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- MADHYA PRADESH BHOJ OPEN UNIVERSITY
BHOPAL (M.P.)
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M.Sc.Chemistry (Previous)
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Sri Sathya Sai College for Women
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Sri Sathya Sai College for Women, Bhopal
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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.
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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 1m 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 50m 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:
preproinsulinproinsulininsulin).
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 0.5m 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 500m. 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 190m 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
ERagranular ERGolgi membranelysosomes, 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 1.5m (0.2 and 0.25 m diameter in most
mammalian tissues : 0.5m 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 1.5m (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-(14)-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(14)
glycosidic linkage; its full name
is thus O-β-D-glucopyranosyl(14)-β-Dglucopyranose.
Fig- 9
In
120
gentiobiose, the glycosidic linkage is β (16). Since both these disaccharides have a
free anomeric carbon, they are reducing sugars.
The disaccharide lactose [O- β -D-glucopyranosyl-(14)- 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-(21)- 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(14) 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 (14) 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(14). 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 (14) but also that there are no branch points. The only
chemical difference between starch and cellulose, both homopolysaccharides of Dglucose, is that starch α (14) linkages and cellulose β (14). Cellulose is not
attacked by either α
β
β
(14) 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(14) 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 (14)
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 (14), but the branch points are α (16)
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(14)]-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(14) 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(14)-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 (16) 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 (16)-glucan 6-glucanohydrolase, also called α (16)glucosidase] can hydrolyze the α (16) linkages at the branch points. The combined
action of a β -amylase and an α(16)-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 (14) 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 (16). Glycogen can be isolated from animal tissues by digesting
128
them with hot KOH solutions, in which the nonreducing α (14) and α (16)
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 (14). Found as
storage polysaccharides in yeasts and bacteria, they vary in their branch points,
which may be 12, 13, 14, or 16 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 (21) 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
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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
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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
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----------------------------------------------------------------------------------------------------------------
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, 41, 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.
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234
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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
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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.
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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.
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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
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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
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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.
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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
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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.
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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
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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
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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.
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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:
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(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.
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(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.
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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
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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.
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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.
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(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 53‟ 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.
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(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
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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.
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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
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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.
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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.
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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
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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
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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
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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.
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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.
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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.
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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
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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.
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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.
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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
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2. Protein Synthesis
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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.
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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.
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 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.
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 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
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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
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 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 53‘ direction.
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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
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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
.
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