy kynurenine is then cleaved to yield 3-hydroxyanthranilate and alanine; the reaction is catalysed by the enzyme kynureninase which requires pyridoxal phosphate (vitamin B6) as coenzyme. 3-hydrxyanthranilate is subjected to another oxidative ring opening adjacent to the hydroxyl group to form 2-acroleyl-3-amino-fumarate. However, in case of vitamin B6 deficiency, kynurenine derivatives are not cleaved and thus reach various extrahepatic tissues (e.g. kidney) where they are converted to xanthurenic acid (an abnormal metabolic of tryptophan) which has been identified in human urine. 2-Acroleyl 3-aminopfumarate is very unstable and may undergo catabolism mainly in two important ways to form different products. Check your progress- 2 1. Write your answer in the space given below. 259 2. Check your answer with the one at the end of the unit. Fill in the blanks:1. An example of α helix of protein---------------------------. 2. An example of β sheets of protein--------------------------. 3. ----------------------- is an example of quarternary structure of protein. 4. Hormone oxytoxin is ------------------------------------------. 1.7 LET’S SUM UP Proteins are regarded as the most important constituents of all living organisms as they play important role in their life. The important functions of proteins are (a) They form the structural framework of the cell, (b) Maintain osmotic balance, (c) catalyse biochemical reactions, (d) regulate metabolism, (e) help in stroge of some elements, (f) act as oxygen carriers, (g) from the colloidal system in protoplasm, (h) transport lipids as lipoproteins, and (i) act as storage proteins ( proteinoplasts). They are polymers of amino acids. The amino acids are derivatives of carboxylic acids in which a hydrogen atom in a α-carbon chain is replaced by an amino group (-NH2). They are represented by a general formula shown below. H | α R―C―COOH | NH2 ( where R-represents a great variety of structures). Proteins, on hydrolysis yield a mixture of α-amino acids.They are produced from natural proteins by the action of enzymes, acids or alkalies. Primary Structure 260 The sequential arrangement of the various amino acids in a protein (Polypeptide chain) through the peptide bonds is known as the primary structure. Each protein molecule consists of one or more polypeptide chains in which the aminoacids are linked by peptide linkages. Myoglobin, a protein, consists of only single polypeptide chain, whereas hemoglobin molecule consists of four polypeptide chains. The covalent bonds and disulphide (―S―S―) bonds are again characteristics of the primary structure of proteins. The disulphide bond is generally established between cysteine residues, as in insulin and ribonucleases.If the protein has only one polypeptide chain, it can have only one free α-amino group(-NH2 terminal) and one free carboxyl (-C-terminal) group. In the determination of primary structure of protein, it is essential to know what amino acids are a N-terminal and C-terminal ends. The free α-amino group can react with the reagents like dinitrofluorobenzenes to give a dinitrophenyl (DNP) derivative which on hydrolysis yields the yellow coloured DNP–amino acids. These can be isolated chromatographically. Secondary Structure of Protein The covalent backbone of a polypeptide chain is formally single-bonded. We would therefore expect the backbone of a polypeptide chain to have an infinite number of possible conformations and the conformation of any given polypeptide to undergo constant change because of thermal motion. However, it is now known that the polypeptide chain of a protein has only one conformation (or a very few) under normal biological conditions of temperature and pH. This native conformation, which confers biological activity, is sufficiently stable so that the protein can be isolated and retained in its native state. This fact therefore impiles that the single bonds in the backbone of native proteins cannot rotate freely. When the long polypeptide 261 chains in a protein undergo folding, they form the secondarystructure or helical structure. The secondary structure is determined by hydrogen bonding between the components of the peptide chain itself. The hydrogen bonds can occur either within one polypeptide chain or between different polypeptide chains of the protein molecule.Thus, the secondary structure of proteins is represented by helical structures which are ultimately formed by the hydrogen bonding between the chains or chain. Fine Structure of Proteins α-Structure: Fine structure of proteins was discovered by Pauling and Corey (1951) of California Institute of Technology and were awarded Nobel Prize in chemistry for this discovery. They proposed the α-helical conformation of protein based on theoretical grounds. β-Structure : Asbury and Street (1933) proposed β-Structure of proteins which was later modified by Pauling and Corey. The β-Structure is represented by parallel zig-zag polypeptide chain which form a pleated sheet-like structure. The hydrogen bonds are formed between NH and C=O groups on the neighbouring chains which stabilize the β-Structure of proteins. The side chain attached to the amino acid residues lies above and below the hydrogen bonded sheets. This structure is a stable arrangement where side chains are small and do not cause distortion of the pleated structure. In fibroin, the chains run anti-parallel to each other, i.e the free amino (or carboxyl) groups are at opposite ends of neighbouring chains. βStructure is found in milk and keratin. Tertiary Structure Very few protein molecules exist as a simple α-helix. Further degrees of folding or coiling of polypeptide chains in α-helix give a complex three- dimensional structure (tertiary structure) which often contains helical and 262 non-helical regions. Folding of the α-helix occurs where the amino acid proline has an imino group instead of an amino group which causes unstability in the α-helix by producing hindrance in the regular internal hydrogen bonding. Three main types of bonds, ionic, hydrogen and hydrophobic, are responsible for the formation of the tertiary structure of a protein. Dipole-dipole interaction and disulphide linkages are also responsible for the formation of tertiary structure. Tertiary structure of proteins is probably thermodynamically the most stable and is of most stable and is of much importance, because the enzymatic properties of a protein depend on it. Quaternary Structure This defines the degree of polymerization of a protein unit. The quaternary structure is exhibited by haemoglobin molecule which was determined by Perutz and coworkers (1960). They showed that this protein undergoes further organization, being made up of 4-polypeptide chains. This further organization is known as the quaternary structure. The chains undergo secondary folding, two of the structures consisting of α-chain and other two of β-chain. All 4-chains fit together in a compact tetrahedral arrangement to form a complete haemoglobin molecule. The forces maintaining the quaternary structure are similar to those involved in tertiary structure, but the association of the sub-units, in general is more flexible. 263 1.8 CHECK YOUR PROGRESS:- THE KEY Check your progress- 1 :- The Key 1. a) refer point:-α-helix b) refer point:-β- Conformation c) refer point;- Heamoglobin 2. Name of scientist, step to determine terminal residue end, terminal residue reagent used , etc. Check your progress- 2 :- The Key 1) horns, nails 2) fibres spun by spiders & silkworms. 3) Haemoglobin. 4) A polypeptide hormone 1.9 Assignment/ Activity 1. Draw parallel & antiparallel β sheets of keratins. 2. Write a detail note of primary, secondary , tertiary & quaternary structure of proteins. 2.0 REFERENCES. 1. Stryer lubert , Biochemistry. 264 2. Lehninger, Biochemistry , Kalyani Publication 3. Jain, J-L , Biochemistry , S.Chand Publication. 4. Agarwal‟s , A text book of biochemistry. Sterioisomerism ↓ ----------------------------------------------------------------------------- 265 ↓ Geometrical This occurs due to occurrence of two different atoms at two valancies of carbon ↓ ------------------------- ↓ ↓ ---------------------------------↓ Conformational ↓ Cis ↓ Interconvertible Optical ↓ Configurational ↓ trans ↓ Arrangement that Arrangement changed. 266 can‟t be UNIT V Nucleic acids 5.1 Introduction 5.2 Objectives 5.3 The chemical basis of heredity 5.4 Purine and pyrimidine bases of nucleic acids. 5.5 Double helical model of DNA(Watson & Crick’s model) and forces responsible for holding it. 5.6 Chemical and enzymatic hydrolysis of nucleic acids. 5.7 Structure of RNA 5.8 Replication of DNA 5.9 Transcription 5.10 Translation and genetic code 5.11 Chemical synthesis of mono and trinucleosides 5.12 Let us sum up 5.13 Check your progress key 5.14 Assignment. 5.15 References 267 3.1INTRODUCTION The nucleic acids are of considerable importance in biological systems.Two types of nucleic acids are found in the cells of all living organisms. These are: 1. Deoxyribonucleic acid - DNA 2. Ribonucleic acid - RNA The nucleic acid was first isolated by Friedrich Miescher in 1868 from the nuclei of pus cells and was named nuclein. At that time its biological significance was barely understood. The name nucleic acid was given to it after knowing its acidic property. They are of two types; (1) Ribose nucleic acid, and(2) Deoxyribose nucleic acid . The basic chemical subunits of the nucleic acids are nucleotides. The nucleotides are made up of three components: (i) A heterocyclic ring containing nitrogen, known as a nitrogenous base, (ii) a five carbon pentose sugar, and (iii) A phosphate group. The bases found in nucleic acid are of two kinds- purines and pyrimidines.Adenine and guanine are purine and cytosine, uracil and thymine are pyrimidine bases. The nucleotides found in nucleic acids are much fewer in number than the α-amino acids. DNA is found in almost all the cells as a major component of chromosomes of the nucleus. In 1962 the presence of chloroplast DNA was reported by Ris and Plant from Chlamydomonas. Segments of DNA as long as 150μ have been reported from chloroplasts by Woodcock and Fernandez-Morgan (1968). In 1963 M.M.K. Nass and S.Nass reported the presence of DNA from mitochondria. Certain viruses, including many of the bacterial viruses or bacteriophages, are DNA-protein particles. Mostly the plant viruses are RNA-protein particles. Ribose nucleic acid (RNA) is also of common occurrence in plants as well as animals. It is of three types- (i) ribosomal RNA (r-RNA); (ii) soluble RNA or transfer RNA (t-RNA) and (iii) messenger RNA (m-RNA). Ribosomal-RNA is found in small sub-cellular particles, the ribosomes. RNAs with sendimentation Coefficient value, 5S, 16S and 23S have been reported from 70S ribosomes, while 268 18S, 28S, 5.8S and 5S r-RNAs have been reported from 80S ribosomes. T-RNA is found in free from in the cytoplasm. M-RNA is found in small quantities in association with ribosomes. 3.2 OBJECTIVES This unit emphasizes on understanding the;1. Structure of purines, pyrimidines, & Nucleic acid. 2. Structural and conformational details of DNA and RNAs. 3. The process of DNA replication & protein synthesis 4. Basic requirement to move a step towards molecular and genetic studies. 3.3 THE CHEMICAL BASIS OF HEREDITY DNAs are present mainly in the nucleus (in chromosomes) of the cell so they are the carrier of gnetic information because the DNA molecule can produce a copy of itself each going to one cell i.e. the parent DNA molecule gives rise to two identical daughter molecules each going to one cell and thus each daughter cell receives exactly the same complement of DNA (both qualitatively and quantitatively as the parent cell. DNA as the bearer of genetic information in the cell is strongly supported by the Watson-Crick structure for this compound which explains beautifully the phenomenon of replication (and hence genetic continuity) of DNA. This phenomenon of DNA replication can be explained as below: the double helix of DNA separates into two strands: the individual strands combine in sequence with their complementary free nucleotides (present in nuclear sap) through specific hydrogen bonding (Adenine….Thymine, Guanine…Cytisine) and now phosphate ester linkages are formed between two nucleotides by the enzyme catalase. 269 Thus DNA in animals, plants, bacterial cells, and some viruses maintain the genetic continuity. The direct evidence in favour of the genetic role of DNA is derived from the process of bacterial transformation. If an extract of the strain of pneumococcus, possessing capsules having specific polysaccharides, is added to the culture of strain of pneumococcus not having the above mentained capsules, the latter is transformed to the former. The active agent (transforming factor) is a DNA molecule which endows the bacterial cell with the capacity for synthesizing an enzyme or enzyme system not present previously in non-encapsulated strain. This enzyme in turn catayzes the formation of the specific capsular polysaccharide. DEOXYRIBONUCLEIC ACID - DNA Occurrence 270 DNA is found in the cells of all living organisms except plant viruses, where RNA forms the genetic material and DNA is absent. In bacteriophages and viruses there is a single molecule of DNA, which remains coiled and is enclosed in the protein coat. In bacteria, mitochondria and plastids of eukaryotic cells DNA is circular and lies naked in the cytoplasm. In the nuclei of eukaryotic cells DNA occurs in the form long spirally coiled and unbranched threads. The number of DNA molecules is equivalent to the number of chromosomes per cell. In them DNA is found in combination with proteins forming nucleoproteins or the chromatin material and is enclosed in the nucleus. 3.4 PURINE AND PYRIMIDINE BASES OF NUCLEIC ACID. Chemical composition The Chemical analysis has indicated that DNA is composed of three different types of compounds: 4. Sugar Molecule represented by a pentose sugar, the deoxyribose or 2‘deoxyribose. 5. Phosphoric Acid. 6. Nitrogenous Bases: These are nitrogen containing organic ring compounds. These are of the following four types: I. Adenine represented by –A II. Thymine represented by –T III. Cytosine represented by –C IV. Guanine represented by –G 271 DEOXYRIBONUCLEIC ACID or DNA These four nitrogenous bases are separated into two categories: (c) Purines: These are two-ringed nitrogen compounds. Adenine and guanine are the two purines found in DNA. Their structural formulae are represented in fig.2. (d) Pyrimidines: These are formed of one ring only and include cytosine and thymine. Chemical analysis of DNA further reveals three fundamental features described by Chargaff and is called Chargaff’s base ratio. Molecular Structure 272 The constituents of DNA were isolated quite early but how these are arranged so as to carry out their cytological and genetical activities was not known for long. DNA is a long chain polymer was clearly understood in late 1930s. However, in 1953, D.S. Watson and F.H.C. Crick presented a working model of DNA. This model illustrates not only its chemical structure but also the mechanism by which it duplicates itself. 1.Nucleosides A nitrogenous base with a molecule of deoxyribose (without phosphate group) is known as nucleoside. The nitrogenous base is attached to first carbon atom C-1 of deoxyribose N-glycosidic bond. In all, there are four nucleosides in a DNA molecule. These are: 1. Adenosine – Adenine + Deoxyribose 2. Guanosine – Guanine + Deoxyribose 3. Cytidine – Cytosine + Deoxyribose 4. Thymidine – Thymine + Deoxyribose 2. Nucleotides ( The Monomers of DNA) A nucleotide is formed of one molecule of deoxyribose, one molecule of phosphoric acid and one of the four nitrogenous bases. Since there are four nitrogenous bases, there are four type of nucleotides namely: 5. Deoxyadenylic acid -Adenine + Deoxiribose + Phosphoric acid 6. Deoxyguanylic acid -Guanine + Deoxiribose + Phosphoric acid 7. Deoxycytidylic acid -Cytosine + Deoxiribose + Phosphoric acid 8. Deoxythymidylic acid -Thymine + Deoxiribose + Phosphoric acid 1. Polynucleotide Chain (Linking of Nucleotides in a DNA Molecule) DNA is a macromolecule formed by the linking of several thousand nucleotides. These are called monomers or building blocks of DNA. In a nucleotide the phosphate (phosphoric acid) molecule is attached to fifth carbon atom (C-5) of the deoxyribose molecule through a phosphodiester linkage. The adjacent nucleotides 273 are connected together forming the sugar phosphate chain in which sugar and phosphate molecule are arranged in alternate fashion. The phosphate molecule of a nucleotide is joined to the third carbon atom of the deoxyribose. These are directed at right angles to the long axis of the polynucleotide chain and are stacked one above the other. Marked Ends of Polynculeotide Chain: Each polynucleotide chain has marked ends. Its top end has a sugar residue with free 5‘ carbon atom which is not linked to another nucleotide. The triphosphate group is still attached to it. This end is called the 5’ or 5’-P terminus. The other end of the chain ends in a sugar residue with C-3 carbon atom not linked. It bears 3‘-OH group. This end of polypeptide chain is called 3’ end or 3’-OH terminus. It means the poplypeptide chains have direction and are marked as 3‘-5‘. 3.5 DOUBLE HELICAL MODEL OF DNA (WATSON & CRICK’S MODEL) AND FORCES RESPONSIBLE FOR HOLDING IT. .Watson and Crick’s Model of DNA Watson and Crick suggested that in a DNA molecule ther are two such polynucleotide chains arranged antiparallel or in opposite directions i.e., one polynucleotide chain runs in 5‘-3‘ direction, the other in 3‘-5‘ direction. It means the 3‘end of one chain lies beside the 5‘ end of other. In such a structure the phosphate groups of nucleotides in each polynucleotide chain or strand lie on the outside of the deoxyribose and the nitrogenous bases are directed inward. The nitrogenous bases of the two chains are linked through hydrogen bonds formed between oxygen and nitrogen atoms of the adjacent bases. The unique feature of pairing between bases is: 1. Purine (adenine and guanine) pairs with pyrimidine (cytosine and thymine), and 2. Adenine pairs with thymine and cytosine pairs with guanine. There are definite reasons for such a specific pairing : 274 1. Such pairing forms a perfect match between hydrogen donor and hydrogen acceptor sites on the two molecules. Adinine and thymine share two hydrogen atoms, whereas cytosine and guanine are joined by three hydrogen bonds. 2. Such a pairing is further supported by the occurrence of constant diameter of DNA. In a limited area a two-ringed molecule (purine) joins a single-ringed molecule maintaining a constant and roughly equal distance. A and G pair will be rather too large to fit inside the helix and C and T would appear to be far apart. Due to this type of base pairing the two strands are complementary to each other. It means if a chain has a region with a sequence of nitrogenous bases, thyminecytosine-adenine-cytosine-guanine, then the corresponding region in the complementary chain will have the base sequence adenine-guanine-thymineguanine –cytosine. DNA consists of two complementary chains twisted around each other forming a right-handed helix. One turn of helix measures about 34 Å. It contains 10 pairs of nucleotides placed at regular intervals of 3.4 Å. The diameter of the helix is roughly 20Å. A narrow helical groove and a wide helical groove run along the length of DNA helix. The narrow groove is the distance between the paired molecules while the wide groove is the space between successive turns when the pair is wound into a helix. In 1953, James Watson and Francis Crick deduced the three dimensional structure of DNA and immediately inferred its mechanism of replication, Watson and Crick analyzed X-ray diffraction photographs of DNA fibres taken by Rosalind Franklin and Maurice Klilkins and derived a structural model that has proved to be essentially correct. The salient features of their model are:275 9. Two helical polynucleotide chains are coiled around common axis, the chains run in the opposite directions. 10.The purine and pyrimidine bases are on the inside of the helix, where as the phosphate and deoxyribose units are on the outside the planes of the bases are perpendicular to the helix axis. The planes of the sugars are nearly at right angles to those of the bases. 11.The diameter of the helix is 20 A0, adjacent bases are separated by 34 A0 along the helix and related by a rotation of 360, hence the helical structure repeats after in residues on each chain, i.e., at interval of 34A0. 12.The two chains are held together by hydrogen bonds between the pairs of bases adenine is always paired with thymine guanine is always paired width cytosine. 13.The sequence of bases along a polynucleotide chain is not restricted in any way, the precise sequence of bases carries the genetic information. 14.The ratio of A+G/C+T always equals to one 15.In every organism, the sequence of nucleotides in constant. The ratio of A=T/G=C is also specific to organisms. 16.Each pitch of DNA has two major and two minor groves. Fig. DNA Structure The most importa 276 nt, aspect of the DNA double helix is the specificity of the pairing of the bases. Watson and Crick deduced that adenine must pair with thymine and guanine with cytosine. The steric and hydrogen bonding factors restriction is imposed by the regular helical nature of the sugar-phosphate backbone of each polynucleotide chain the glycosidic bonds that are attached to a bonded pair of bases are always 10.85 A apart a pair of pyrimidine base pair fits perfectly in this shape, in contrast, there is insufficient room for two purines. There is more than enough space for two pyrimidine but they would be too far apart to form hydrogen bonds Hence, one member of a base pair in a DNA helix must always be a purine the other a pyrimidine, because of stric factors. The base pairing is further restricted by hydrogen bonding requirements. The hydrogen atoms in the purine and pyrimidine bases have well defined positions. Adenine can not pair with cytosine because there would be two hydrogen near one of the bonding positions and none at the other like wise guanine can‘t pair with thymine, where as guanine forms three bonds with cytosine. The orientation and distance of those hydrogen bonds are optimal for achieving strong interaction between the bases. The base pairing scheme was strongly supported by the base compositions of DNA‘s from different types. In 1950, Erwin chargaff found that the ratios of adenine to thymine and guanine to cytosine were nearly 1 in all the samples studied. 277 Check your progress-1 1. Note :- Write your answer in the space given 2. Compare your answer with the one given at the end of the unit. a. Name purines & Pyrimedines bases of DNA. b. What does Chargaff rule says ? c. Explain the structure of DNA. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 278 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 3.6 CHEMICAL AND ENZYMATIC HYDROLYSIS OF NUCLEIC ACIDS. Hydrolysis or Degradation of RNA Like DNA, RNA can also be hydrolysed under different conditions. The acid hydrolysis yields the different components which actually form it. The various components are free purines and pyrimidines, ribose sugar and phosphoric acid 279 Showing hydrolysis of DNA 3.7 STRUCTURE OF RNA In all other organisms, where DNA is the hereditary material, different types of RNA are nongenetic. In general, three types of RNAs have been distinguished: 1. Messenger RNA or nuclear RNA (mRNA) 2. Ribosomal RNA (rRNA) 3. Transfer RNA (t RNA) Messenger RNA or Nuclear RNA 280 mRNA is synthesised inside the nucleus as a complementary strand to DNA and carries genetic informations from chromocomal DNA to the cytoplasm for the synthesis of proteins. For this reason only, it was named messenger RNA (mRNA) by Jacob and monod in 1961. It has following characteristics. (a) It is formed as a complementary strand to one of the two strands of a DNA. (b)The thymine of DNA is substituted by uracil in mRNA. mRNA, therefore, contains the same information as coded in that part of DNA. (c)After synthesis it immediately diffuses out of the nucleus into the cytoplasm, where it is deposited on certain number of ribosomes. (d)Here mRNA acts as a template for protein synthesis. (e)It has a short life span and withers away few translations. Monocistronic mRNA molecule containing the codons of a single cistron, which codes for one complete molecule of protein. Polycistronic mRNA molecule containing the codons for more than one cistron which may lie close together. This type of mRNA synthesises more than one protein chains. For example, mRNA molecule which governs the metabolism of histidine codes for the synthesis of ten specific enzymes. Transfer RNA (tRNA) The transfer RNA is a family of about 60 small sized ribonucleic acids which can recognize the codons of mRNA and exhibit high affinity for 21 activated amino acids, combine with them and carry them to the site of protein synthesis. tRNA molecules have been variously termed as soluble RNA or supernatant RNA or adaptor RNA. tRNA is about 10-15 percent of total weight of RNA of the cell. Its molecules have following characteristics; 1. tRNA molecules are smallest, containing 75 to 85 nucleotides. 2. Its polynucleotide chain is bent in the middle and folded back on itself ( clover leaf model) and the two arms coiled over one another. 281 3.The 3‘ end of the polynucleotide chain ends in CCA base sequence. This represents site for the attachment of activated amino acid. The end of the chain terminates with guanine base. 4.The band in chain of each tRNA molecule contains a definite sequence of three nitrogenous bases which constitute the anticodon. It recognizes the codon on mRNA. 5. Four different regions or special sites can be recognized in the molecule of tRNA. These are: (a) Amino acid attachment site. (b) Recognition site. (c) Anticodon or codon recognition site. (d) Ribosome recognition site RIBOSOMAL RNA or rRNA 282 The RNA, which is found in ribosomes, is called ribosomal RNA. Ribosomes are chemically ribonucleoprotein as they consist of RNA and proteins. It is known as soluble RNA. Its quantity in a cell is much higher than that of mRNA and tRNA. It constitutes about 80% of total RNA. On the basis of their sedimentation coefficient or rate of sedimentation, rRNA molecules may be classified into following categories: 28S-rRNA: It has molecular weight more than 10,00000. Sedimentation coefficient is between 21S and 29S. It is found in 60S subunit of eukaryotic ribosomes. 18s-rRNA: It molecular weight is less than a millions. Sedimentaion varies between 12S to 18S. It is found in 40S subunit of ribosomes. 5S-rRNA: It has much lower molecular weight and is found in 30S unit of ribosomes. Structure of rRNA Ribosomal RNA molecules are single stranded but in the solution of high ionic concentration, irregular spiral coiling of rRNA is formed. As the ionic concentration of the solution increases, the degree of irregular coiling of rRNA also increases. In this coiling the intramolecular bases show base pairing. The pairing is normal as A pairs with U and C pairs with G. 283 Check your progress-2 1. Note :- Write your answer in the space given 2. Compare your answer with the one given at the end of the unit. Write notes on A. DNA replication B. Protein Synthesis -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------284 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 3.8 REPLICATION OF DNA Semi-conservative replication of Chromosomes in eukaryotes: Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells were first published in 1957. They reported that DNA in all the organisms has the inherent capacity of self-replication. The mechanism of DNA replication is so precise that all the cells derived from a zygote contain exactly similar DNA both in terms of quality and quantity. The replication takes place in interphase after every cell division. Theoretically, there may be following three possible modes of DNA replication: Dispersive Method Conservative Method Semiconservative Method Semiconservative mode is the most accepted of all. Semiconservative Method: - During replication, the two strands of the DNA molecule uncoil with the help of some proteins and enzymes. The unpaired bases in the single stranded regions of the two strands binds with their complementary bases in the single stranded regions of the two stands bind with their complementary bases present in the cytoplasm in the form of 285 nucleotides. These nucleotides become joined by phosphodiester linkages generating complementary strands on the old ones. This provides for an almost error free, high fidelity replication of DNA. Fig. Semiconservative replication. Detailed mechanism of semiconservative mode of DNA replication was given by Kornberg. He proposed that following enzymes are important for functional replication of DNA. 286 Nucleases Unwinding Proteins DNA Polymerases DNA Ligases RNA primer Primases or RNA polymerases. Out of the above six enzymes/proteins, the three; nucleases, RNA polymerases and DNA polymerases are known as “Kornberg Enzyme”. 1. NUCLEASES These are the enzymes which digest or breakdown nucleic acid molecules. These attack on phosphodiester bonds of the nucleic acid backbone and release nucleotides through hydrolysis. On the basis of their mode of function, nucleases may be classified into following two: (i) Exonucleases (ii) Endonucleases. Exonucleases function on phosphodiester bonds of the DNA at both the terminus. The endonucleases, on the other hand attack the phosphodiester bonds at intercalary regions of the DNA breaking it into as many parts as the site of function. 2. UNWINDING PROTEINS. Unwinding proteins are those proteins or enzymes, which uncoil the DNA helix and separate the two DNA strands by breaking hydrogen bonds between them. Due to this function these are known as unwinding proteins. 287 Due to separation, the two strands form a „Y‟ or fork like structure, which is known as replication fork. Usually two types of unwinding proteins have been recognized: (i) DNA helicases : These enzymes or proteins uncoil the helix of DNA which may now appear as ladder. (ii) DNA gyrases: These enzymes breakdown the hydrogen bonds (A=T, C=G) between two strands of a DNA molecule. E.g., DNA topoisomerase. 3. DNA Polymerases or Replicase Enzyme DNA polymerases are the first enzymes suggested to be implicated in DNA replication. It mainly function in the polymerazation of nucleotides on the DNA template producing thereby the polynucleotide chain. In eukaryotic cells, these three enzymes have been name as DNA polymerase, β-DNA polymerase and -DNA polymerase. Due to their role in DNA replication, DNA polymerases are also known as DNA replicases. (1) DNA POLYMERASE-I: This enzyme was first discovered by Arthur Kornberg in E.coli. After the name of the scienctist, DNA polymerase-I is also known as „Kornberg enzyme‟ or „Kornberg polymerase‟. It is the most extensively studied DNA polymerase studied in DNA replication machinery of E.coli. Nowadays, it is believed that this enzyme is not responsible for DNA replication and it mainly function in DNA repair. Structurally, a molecule of DNA polymerase-I consists of a single polypeptide chain having the molecular wt. 109,000. An atom of Zn is associated with each molecule of this enzyme. Following sites have been reported to be present on its surface: 288 (a) Template Site: it is occupied the DNA strand. (b) Primer Site: The site at which the growth of DNA chain occurs. (c) Nucleotide triphosphate site: The site where the incoming nucleotide triphosphate is received. (d) Primer terminus site: This site, which is used for removing any mismatched nucleotide at the end of growing chain. (e) Site for 5’ 3’ cleavage: The site which is used for removing any strand coming in the path of growing primer. DNA polymerase-I function in following activities within cells. (i) 5’ 3’ polymerization activity: Attachment of nucleotides with each other by the activity of DNA polymerase forming a polynucleotide chain is called polymerization. The rate of polymerization in E.coli at 370 C has been noted to be 1000 nucleotides per minute. It occurs in 5‟ 3‟ direction. It forms small DNA segments through polymerization, which is used in repair mechanism. (ii) 3’ 5’ Exonuclease Activity: The activity of DNA polymerase-I is performed to remove any nucleotide which mispair during elongation of growing strand. (iii) 5’ 3’ Exonuclease Activity: This activity is performed by this enzyme to remove any DNA segment which comes as an obstruction in the way of growing DNA Strand. (iv) Removal of Thymine Dimmers: DNA polymerase-I function in the removal of thymine dimers from the DNA strand. Such thymine dimmers are produced to UV irradiation. After removal of diers, it also fills the gap, formed dut to excision. 289 (2) DNA Polymerase-II : This enzyme has also been isolated from E.coli and has the molecular wt. 90,000. Polymerization rate DNA polymerase-II is much slower than polymerase-I. It is only 50 nucleotides per minute in E.coli, it has 3‟ 5‟ exonuclease activity but lacks 5‟3‟ exonuclease activity unlike polymerase-I. (3) DNA- Polymerase-III : This is the main DNA polymerase involved in DNA replication. This polymerase enzyme was originally discovered in a lethal mutant of E.coli having mutation at dnaE locus. The enzyme has higher affinity for nucleotides than polymerase-I and II have. The rate of polymerization by polymerase-III is approximately 10-15 times higher than the polymerase-I. Structurally, DNA polymerase molecule consists of two polypeptide chains each having the molecular wt. of 90,000. This dimeric enzyme does not function unless it associates with two more chains of copolymerase-III each having the molecular wt. of 77,000. The holoenzyme may be represented as α2β2 where α2 polymerase-III and β2 copolymerase-III. ATP is also needed for the growth of polynucleotide chain. 290 Fig.- Enzyme polymerase. The enzyme DNA polymerase-III has 3‟5‟ exonuclease activity and 5‟3‟ polymerase activity. 4. DNA LIGASES Through polymerase activity by DNA polymerase-III, polynucleotide chains is formed in the form of small fragments which are known as Okazaki fragments. In order to form a complete chain complementary to that of the template, ligation of okazaki fragments is essential. The ligation reaction is performed by RNA ligases. 5. RNA PRIMER DNA replication really starts with the formation of a RNA fragment known as RNA primer. It is formed at the point of origin. The primer is formed through polymerization activity by RNA polymerase. Due to this reason, RNA polymerase is also needed for functional replication of DNA. 6. PRIMASES This is the group of enzymes, which are involved in RNA primer synthesis. RNA polymerase is an example of primases. Detailed molecular biological studies of the process of DNA replication now have revealed that the process is much complex and requires a multienzyme complex. About two dozens of enzymes are involved in this complex. The complex is known as „replisome‟ STEPS OF DNA REPLICATION 291 Before studying the mechanism of DNA replication, we must be familiar with following terms: 1. Replicon: A replicon is the unit of DNA in which individual act of replication takes place. It has capacity of DNA replication independent of other segments. Therefore, each replicon has its own origin and terminus, at which DNA replication stops. 2. Origin: This is the sequence of a replicon which supports initiation of DNA replication and also regulates the frequency of replication initiation. A general feature of origin is that it is A=T rich. An origin in E.coli, oric has been identified to 250 base pairs long. 3. Terminus: In most of prokaryotes, replicons has a specific site at the extreme downstream of the strands. This site stops replication fork movement and thereby terminates DNA replication. In order to understand the exact mechanism of DNA replication. The process must be studied in stepwise manner. The overall process is completed in following steps: (1) Before the start of DNA replication and formation of origin point, the enzyme DNA helicase associates with the site of DNA. Its molecules unwind the two strands of the DNA. Another enzyme, DNA gyrase or DNA topoisomerase breaks the hydrogen bonds between the two strands and separate them from each other forming a „Y‟ shaped replication fork. (2) The two strands of a DNA molecule separated in the way explained in the first step function as template. It should be noted here that template is the single strand of DNA on which polymerization of nucleotides forming a new strands takes place. 292 In eukaryotes, evidences for bi-directional DNA replication are available. DNA replication starts at many points each of which start as a loop and can be seen as expanding bubbles or eyes in electron micrograph. The number of eyes or bubbles indicates the number of replicons. Figure- DNA Replication (2) Formation of RNA Primer: Before the actual replication of DNA starts at origin, a short fragment of RNA is synthesized with the help of RNA polymerase. This RNA fragment is called RNA primer. It is believed that it provides safety to the new DNA strands, which is synthesized extending the RNA primer itself. 293 (3) Synthesis of Complementary Strand of DNA: DNA replication or synthesis of a new DNA strand complementary to the template is catalysed by the enzyme, DNA polymerase-III. It starts at the end RNA primer in 5‟3‟ direction. The nucleotide sequence in the new strand is always complementary to the sequence of nucleotides in the template. Some features of DNA replication are as follows: (i) DNA Replication is Bi-directional: Johan Cairsn on the basis of his experiments on atoradigraphy concluded that DNA synthesis starts at a fixed point on the chromosome and proceeds in one direction only. Subsequently, it was realized that Cairns results could be interpreted in terms of bidirectional replication also. On the basis of many other experiments it has convincingly been demonstrated that DNA replication begin at a unique site at origin and proceeds in both the direction on a strand. It takes place in the form of pieces called „Okazaki fragment which are joined with each other with the help of DNA ligases. (ii) The two strands of the parent DNA at the point of replication fork or origin replicate together with each other. (iii) Replication of 3‟5‟ strand of DNA molecule is continuous and the new strand grows in 5‟3‟direction. Replication in the second strand of the DNA molecule is discontinuous. Replication of this strand starts somewhat later than that of strand. Consequently, a given segment of 53‟ strand always replicates i.e., the 3‟5‟ strand. Therefore, the 3‟5‟ strand of the parent DNA molecule is known as the leading strand while the 5‟3‟ strand is termed as the lagging strand. 294 (iv) Formation of RNA primer takes place in the beginning of each and every okazaki fragments. 4. Termination of DNA Replication: The termination of DNA replication so signaled by specific sequences, the ter-elements. E.coli, the ter-element of R6 K plasmid has a 23 base pair sequence. This site functions as the binding site of Tus, a 36 K dal protein necessary for termination. This stops the replication fork movement and thereby stops DNA replication. 5. Removal of RNA Primer: After the whole DNA molecule is replicated on both the strands, the RNA primer on all the segments are removed or degraded with the help of DNA polymerase-I through its nuclease activity. 6. Synthesis of DNA Strand at the place of RNA Primer: in order to have replication of a complete DNA molecule, replication of the segment at the place of RNA primer is necessary. This process is performed with the help of DNA polymerase-I instead of polymerase-III through polymerization of deoxyribonucleotides. After DNA replication at the place of RNA primer, the replication process is completed. 7. Proof Reading and Repair Mechanism: The complementary base pairing during DNA replication is much accurate and precise, however, there are chances of error in this process of base pairing. It is mainly due to various physical and chemical forces involved in rate of error in E.coli are 5 x 10-8 to 5 x 10-10. 8. The proof reading of newly synthesized DNA strand is done by DNA polymerase-III through correction of mismatched base pairing. It deletes the wrong base and replaces it by a correct base. The process of proof reading 295 also takes place in 5‟3‟ direction. In E.coli, mutants defective in proof reading show an increase in mutation frequency by over 1000 times. 3.9 TRANSCRIPTION The production of RNA copies from a DNA template is known as transcription. It is catalysed by a specific enzyme RNA polymerase or transcriptase. During this process, only one strand of DNA duplex is known as template strand or antisence strand. This results into the production of mRNA molecule having base sequence complementary to the template DNA strand. It should be noted here that the sense. strand or coding strand of DNA is now copied and has the same base sequence as the RNA produced by the antisense strand. The RNA polymerase is a complex enzyme and usually consists of a larger protein part (apoenzyme), which is known as core enzyme and a cofactor, which is known as sigma factor. The two combines to produce the complete enzyme of holoenzyme. Unless and until the two parts of RNA polymerase do not combine with each other, it is not functional. As far as the nature of RNA polymerase in prokaryotes and eukaryotes is concerned, it shows much diversity. While in prokaryotes like E.coli a single species of this enzyme is found, at least three distinct RNA polymerases have been reported in nuclei of most of eukaryotes. These have been named as : 1. RNA polymerase-I or A, 2. RNA polymerase-II or B and 3. RNA Polymerase-III or C. They have different functions as: RNA polymerase-I or A: It is located in the nucleolus and responsible for the synthesis of rRNA. 296 RNA polymerase-II or B: It is found in the nucleoplasm and is responsibe for the synthesis of hnRNA which is a precursor of mRNA. RNA polymerase-III or C: It is also found in the nucleoplasm. It is responsible for the production of 5s rRNA and tRNA. 1. A Promoters for RNA Polymerase. Promoters for RNA polymerase I have atleast two elements: A GC-rich upstream (-180 to -107) control element. A core region that overlaps the transcription start site (-45 to +20). Protein coding structural genes in higher eukaryotes are transcribed in the nucleus, but the primary RNA transcripts in the nucleus differ from mRNAs used in the cytoplasm for translation. The RNA transcripts in the nucleus are collectively described as heterogeneous nuclear RNA or pre-mRNA molecules each of which is generally much larger than its corresponding mRNA. The hnRNA molecules, which are destined to produce mRNA, undergo RNA processing which includes the following events: (i) Modification of 5‟ end by capping and modification of 3‟ end by a tail after enzymatic cleavage; (ii) Splicing out of intron sequences from RNA transcripts of interrupted genes. Cleavage and polyadenylation usually proceed RNA splicing. Promoter, enhancer and silencer sites for initiation of transcription in eukaryotes In eukaryotes there are three RNA polymerases: RNA polymerase I or RNAPI for synthesis of pre-rRNA; RNA polymerase II or RNAPII for synthesis of re-mRNA or hnRNA and several snRNAs, and RNA polymerase III or 297 RNAPIII for synthesis of 5S RNA, tRNA. Different promoter sequences have been identified for different RNA polymerases. MECHANISM OF TRANSCRIPTION: The overall process of transcription is completed in following steps: Formation of holoenzyme: The core enzyme of RNA polymerase cannot start the polymerization process producing RNA. It first combines with the sigma factor and produce the holoenzyme, It is assumed that the sigma factor helps the enzyme in recognition of the initiation site on the DNA template. Attachment of holoenzyme on DNA duplex: The holoenzyme first binds at the promoter site of DNA forming the closed promotor complex or „closed binary complex‟. In this stage the DNA still remains in the form of double helical. Unwinding of DNA: It includes strand separation in the DNA duplex in a stretch of the DNA bound with RNA polymerase; It extends just beyond the start point so that the template becomes available for transcription initiation. The open DNA strands form the „open binary complex‟ Synthesis of RNA: After the open binary complex is formed on DNA, synthesis of RNA starts. Once the template or antisense strand of DNA becomes available, the enzyme begins to incorporate RNA nucleotides beginning at the start points. The polymerization of these nucleotides takes place in 5‟ 3‟ direction. As the enzyme molecule move ahead in this direction, phosphodiester linkage or bond is formed between two adjacent nucleotides. 298 The process of elongation of RNA synthesis take place when the holoenzyme leave the promoter region and move ahead in 5‟3‟ direction. Together with the movement of the holoenzyme, the trancription bubble also moves in the same direction. The transcription bubble represents the region of the DNA duplex in which the two strands are separated from each other. The length of the bubble ranges from 12 to 20 base pairs. The bubble movement and sequential adding of correct nucleotides on RNA chain take place simultaneously. The 5‟ end of the newly synthesized RNA progressively separate from the DNA template DNA. In the back of the bubble, the two DNA strands reassociate to form DNA duplex. Termination of RNA formation: Specially in prokaryotes, termination of transcription or RNA formation is brought about by certain termination signals on DNA The termination may be of two types: Rho Independent Terminations: This types of RNA synthesis termination is due to specific sequences on DNA. A typical hairpin like structure is formed on DNA template due to which the movement of RNA polymerase on the template is obstructed. The hairpin structure is formed due to inverted repeat sequences on DNA. The hairpin or stem-loop is followed by a run of adenine residues in DNA and U residues in mRNA in the downstream. Rho Dependent Termination: This type of termination is due to presence of special factor, which is called Rho factor. It has a mol. wt. of 60,000 and is not a part of RNA polymerase. After the synthesis of mRNA on template DNA is completed, it attaches with the template. The site for its attachment is characterized by 5‟-CAATCAA-3‟. The actual and precise mechanism of the function of factor is not known. 299 In eukaryotes, the termination process is more completed. The termination sites similar to prokaryotes are also operative in eukaryotes but these sites are believed to be present away up to 1 kb from the site of the 3‟end of the mRNA. AAUAAA sequence on mRNA and „snurp‟ are assumed to play important role in termination of the process in eukaryotes. Maturation of mRNA from hnRNA in eukaryotes: The mature mRNA molecules very often have much lower molecular wt. and base sequence length in comparison to the DNA segment from which it is transcribed. The primary RNA transcript of a structural gene is called pre-mRNA. It is also known as the heterogeneous RNA, high molecular wt. RNA. It is much bigger in size than mRNA. The later is formed by splicing of hnRNA followed by some other modifications. The heteronuclear mRNA undergoes following modifications: Addition of Cap (m7G) and Tail (Poly A) for mRNA in Eukaryotes Addition of methylated cap at the 5’ end The initial RNA transcript, derived from genes coding for proteins, gets modified so that its 5‟ end gains a methylated guanine and its 3‟ end is polyadenylated. Capping at 5‟ end occurs rapidly after the start of transcription and much before completion of transcription. Transcription starts with a nucleoside triphosphate, and a 5‟ triphosphate group is retained at this first position. The initial sequence at 5‟ end of hnRNA is therefore 5‟ pppApNpNp…3‟. To the 5‟ end is added a terminal G with the help of an enzyme guanyl transferase as follow: 5‟Gppp+5‟pppApNpNp 5‟Gppp5‟ApNpNp+pp+p 300 The new G residue is in the reverse orientation with respect to all other nucleotides and undergoes methylation at its 7th position. The cap with a single methyl group at this terminal guanine residue is found in unicellular eukaryotes and described as cap0, but in most eukaryotes, methyl group may also be present on the penultimate base at 2‟ position of sugar moiety, so that nucleotides, it is now described as cap1. Removal of cap leads to loss of translation activity due to loss of the formation of mRNA-ribosome complex. It suggests that the „cap‟ helps in recognition of ribosome. Only in some eukaryotic nRNAs, caps may be absent and may not be required for translation. 2. Polyadenylation and the generation of 3’ end in eukaryotes The 3‟end of n RNA is generated in two steps (i) Nuclease activity cuts the transcript at an appropriate location. (ii) Poly (A) is added to the newly generated end by an enzyme, poly (A) polymerase (PAP), utilizing ATP as a substrate. Ordinarily 30% of hnRNA and 70% of mRNA are polyadenylated. In addition to AAUAAA, there are following consensus sequences, that are involved in polyadenylation: (i) a G-U rich element is present downstream to the site of cleavage, and is important for efficient processing for polyadenylation: (ii) a G-A sequence immediately5‟ of the cleavage site;(iii) consensus upstream element situated 5‟ of a poly A signal or AAUAAA. Splicing of RNA parts coded by introns: Self splicing is a very common phenomenon found in hnRNA. In this process generally those parts of the RNA are removed or spliced out, which have been transcribed from intron regions of the template DNA. These regions have short consensus sequences which pair to formstem-loop like secondary structure. These are 301 helpful in self or autosplicing. Stem-loop like structures were observed for the first time in the hnRNA of Tetrahymena thermophila. Editing of RNA: Theoretically, the base sequence of a mRNA is just complementary to the base sequence of the segment of the template DNA from which it is transcribed. However, in many cases, the base sequence of mRNA has been found to be changed after transcription at the level of RNA. This process of change in the base sequence of mRNA is known as RNA editing. It may be confined to a single base or may affect the entire mRNA. 3.12 RIBOSOME STRUCTURE, r-RNA AND BIOSYNTHESIS Ribosomes are round, granular and membraneless cell organelle which are chemically nucleoprotein and found enormously in all the prokaryotic and eukaryotic cells. These were discovered first by Claude in 1943 and were named as „microsomes‟. Robinson and Brown isolated ribosomes from root cells of broad bean. Palade coined the term „ribosomes‟ and isolated it from animal cells. After his name ribosomes are laso called „Palade granules.‟ Ribosomes may be defined as “The smallest known electron microscopic, ribonucleoprotein particles attached the on RER or floating freely in the cytoplasm and are the sites of protein biosynthesis”. OCCURRENCE: Ribosomes are generally found in all known prokaryotic and eukaryotic organisms except mature RBCs. In prokaryotes these are found only in free form in the cytoplasm while in eukaryotes these are found both in the cytoplasm and on the surface of RER. The former is called cytoplasmic and later is called bound form of ribosomes. These may also be found on the surface of nuclear membrane. Some organelles like 302 mitochondria and chloroplast contain ribosomes in the matrix. These are called organellar ribosomes and reffered as „mitoribosomes‟ and „plastidoribosomes‟. The ribosomes found on the surface of RER is bound with the membrane with special proteins called ribo-phorines. Number: The number of ribosomes in a cell depends on the content of RNA. These are more in number in metabolically active cells like plasma cells, livercells, nissl‟s granules of nerve cells, meristematic cells, cancer cells, endocrine cells etc. In a cell of E.coli, the number of ribosomes vary from 10,000-20,000. Structure: Ribosomes are globular structures having the diameter of 150250 Å. Each ribosome is made up of two subunits one is smaller and another is larger in size. The later in dome shaped and is covered by cap like smaller unit. In 70S type of ribosome the larger and smaller units are 50S and 30S type. On the other hand, in 80S type, these are of 60S and 40S type, respectively. The two subunits of ribosomes are freely distributed in the cytoplasm. The two subunits unite to form a complete ribosome. Likewise, the two subunits dissociate with each other when the concentration of Mg ++ ion decreases in the cytoplasm. During protein synthesis many ribosomes become attached with mRNA forming a peculiar structure called polyribosome. 303 Location of antigenic sites of ribosomal proteins. Stoffler and Wittmann’s model Ultrastructure of Ribosomes The last point about the ultra structure of ribosomes has not been said till date. The credit of giving the present knowledge of the ultrastructure of ribosomes goes to Nauninga. According to him, the size of larger (50S) subunit of 70S type of ribosome is 160 to 180 Å which is pentagonal in 304 shape. This unit has a groove of 40-60 Å size in which the smaller subunit is attached during association. The smaller subunit has a platform, cleft, head, base and also a binding site for nRNA. The smaller unit of 70S and 80S type of ribosomes does not have a definite shape. Florendo in 1968reported a pore like transparent area on the larger unit of 50S of 70S ribosomes. In between two subunits of ribosomes, mRNA is found. t-RNA molecule is found in the side of nRNA. The new-formed polypeptide chain being synthesized on the ribosome mRNA complex has been seen passing through the transparent pore on the larger unit. It also has a protuberance, a ridge and stalk. Two binding sites, peptidyl and amino acyl sites are found on the larger units. The 50S and 30S subunits have been reported to have the molecular weight of 1.8 X 106 Daltons and 0.9 X 106 Daltson, respectively. It must be noted here that size and type of ribosomes and their subunit are determined on the basis of their sedimentation coefficient. Types of Ribosomes On the basis of their sedimentation coefficient, ribosomes have been classified into two main types: 70S ribosomes: These are found in prokaryotes and mitochondria and plastids of eukaryotes. Each 70S ribosome is about 200-290 Å in size and 2.7 X 106 Daltons in its mol. Weight. It consists of two subunits of 50S and 30S size. Both of these units are made up of ribosomal RNA and ribosomal proteins. The 50S subunits again consist of 23S and 5S rRNA and 30 types of proteins. Similarly, the smaller unit is made up of 16S type of rRNA and 20 types of proteins. 305 80S ribosomes: These are the characteristic of eukaryotic cells and found. In their cytoplasm. It consists of two subunits. The size of larger subunits is 60S and that of smaller subunit is 40S. It is also made up rRNA and proteins. The 60S subunits consists of 28S rRNA, 5.8 S rRNA and 5S rRNA and about 50 types of proteins. The smaller subunits is similarly made up of 18S rRNA and 30 different proteins. Polyribosomes or Polysomes: When many ribosomes are attached to same mRNA strand, it is called polyribosomes or polysome. It is formed when a simple protein is required in high quantity. The number of ribosomes in a polysome depends on the length of mRNA. The distance between two adjacent ribosome is about 90 nucleotids. Origin of ribosomes: We have studied that ribosomes are solely made up of rRNA and proteins. The former is formed inside the nucleus and the later is produced in the cytoplasm. Therefore, these are partly nuclear and partly cytoplasmic is nature. However, in prokaryotes, since there in no nucleus, ribosomes are totally cytoplasmic in nature. Functions of Ribosomes. Ribomes are called factories of proteins or engineers of the cell because these are the side of protein synthesis. Sometimes rRNA of ribosomes has been found to function as enzymes controlling the cellular functions. These are called ribozymes. The process of translation of genetic language into the language of enzymes or protein take place at ribosomes. It takes place with the help of rRNA, which, is produced during transcription of nuclear DNA. 306 In general the ribosomes bound on RER synthesise enzymes for extracellular use e.g., pancreatic cells, chief cells of gastric glands, liver cells etc. Ribosomes temporarily store proteins. Ribosomes keep the mRNA molecules functionally alive. RIBOSOMAL RNA or rRNA The RNA, which is found in ribosomes, is called ribosomal RNA. Ribosomes are chemically ribonucleoprotein as they consist of RNA and proteins. It is known as soluble RNA. Its quantity in a cell is much higher than that of mRNA and tRNA. It constitutes about 80% of total RNA. On the basis of their sedimentation coefficient or rate of sedimentation, rRNA molecules may be classified into following categories: 28S-rRNA: It has molecular weight more than 10,00000. Sedimentation coefficient is between 21S and 29S. It is found in 60S subunit of eukaryotic ribosomes. 18s-rRNA: It molecular weight is less than a millions. Sedimentaion varies between 12S to 18S. It is found in 40S subunit of ribosomes. 5S-rRNA: It has much lower molecular weight and is found in 30S unit of ribosomes. Structure of rRNA Ribosomal RNA molecules are single stranded but in the solution of high ionic concentration, irregular spiral coiling of rRNA is formed. As the ionic 307 concentration of the solution increases, the degree of irregular coiling of rRNA also increases. In this coiling the intramolecular bases show base pairing. The pairing is normal as A pairs with U and C pairs with G. Function of rRNA The main function of rRNA may be summarized as below: In many viruses specially in plant viruses, RNA function as genetic material and carry genetic information from generation to generation. Different RNAs function as structural component of a cells mRNA are the site of protein synthesis where polymerization of amino acids takes place through peptide bond formation between amino acid molecules during translation process. A tRNA molecule has anticodon site and has the capacity of attachment with the complementary condon on mRNA. tRNAs further carry activated amino acids to the mRNA and catalyse peptide bond formation between two amino acid molecules.Ribosomal RNA(rRNA) is the constituent unit of ribosomes. TRANSLATION AND GENETIC CODE The synthesis of protein from mRNA involves translation of the language of nucleic acids into language of proteins. For initiation and elongation of a polypeptide, the formation of aminoacyl transfer RNAs is a prerequisite, Formation of Aminoacyl rRNA Activation of amino acid This reaction is brought about by the binding about by the binding of an amino acid with ATP and is mediated by specific activating enzymes known as amino acyl tRNA syntehtases or aaRs. As a result of this reaction between amino acid and adenosine triphosphate, mediated by specific enzyme, a complex (amino acyl-AMP- enzyme complex) is formed. Amino 308 acyl-RNA synthetases are specific with respect to amino acids. For different amino acids, different enzymes would be required. Aa1 + ATP (Enzl) (aa1-AMP) Enz1 + PP Activation of amino acid The transfer of amino acid to rRNA The amino acyl-AMP-enzyme complex, formed during the step outlined above, reacts with a particular tRNA and transfers the amino acid to the tRNA. A particular amino acid would require a particular enzyme and a particular species of tRNA. This would mean that for 20 amino acids, at least 20 different enzymes and also atleast 20 different t-RNA species would be required. (aa1-AMP) Enz1 + t-RNA1 aa1- t-RNA1 + AMP + Enz1 309 Transfer of amino acid to tRNA Initiation of Polypeptide The initiation of polypeptide chain is always brought about by the amino acid methionine, which is regularly coded by the condon AUG, In eukaryotes, formylation of initiating methionine is not brought about due to the absence of tRNAfmet in plants and animals. Initiation in higher organisms will therefore, take place without formylation. Initiation in eukaryotes Initiation of polypeptide chain in eukaryotes is similar to that is prokaryotes, except the following minor differences. (i) In eukaryotes there are more initiation factors. They are named by putting a prefix „e‟ to signify their eukaryotic origin. These factors are eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF4D, eIF4F, eIF5 and eIF6. (ii) In eukaryotes, formylation of methionine does not take place. (iii) In eukaryotes, smaller subunit associates with initiator tRNAimet, without the help of mRNA, while in prokaryotes, generally the 30S-mRNA complex is first formed which then associates with f-mettRNAfmet. Kozak’s ribosome scanning hypothesis for translation in eukaryotes In 1983, Marilyn Kozak proposed a hypothesis for initiation of translation by eukaryotic ribosome. According to this hypothesis, 40S smaller subunit of a eukaryotic ribosome with its associated met-tRNA moves down the mRNA from 5‟ end, until it encounters the first AUG. At this point, the 60S subunits join and the translation begins. The 80S ribosome, after reaching termination, releases protein and dissociates in two subunits. Elognation of Polypeptide 310 The following three steps are important in the elongation process. Binding of AA-rRNA at site ‘A’of ribosome (classical vs hybrid state models for translation) In earlier classical model, each ribosome had two cavities, in which tRNA could be inserted. These were ‘P’ site and ‘A’ site. However, later a third cavity was suggested. F-met-tRNAfmef comes on ‘E’ site, to make „A‟ site available for the next amino acyl tRNA (AA-rRNA). Various steps of protein synthesis: (A-B) Attachment of tRNA-fmet-mRNA and smaller unit of ribosome, (C) Union of subunits of ribosomes, (D) Union of second. Another site called „R‟ sites located on smaller subunit of ribosome, was proposed „R‟ site plays a role in the improvement of accuracy 311 of translation. The aminoacyl rRNA first binds to R site involving codonanticodon pairing. Later aminoacyl rRNA is flipped to „A‟ site using energy from GTP molecule. During this flipping, tRNA is held only by condonanticodon pairing. After formation of 70S initiation complex, the next amino acyl tRNA enters „A‟ site. Elongation factors EF-Tu and EF-Ts participate. The elongation factor EF-Tu first combines with GTP and changes to an active binary complex, which binds with aa-tRNA, to form a ternary complex. EF-Tu-Ts+GTP EF+Tu.GTP+EF-Ts Binary Complex EF-Tu.GTP+aa-tRNA EF-Tu..GTP.aa-tRNA (Ternary Complex) (Hybrid states Models), it has been shown that above ternary complex actually binds in an A/P hybrid state, the anticodon binding to the A-site of the 30S subunit and the CCA end binding to the P-site of the 50S subunit as well as to the 30S subunit. The „P‟ site is already occupied by f-met. tRNAfmet or by a peptidyl tRNA. Following the GTP hydrolysis, EF-Tu.GDP+P are released from the ternary complex, permitting movement of CCA end of aatRNA into A site of the large 50S subunit. EF-Ts now displaces GDP in the EF-Tu.GDP binary complex and associates with EF-Tu, so that GTP can again associate with EF-Tu to start another cycle for the binding of aa-tRNA. Formation of peptide bond This is a catalytic reaction during which a peptide bond is formed between the free carboxyl group of the peptidyl tRNA at the „P‟ site and the free amino group present with amino acyl tRNA, which is available at the A site. The 50S 312 rRNA have peptidyl transferase activity, so that the ribosome is described as a ribozyme. According to this displacement model, peotidyl chain remains in a constant position relative to ribosome, while the tRNA moves during the peptide reaction. After the formation of peptide bond, the tRNA at „P‟ site is deacylated and the tRNA at „A‟ site now carries the polypeptide. Translocation of peptidyl tRNA From ‘A’ to ‘P’ site. The peptidyl tRNA present at ‗A‘ site is now Translocated to ‗P‘ site. For translocation of peptidyl tRNA from ‗A‘ site to P site, there are two models available: (i) According to two sites model, deacylated tRNA is liberated from ‗P‘ site, and with the help of one GTP molecule and an elongation factor EF-G, the peptidyl tRNA is translocated from ‗A‘ to ‘P‘ site. Thus according to this model, tRNA is either entirely in the A site or entirely in the P Site 313 Different stages in translation The requirement of EF-G and GTP for translocation was revealed by the use of antibiotic. The elongation factor EF-G binds to ribosome and is released on hydrolysis of GTP, which is a ribosomal function. EF-G and EF-Tu cannot bind to ribosome simultaneously, so that the entry of a fresh aa-tRNA on ‗A‘ site and the translocation of peptidyl tRNA from ‗A‘ to ‗P‘ site has to follow each other and cannot occur simultaneously. 314 In eukaryotes, the elongation factor needed for translocation is called eEF-2, for the formation of one peptide bond. One ATP molecule and two GTP molecules (one for transfer of aa-tRNA to ‗A‘ site and the other for translocation of peptidyl tRNA from ‗A‘ to ‗P‘ site) are required. Termination of Polypeptide Terminations in mRNA with stop condon Termination of the polypeptide chain is brought about by the presence of any one of the three combination condons, namely UAA, UAG and UGA. These termination condons are recognized by one of the two release factors RF1 and RF2. The release factors to act on ‗A‘ site, since suppressor rRNA capable of recognizing by entry at ‗A‘ site. A third release factor RF3 stimulate the action of RF1 and RF2 in a GTPdependent and condon independent manner GTP molecule is hydrolysed during release of a polypeptide, when RF3 stimulates RF1 and RF2. For release reaction, the polypeptidyl tRNA must be present on ‗P‘ site and the release factors help in splitting of the carboxyl group between the polypeptide and the last tRNA carrying this chain. Polypeptide is thus released and the ribosome dissociates into two subunits with the help of ribosome release factor or RRF. It has been shown that the translation apparatus in chloroplasts and mitochondria differs from that in cytoplasm in eukaryotes in the following respects. (i) Ribosomes in these organelles are smaller in size than these in cytoplasm. (ii) The tRNAs are specific and differ, the number of tRNAs in mitochondria being 22 as against 55 in cytoplasm. (iii) Initiation of translation takes place by formyl-methionyl tRNA both in chloroplasts and mitochondria, although no formylation takes place in cytoplasm. (iv) Translation in chloroplasts and mitochondria can be inhibited by chloramphenicol. 315 Modification of Folding of Released Polypeptide Modification of released polypeptide After translation, the released polypeptide is modified in various ways. Due to the action of certain other enzymes, exo-amino-peptidases, amino acids may be removed from either the N-terminal end or the C-terminal end or both. The polypeptide chain singly or in association with other chains also folds into a tertiary structure. This problem of protein folding is sometimes described as ‗Second Half of the Genetic Code‘. Genetic code : Several theories were proposed to explain the mechanism by which a particular sequence of nitrogenous bases in DNA by transcribing complementary bases in mRNA determines the position of specific amino acid in the protein molecule. The theory which is widely accepted till now was proposed by F.H.C. Crick. The theory holds the existence of a genetic code and its smallest unit which codes for one amino 316 acid is known as codon. A codon (code word) is the nucleotide or nucleotide sequence in mRNA which codes for particular amino acid, whereas the genetic code is the sequence of nitrogenous bases in mRNA molecule, which encloses information for the synthesis of protein molecules. Essential features of genetic Code. 1. Triplet : A codon of the modern genetic code comprises of three nitrogenous bases of mRNA in a specific sequence. 2. Commaless : There is no punctuation (comma) between the adjacent codons i.e., each codon is immediately followed by the next codon with no intervening spaces of letters for comma. 3. Non-overlapping : Under the overlapping triplet code the number of codons could be reduced to twenty. But evidences have been gathered in support of the existence of non-overlapping code. 4. Ambiguity : The genetic code inside the cell medium (in vivo) is said to be nonambiguous, because a particular codon always codes for the same amino acid. No doubt the same amino acid may be coded by more than one codon (degeneracy), but one codon never codes for two different amino acids. 5. Universality : The same genetic code is said to be present in all kinds of living organisms including viruses, bacteria, unicellular and multicellular organisms. 6. Collinearity : The codons in DNA and mRNA and the corresponding amino acid residues in the polypeptide chain have a linear arrangement which has been demonstrated by the studies of T4 mutants. These produce incomplete head protein molecules. These mutants can be shown to map in linear sequence by recombination technique. This suggests that the code is collinear. 317 The multiple system of coding is known as degenerate system or degenerate code and provides protection to organisms against many harmful mutations. The major degeneracy occurs at the third position(3‘ end of the triplet codon). When first two bases are specified, the same amino acid may be coded for whether the third base is U, C, A, or G. This base is described as ‘Wobbly base’. Indian born biochemist, Dr. H.G.Khorana, devised an ingenious technique for artificially synthesizing mRNA with repeated sequences of known nucleotides. For this valuable contribution he was awarded Noble Prize in 1968. 318 By using synthetic DNA, Khorana and his coworkers prepared chains of polyribonucleotides with known repeating sequences of two or three nucleotides as follows; (a) Poly CUC UCU CUC UCU……………. (b) Poly CUA CUA CUA CUA…………… In first case CUC and UCU are two codons arranged alternately in the polynucleotide chain. This dictates the formation of polypeptide chain having two amino acids (leucine and serine) arranged alternately. The second case is an example of homopolymer chain. The polynucleotide chain is formed of repeated linkage of codon CUA. This dictates the formation of a polypeptide chain consisting of only one amino acid leucine. KEY TERMS Genetic code Termination codon Degeneracy codon Triplet codon Initiation codon Ambiguity of genetic code Wobble hypothesis The multiple system of coding is known as degenerate system or degenerate code and provides protection to organisms against many harmful mutations. The major degeneracy occurs at the third position(3‘ end of the triplet codon). When first two bases are specified, the same amino acid may be coded for whether the third base is U, C, A, or G. This base is described as ‘Wobbly base’. Check your progress-2 1. Note :- Write your answer in the space given 2. Compare your answer with the one given at the end of the unit. Write notes on 1. DNA replication 319 2. Protein Synthesis 320 1.12 LET US SUM UP:Two types of nucleic acids are found in the cells of all living organisms. These are: 1. Deoxyribonucleic acid - DNA 2. Ribonucleic acid - RNA The Chemical analysis has indicated that DNA is composed of three different types of compounds: 7. Sugar Molecule represented by a pentose sugar, the deoxyribose or 2‘deoxyribose. 8. Phosphoric Acid. 9. Nitrogenous Bases: These are nitrogen containing organic ring compounds. These are of the following four types: I. Adenine represented by –A II. Thymine represented by –T III. Cytosine represented by –C IV. Guanine represented by –G These four nitrogenous bases are separated into two categories: Purines: These are two-ringed nitrogen compounds. Adenine and guanine are the two purines found in DNA. Their structural formulae are represented in fig.2. 321 Pyrimidines: These are formed of one ring only and include cytosine and thymine Nucleotides ( The Monomers of DNA) A nucleotide is formed of one molecule of deoxyribose, one molecule of phosphoric acid and one of the four nitrogenous bases. Since there are four nitrogenous bases, there are four type of nucleotides namely: 9. Deoxyadenylic acid -Adenine + Deoxiribose + Phosphoric acid 10.Deoxyguanylic acid -Guanine + Deoxiribose + Phosphoric acid 11.Deoxycytidylic acid -Cytosine + Deoxiribose + Phosphoric acid 12.Deoxythymidylic acid -Thymine + Deoxiribose + Phosphoric acid DNA Structure & Forms. The Watson & Cricks model of DNA, actually describes B form of DNA. Two Polynucleolide chains run antiparallely. Purines and Pyremedines are on inside while phosphate and deoxyribose units are on outside. The diameter of Helix is 20 A0, Adjacent basee are separted by 3.4 A0 along the helix and are related by a rotation of 360o. The helical structure repeats after ten residues at interval of 34 A0. G = C & A = T are hydrogen bonds. G always pairs with C & A with T Replication of DNA: -Basic Rules of Replication Replication is a semi conservative process. Replication has direction. It could be unidirectional or bi-directional fork. Unidirectional – Mitochondrial DNA, ø Bidirectional - In E.coli & Eukaryotic Chromosome. Replication starts at a unique point on bacterial and viral chromosome. 322 Replication of both strands proceed by the addition of nucleotide monomers in the 5‘ 3‘directional Replication starts in short discontinuous pulses. Replication at the level of short fragments in initiated by the production of a short segment of RNA to serve as a primer for DNA polymerase. Replication of viral DNA is circular but progeny has linear DNA, Replication time is 30 Minutes in E.Coli. Enzymes of Replication RNA Polymerase or Primerases DNA Polymerase Nucleases (Endonucleases & Exonucleases) DNA Ligases Restriction Enzymes Swivelases Unwinding enzymes and proteins Steps: Binding of Unwinding proteins Initiation of chain Elongation Termination Binding or joining by ligases. Transcription: Transcription is the synthesis of mRNA and stable RNA molecules from a DNA Template by RNA Polymerase, using ribonucleotide triphosphates as precursor. 323 Bacterial cells contain only on RNA Polymerase where as eukaryotes have at least three viz. RNA Poly. I, II & III. The three stages of transcription are Initiation Elongation Termination Promoters consist of conserved seq. necessary for the initiation of transcription. There are many different types of promoters. The promoter sites for RNA polymerase I and II are located before the start site for transcription. RNA Polymerase III recognizes sites within the gene itself. In eukaryotes the promoters sites for RNA Polymerases I and II are located at the 5‘ end of the gene but the polymerase III promoter lies with in the gene. RNA Polymerase II promoters have TATA box with consensus seq. of TATAAA. Main features of eukaryotic Transcription The template for transcription is a completes of DNA and Protein That has beaded appearance. Eukaryotic RNA synthesis starts at precise promoter seq. As a Result genes that is very actively transcribed and show ―fern leaf‖ or ―Christmas Tree‖ configuration in tRNA and lampbrush chromosome. At any one time, only a very small fraction of the total chromatin is transcribed.The nascent RNA gets associated with protein as it is being transcribed, producing ribonucleus protein particles (RNP). In eukaryotes the nuclear envelope introduces as barrier between 324 transcription and protein synthesis Eukaryotic mRNA Eukaryotic mRNA is metabolically stable as compared to mRNA of pro. As they have comparatively longer half-life. It is monocistronic. The 5‘ end of blocked by 7-methyl –G.] The 3‘ end of euk. mRNA ends with A poly A segment./ Eukaryotic genes. Frequently contain insertions of non-coding DNA. Heterogeneous Nuclear RNAs are mRNA precursors containing intervening sequences. Eukaryotic mRNA are associated with proteins Translation: Main steps of translation are: 1. Activation of amino acids 2. Transfer of amino acid to t-RNA. 3. Initiation of polypeptide chain 4. Elongation of polypeptide chain 5. Termination of polypeptide chain I. Activation of amino acids It includes screening of amino acids. There activation at carboxyl gr. Enzyme is amino acyl tRNA synthetase II. Transfer of a.a to t-RNA t-RNA are specific and named after amino acids Ester bonds are formed between amino acids & t-RNA III Initiation 325 In 1975 Anderson reported IF- MP, IF –M1, IF-M2A, IF- - -M3. Formylation of methionine does not occur in rule. Reaction is catalyzed by transformylase enzymes Initiation complex is formed by mRNA + 40S ribonucleus S.U + tRNA + GTP + 3 Initiation factors. Here, nmet-tRNA binds first to 40s sub unit. met RNA. The P and A sites are located on 70s. subunit. Met RNA binds to the P site. AU other tRNA as first build to A site, then shift to P site. 60s& 40s sub units join to form 80s subunit elongation of polypeptide chain. EF1& EF2 are required for elongation of polypeptide chair. In the presence of elongation factor, amino acyl tRNA bends to A site (by using GTP) on ribosome. Thus ternary complex is formed. Peptide bond formation: First amino acid is now united by peptide bond formation with second amino acids. Peptide bond formation does not require external energy source. This reaction is catalyzed by peptide transferase complex located in large subunit Alpha Amino group of one amino acid is bonded to the alpha- carboxyl of other with elimination of H2O. Translocation : The movement of ribosome relative to mRNA is called translocation. It occurs in 53‘ direction. 326 Termination. RNA polymerase recognizes termination signal UAA,UAG & UGA. The termination condons provides signals to the ribosomes for attachment of release factors. RF-1, RF-2, RF-3. 1.13 CHECK YOUR PROGRESS KEY Check your progress Key-1 1. Purines are adenine , guanine, Pyrimidines are Thymine , Uracil , Cytosine. 2. A+G/C+T = 1 3. Watson & Cricks model. Check your progress Key-2 1. Rules of replication , enzymes of replication. 2. Transcription & Translation steps. 1.14 ASSIGNMENT / ACTIVITY Q1. Explain process of DNA replication in eukaryotes. Q2. Prepare a model of DNA. Q3. Write short notes on 327 a. Transcription b. DNA hydrolysis. 1.15 REFERENCES 1. E.D.P De Roberties & E.M.F. De Robertis Jr. - Cell and Molecular Biology. 2. David Friefielder - Molecular Biology. 3. P.K. Gupta - Cell and Molecular Biology. 4. Stanier - Microbiology 5. Benjamin Lewin - Genes. 6. C.B.Powar - Cell Biology . 328 329