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Dr. Gobinath Pandian 214, Environmental Science Building 01049535553 (Phone number) Chapter: 1 Introduction Amino acids Introduction to Proteins & Enzymes INTRODUCTION Amino acids are molecules containing an amine group (NH), a carboxylic acid group (COOH) and a side chain(R) that varies between different amino acids. These molecules contain the key elements of carbon, hydrogen, oxygen, and nitrogen. • Any of a class of organic compounds in which a carbon atom has bonds to an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom (-H), and an organic side group (called -R). • Amino acids are organic compounds made of carbon, hydrogen, oxygen, nitrogen, and (in some cases) sulfur bonded in characteristic formations. • They are therefore both carboxylic acids and amines. The physical and chemical properties unique to each result from the properties of the R group, particularly its tendency to interact with water and its charge (if any). • Amino acids joined linearly by peptide bonds (see covalent bond) in a particular order make up peptides and proteins. • Amino acids are critical to life, and have many functions in metabolism. One particularly important function is to serve as the building blocks of proteins, which are just linear chains of amino acids, or more precisely, amino acid residues. • Every protein is chemically defined by the order of amino acid residues, their primary structure and this, in turn, determines their secondary structure Glycine is the smallest of the amino acids. It is ambivalent, meaning that it can be inside or outside of the protein molecule. In aqueous solution at or near neutral pH, glycine will exist predominantly as the zwitterion The isoelectric point or isoelectric pH of glycine will be centered between the pKas of the two ionizable groups, the amino group and the carboxylic acid group. In estimating the pKa of a functional group, it is important to consider the molecule as a whole. For example, glycine is a derivative of acetic acid, and the pKa of acetic acid is well known. Alternatively, glycine could be considered a derivative of aminoethane. Cysteine is one of two sulfur-containing amino acids; the other is methionine. Cysteine differs from serine in a single atom-- the sulfur of the thiol replaces the oxygen of the alcohol. The amino acids are, however, much more different in their physical and chemical properties than their similarity might suggest. Cysteine also plays a key role in stabilizing extracellular proteins. Cysteine can react with itself to form an oxidized dimer by formation of a disulfide bond. The environment within a cell is too strongly reducing for disulfides to form, but in the extracellular environment, disulfides can form and play a key role in stabilizing many such proteins, such as the digestive enzymes of the small intestine. • Methionine, an essential amino acid, is one of the two sulfurcontaining amino acids. The side chain is quite hydrophobic and methionine is. usually found buried within proteins. • Unlike cysteine, the sulfur of methionine is not highly nucleophilic, although it will react with some electrophilic centers. It is generally not a participant in the covalent chemistry that occurs in the active centers of enzymes. • Methionine as the free amino acid plays several important roles in metabolism. It can react to form S-Adenosyl-L-Methionine (SAM) which servers at a methyl donor in reactions • Alanine is a hydrophobic molecule. It is ambivalent, meaning that it can be inside or outside of the protein molecule. The α carbon of alanine is optically active; in proteins, only the L-isomer is found. • Note that alanine is the α-amino acid analog of the αketo acid pyruvate, an intermediate in sugar metabolism. Alanine and pyruvate are interchangeable by a transamination reaction. • Asparagine is the amide of aspartic acid. The amide group does not carry a formal charge under any biologically relevant pH conditions. The amide is rather easily hydrolyzed, converting asparagine to aspartic acid. • Asparagine has a high propensity to hydrogen bond, since the amide group can accept two and donate two hydrogen bonds. • Asparagine is a common site for attachment of carbohydrates in glycoproteins. • Aspartic acid is one of two acidic amino acids. Aspartic acid and glutamic acid play important roles as general acids in enzyme active centers, as well as in maintaining the solubility and ionic character of proteins. • Proteins in the serum are critical to maintaining the pH balance in the body; it is largely the charged amino acids that are involved in the buffering properties of proteins. • Aspartic acid and oxaloacetate are interconvertable by a simple transamination reaction, just as alanine and pyruvate are interconvertible. • Glutamine is the amide of glutamic acid, and is uncharged under all biological conditions. • The additional single methylene group in the side chain relative to asparagine allows glutamine in the free form or as the N-terminus of proteins to spontaneously cyclize and deamidate yielding the six-membered ring structure pyrrolidone carboxylic acid, which is found at the Nterminus of many immunoglobulin polypeptides. This causes obvious difficulties with amino acid sequence determination. • Histidine, an essential amino acid, has as a positively charged imidazole functional group. • The imidazole makes it a common participant in enzyme catalyzed reactions. • The unprotonated imidazole is nucleophilic and can serve as a general base, while the protonated form can serve as a general acid. The residue can also serve a role in stabilizing the folded structures of proteins. • Isoleucine, an essential amino acid, is one of the three amino acids having branched hydrocarbon side chains. It is usually interchangeable with leucine and occasionally with valine in proteins. • The side chains of these amino acids are not reactive and therefore not involved in any covalent chemistry in enzyme active centers. • However, these residues are critically important for ligand binding to proteins, and play central roles in protein stability. Note also that the β carbon of isoleucine is optically active, just as the β carbon of threonine. These two amino acids, isoleucine and threonine, have in common the fact that they have two chiral centers. • Leucine, an essential amino acid, is one of the three amino acid with a branched hydrocarbon side chain. • It has one additional methylene group in its side chain compared with valine. • Like valine, leucine is hydrophobic and generally buried in folded proteins. • Lysine. an essential amino acid, has a positively charged ε-amino group (a primary amine). • Lysine is basically alanine with a propylamine substituent on theβcarbon. The ε-amino group has a significantly higher pKa (about 10.5 in polypeptides) than does the α-amino group. • The amino group is highly reactive and often participates in a reactions at the active centers of enzymes. Proteins only have one α amino group, but numerous ε amino groups • Phenylalanine, an essential amino acid, is a derivative of alanine with a phenyl substituent on the β carbon. Phenylalanine is quite hydrophobic and even the free amino acid is not very soluble in water. • Due to its hydrophobicity, phenylalanine is nearly always found buried within a protein. The π electrons of the phenyl ring can stack with other aromatic systems and often do within folded proteins, adding to the stability of the structure. • Proline shares many properties with the aliphatic group. • Proline is formally NOT an amino acid, but an imino acid. Nonetheless, it is called an amino acid. The primary amine on the α carbon of glutamate semialdehyde forms a Schiff base with the aldehyde which is then reduced, yielding proline. • When proline is in a peptide bond, it does not have a hydrogen on the α amino group, so it cannot donate a hydrogen bond to stabilize an α helix or a β sheet. It is often said, inaccurately, that proline cannot exist in an α helix. When proline is found in an α helix, the helix will have a slight bend due to the lack of the hydrogen bond. • Serine differs from alanine in that one of the methylenic hydrogens is replaced by a hydroxyl group. • Serine is one of two hydroxyl amino acids. Both are commonly considered to by hydrophilic due to the hydrogen bonding capacity of the hydroxyl group. • Threonine, an essential amino acid, is a hydrophilic molecule. • Threonine is an other hydroxyl-containing amino acid. It differs from serine by having a methyl substituent in place of one of the hydrogens on the β carbon and it differs from valine by replacement of a methyl substituent with a hydroxyl group. • Note that both the α and β carbons of threonine are optically active. • Tryptophan, an essential amino acid, is the largest of the amino acids. It is also a derivative of alanine, having an indole substituent on the β carbon. • The indole functional group absorbs strongly in the near ultraviolet part of the spectrum. The indole nitrogen can hydrogen bond donate, and as a result, tryptophan, or at least the nitrogen, is often in contact with solvent in folded proteins. • Tyrosine, an essential amino acid, is also an aromatic amino acid and is derived from phenylalanine by hydroxylation in the para position. • While tyrosine is hydrophobic, it is significantly more soluble that is phenylalanine. The phenolic hydroxyl of tyrosine is significantly more acidic than are the aliphatic hydroxyls of either serine or threonine, having a pKa of about 9.8 in polypeptides. • As with all ionizable groups, the precise pKa will depend to a major degree upon the environment within the protein. • Tyrosines that are on the surface of a protein will generally have a lower pKa than those that are buried within a protein; ionization yielding the phenolate anion would be exceedingly unstable in the hydrophobic interior of a protein. • Valine, an essential amino acid, is hydrophobic, and as expected, is usually found in the interior of proteins. • Valine differs from threonine by replacement of the hydroxyl group with a methyl substituent. Valine is often referred to as one of the amino acids with hydrocarbon side chains, or as a branched chain amino acid. • Note that valine and threonine are of roughly the same shape and volume. It is difficult even in a high resolution structure of a protein to distinguish valine from threonine. • Glutamic acid has one additional methylene group in its side chain than does aspartic acid. The side chain carboxyl of aspartic acid is referred to as the β carboxyl group, while that of glutamic acid is referred to as the γ carboxyl group. • The pKa of the γ carboxyl group for glutamic acid in a polypeptide is about 4.3, significantly higher than that of aspartic acid. • In some proteins, due to a vitamin K dependent carboxylase, some glutamic acids will be dicarboxylic acids, referred to as γ carboxyglutamic acid, that form tight binding sites for calcium ion. • Arginine, an essential amino acid, has a positively charged guanidino group. Arginine is well designed to bind the phosphate anion, and is often found in the active centers of proteins that bind phosphorylated substrates. • As a cation, arginine, as well as lysine, plays a role in maintaining the overall charge balance of a protein. • There are 6 codons in the genetic code for arginine, yet, although this large a number of codons is normally associated with a high frequency of the particular amino acid in proteins, arginine is one of the least frequent amino acids. • The discrepancy between the frequency of the amino acid in proteins and the number of codons is greater for arginine than for any other amino acid. Non-polar amino acids • They have equal number of amino and carboxyl groups and are neutral. • These amino acids are hydrophobic and have no charge on the 'R' group. The amino acids in this group are alanine, valine, leucine, isoleucine, phenyl alanine, glycine, tryptophan, methionine and proline. Non-polar amino acids Polar amino acids with no charge • These amino acids do not have any charge on the 'R' group. These amino acids participate in hydrogen bonding of protein structure. • The amino acids in this group are - serine, threonine, tyrosine, cysteine, glutamine and aspargine Polar amino acids with no charge Polar amino acids with positive charge • Polar amino acids with positive charge have more amino groups as compared to carboxyl groups making it basic. • The amino acids, which have positive charge on the 'R' group are placed in this category. They are lysine, arginine and histidine. • Polar amino acids with negative charge • Polar amino acids with negative charge have more carboxyl groups than amino groups making them acidic. • The amino acids, which have negative charge on the 'R' group are placed in this category. They are called as dicarboxylic mono-amino acids. They are aspartic acid and glutamic acid. Types of Amino acids 1. Aromatic group 2. Aliphatic group 3. Sulphur Containing amino acids 4. Hydrophylic amino acids • Aromatic amino acids are normally hydrophobic and includes phenylalanine, tyrosine and tryptophan. • Aliphatic amino acids are basically hydrophobic and an be located in core of protein. glycine ,valine, alanine, leucine, proline and isoleucine are aliphatic amino acids. • sulphur containing amino acids include sulphur atom and cysteine and methionine are the examples. • Hydrophilc amino acids are further categorized as acidic ,neutral and basic amino acids. Acidic amino acids are highly polar and are always negatively charged. Aspartate and glutamate are the examples. • Basic amino acids contains side chains that are positively charged . lysine,arginine and histidine are the examples. • Neutral amino acids are polar in nature and serine ,threonine, asparagine and glutamine are the examples. Introduction • Proteins are the machines that drive cells and, ultimately, organisms. Proteins are composed of individual units called amino acids. Amino acids all share a similar structure. The difference between them is the so-called "R" group. The "R" group is the cluster of atoms that give an amino acid its particular characteristics. • Proteins are not linear molecules as suggested when we write out a "string" of amino acid sequence, -Lys-Ala-Pro-Met-Gly- etc., for example. • Rather, this "string" folds into an intricate threedimensional structure that is unique to each protein. • It is this three-dimensional structure that allows proteins to function. • Thus in order to understand the details of protein function, one must understand protein structure. Formation of Peptide Bond in AA ↓ → H O ↓ 2 Peptide Bond ↓ Amino Acids • The amino acid residues of proteins are defined by an amino group and a carboxyl group connected to an alpha carbon to which is attached a hydrogen and a side chain group R. • The smallest amino acid, glycine, has a hydrogen atom in place of a side chain. • All other amino acids have distinctive R groups. Because the alpha carbon of the other amino acids have four different constituents, the alpha carbon atom is an asymmetric center and most naturally occurring amino acids are in the L form. (S)-Alanine (left) and (R)-alanine (right) in zwitterionic form at neutral pH (S)- Alanine (left) (R)-alanine (Right) • Any number of amino acids can be joined together to form peptides of any length. • Small peptides (containing less than a couple of dozen amino acids) are sometimes called oligopeptides. Longer peptides are many times called polypeptides. • Notice that peptides have a "polarity"; each peptide has only one free a-amino group (on the amino-terminal residue) and one free (nonsidechain) carboxyl group (on the carboxyterminal residue) • Amino acids fall into several naturally occurring groups including hydrophobic, hydrophilic, charged, basic, acidic, polar but uncharged, small polar, small hydrophobic, large hydrophobic, aromatic, beta-branched, sulfur containing etc. • Hydrophobic amino acids, sometimes called non-polar amino acids, reside primarily on the interior of the protein. • Hydrophilic amino acids, sometimes called polar amino acids, reside primarily on the exterior of the protein. • Many amino acids will fall into more than one group since each amino acid side chain has several properties. Forces determining protein structure • Several covalent and non-covalent determine protein structure. 1) van der Waals interactions 2) Hydrophobic force 3) Electrostatic forces 4) Dipole moments 5) Hydrogen bonds 6) Covalent bond forces 1. Van der Waals interactions • interactions between immediately adjacent atoms: These non-covalent forces result from the attraction of one atoms nucleus for the electrons of another atom in a non-covalent form (no sharing of orbitals). • These forces are much weaker than covalent interactions and the interaction distances are much longer than covalent bonds and much shorter than the other noncovalent interactions. • Van der Waals interactions are non-directional and very weak. However, significant energy of stabilization can be obtained in the central hydrophobic core of proteins by the additive effect of many such interactions. 2.Hydrophobic force • The hydrophobic force is really a negative non-covalent force. • The presence of hydrophobic side chains in aqueous solution induces the formation of structured water (clathrate cages of water molecule form, like miniature ice crystals about the hydrophobic side chains). • The hydrophobic force is one of the largest determinants of protein structure. Most secondary structural elements we will discuss have an amphipathic nature, one hydrophobic side and one hydrophilic surface of the protein. 3.Electrostatic forces • The attraction of oppositely charged side chains can form saltbridges that stabilize secondary and tertiary structures. • The electrostatic force is quite strong, falling off as the square of the distance between the charged atoms. • It also depends heavily on the dielectric constant of the medium in which the protein is dissolved. • It is strongest in a vacuum and 80 fold weaker in water and weaker still at elevated salt solutions. • Water and ions can shield electrostatic interactions reducing both their strength and distance over which they operate. 4.Dipole moments • Dipole moments are caused by pairs of charges separated by a larger distance than permitting a salt- or ion bridge. • The dipole moment gives rise to an electric field along the entire length of a structural element. • Dipole moments are often used by proteins to attract and position charged substrates and products. • The peptide chain naturally has a dipole moment because the Nterminus carries about 1/2 a positive charge and the C-terminus carries about 1/2 unit of negative charge. • The alpha helix is known to carry a partial negative charge at its Cterminus and a positive charge at its N-terminus. • In order to help neutralize this charge distribution, alpha helices often have acidic residues near their N-terminus and a basic residue near their C-terminus. 5. Hydrogen bonds • Hydrogen bonds occur when a pair of nucleophilic atoms such as oxygen and nitrogen share a hydrogen between them. • The hydrogen may be covalently attached to either nucleophilic atom (the H-bond donor) and shared with the other atom (the H-bond receptor). • H-bonds are directional and their strength deteriorates dramatically as the angle changes. • Hydrogen bonds do not, in general, contribute to the net stabilization energy of proteins because the same groups that hydrogen bond to each other in a native protein structure, can hydrogen bond to water in the denatured state. • However, hydrogen bonds are extremely important because of their directionality, they can control and limit the geometry of the interactions between side-chains. • This is shown most dramatically in patterns of hydrogen bonding between the carboxyl groups and the amino groups in the peptide backbone that give rise to alpha helix and beta strand conformations. 6. Covalent bond • The major properties of the covalent bonds hold proteins together are their bond distances and bond angles. • In particular, the bond angles between two adjacent bonds on either side of a single atom, or the dihedral angles between three contiguous bonds and two atoms control the geometry of the protein folding. Primary Structures- Protein • Primary structure refers to the "linear" sequence of amino acids. • Primary structure is sometimes called the "covalent structure" of proteins because, with the exception of disulfide bonds, all of the covalent bonding within proteins defines the primary structure. • Generally, peptides are small 10 or 20 residues; polypeptides might range up to 50 or 60 residues, • Small peptides (containing less than a couple of dozen amino acids) are sometimes called oligopeptides. Longer peptides are many times called polypeptides. • each peptide has only one free a-amino group (on the aminoterminal residue) and one free (non-sidechain) carboxyl group (on the carboxy-terminal residue): Primary Structure of Protein Secondary Structures (SS) • Secondary structure is the initial folding pattern (periodic repeats) of the linear polypeptide. There are 2 main types of secondary structure: α- helix and β-sheet Secondary structures are stabilized by hydrogen bonds. The α-helix • The α-helix is right-handed or clock-wise (for L isoforms left-handed helix is not viable due to steric hindrance) Each turn has 3.6 aa residues and is 5.4 A° high. • The helix is stabilized by H-bonds between –N-H and –C=O groups of every 4th amino acid. • α-helices can wind around each other to form ‘coiled coils’ that are extremely stable and found in fibrous structural proteins such as keratin, myosin (muscle fibers) etc The α-helix Structure Five different kinds of constraints affect the stability of an a helix: • The electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups. • The bulkiness of adjacent R groups. • The interactions between amino acid side chains spaced three (or four) residues apart. • The occurrence of Pro and Gly residues. • The interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the a helix. β-Pleated Sheet • Extended stretches of 5 or more aa are called β- strands. β-strands organized next to each other make β-sheets. • If adjacent strands are oriented in the same direction (Nend to C-end), it is a parallel β-sheet, if adjacent strands run opposite to each other, it is an antiparallel β-sheet. • There can also be mixed β-sheets. The H-bonding pattern varies depending on type of sheet. • β-sheets are usually twisted rather than flat. • Fatty acid binding proteins are made almost entirely of β-sheets β-Pleated Sheet Tertiary Structure • 3D folding or ‘bundling up’ of the protein is the tertiary structure of the proteins. • Non-polar residues are buried inside, polar residues are exposed outwards to aqueous environment. • Many proteins are organized into multiple ‘domains’ which are compact globular units that are connected by a flexible segment of the polypeptide. • Each domain contributes a specific function to the protein. Different proteins may share similar domain structures, eg: kinase-, cysteine-rich-, globin-domains. Tertiary Structures The protein then can compact and twist on itself to form a mass called it’s Tertiary Structure 5 kinds of bonds stabilize tertiary structure • • • • • H-bonds, van der waals interactions, hydrophobic interactions, ionic interactions and disulphide linkages In disulphide linkages, the SH groups of two neighboring cysteines form a –S-S- bond called as a disulphide linkage. It is a covalent bond, but readily cleaved by reducing agents that supply the protons to form the SH groups again. Quaternary Structure. • Quaternary structure is a larger assembly of several protein molecules or polypeptide chains, usually called subunits in this context. • The quaternary structure is stabilized by the same non-covalent interactions and disulfide bonds as the tertiary structure. • Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. • Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. • The subunits are frequently related to one another by symmetry operations, such as a 2-fold axis in a dimer. • Multimers made up of identical subunits are referred to with a prefix of different subunits are referred to "hetero-" (e.g. a heterotetramer, such as the two alpha and two beta chains of hemoglobin). • Many proteins do not have the quaternary structure and function as monomers. Protein Structures Overview Several Proteins then can combine and form a protein’s Quaternary Structure Functions of Protein What does Protein Do? • Protein has a large number of important functions in the human body—and in fact, the human body is about 45% protein. It’s an essential macromolecule without which our bodies would be unable to repair, regulate, or protect themselves. • Proteins are, in effect, the main actioners in cells and in an entire organism. Without proteins the most basic functions of life could not be carried out. Respiration, for example, requires muscle contractions, and muscle contractions require proteins. Protein has a range of essential functions in the body, including the following: • Required for building and repair of body tissues (including muscle) • Enzymes, hormones, and many immune molecules are proteins • Essential body processes such as water balancing, nutrient transport, and muscle contractions require protein to function. • Protein is a source of energy. • Protein helps keep skin, hair, and nails healthy. • Protein, like most other essential nutrients, is absolutely crucial for overall good health. Proteins as Enzymes • The function of proteins as enzymes is perhaps their bestknown function. Enzymes are catalysts—they initiate a reaction between themselves and another protein, working on the molecule to change it in some way. • The enzyme, however, is itself unchanged at the end of the reaction. • Enzymes are responsible for catalyzing reactions in processes such as metabolism, DNA replication, and digestion. • In fact, enzymes are known to be involved in some 4,000 bodily reactions. Proteins in Cellular Signaling and Molecular Transport • Cells signal one another for an enormous variety of reasons, the most basic of which is simply to coordinate cellular activities. Signaling is how cells communicate with one another, allowing such essential processes as the contraction of the heart muscle to take place. • Proteins are important in these processes due to their ability to bind other molecules—a protein produced by one cell may bind to a molecule produced by another, thus providing a chemical signal which allows the cells to provide information about their state. Proteins are also involved in molecular transport. • A prime example of this is the protein called hemoglobin, which binds iron molecules and transports them in the blood from the lungs to organs and tissues throughout the body. • Structural proteins are those which confer strength and rigidity to biological components which would otherwise be unable to support themselves. • Structural proteins tend to have very specific shapes— long, thin fibers or other shapes which, when allowed to form polymers, provide strength and support. • Structural proteins are essential components of collagen, cartilage, nails and hair, feathers, hooves, and other such components. • Structural proteins are also essential components of muscles, and are necessary to generate the force which allows muscles to contract and move. • Though enzymes exhibit great degrees of specificity, cofactors may serve many apoenzymes. • For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a great number of dehydrogenase reactions in which it acts as a hydrogen acceptor. • Among them are the alcohol dehydrogenase, malate dehydrogenase and lactate dehydrogenase reactions. Enzymes can be classified by the kind of chemical reaction catalyzed • Addition or removal of water – Hydrolases - these include esterases, carbohydrases, nucleases, deaminases, amidases, and proteases – Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase • Transfer of electrons – Oxidases – Dehydrogenases • Transfer of a radical – Transglycosidases - of monosaccharides – Transphosphorylases and phosphomutases - of a phosphate group – Transaminases - of amino group – Transmethylases - of a methyl group – Transacetylases - of an acetyl group • Splitting or forming a C-C bond – Desmolases • Changing geometry or structure of a molecule – Isomerases • Joining two molecules through hydrolysis of pyrophosphate bond in ATP or other triphosphate – Ligases Enzyme Kinetics: Basic Enzyme Reactions • Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. • The basic enzymatic reaction can be represented as follows • where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of the reaction. Enzyme Kinetics: The Enzyme Substrate Complex • A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist Savante Arrhenius in 1888. • He proposed that the substrate and enzyme formed some intermediate substance which is known as the enzyme substrate complex. The reaction can be represented as: • The existence of an intermediate enzymesubstrate complex has been demonstrated in the laboratory, for example, using catalase and a hydrogen peroxide derivative. • This experimental evidence indicates that the enzyme first unites in some way with the substrate and then returns to its original form after the reaction is concluded. The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and Km (which are set by the particular equation). The symbols used in the Michaelis-Menten equation refer to the reaction rate [v1], maximum reaction rate (Vmax), substrate concentration [S] and the Michaelis-Menten constant (Km). Factors Affecting Enzyme Activity • Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to understand the basic enzymatic mechanism and to select a method for enzyme analysis. • The conditions selected to measure the activity of an enzyme would not be the same as those selected to measure the concentration of its substrate. • Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators. Enzyme Concentration • In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. • Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as: • The amount of enzyme present in a reaction is measured by the activity it catalyzes. • The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. • An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. • It is satisfied only when the reaction is zero order. Substrate Concentration • If the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (∆A / ∆T). • It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. • Michaelis constants have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme. • A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. • A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity. • The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes. Effects of Inhibitors on Enzyme Activity • Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, noncompetitive and substrate inhibition. • Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory. Temperature Effects Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. • The reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40°C, most enzyme determinations are carried out somewhat below that temperature. • Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes lose their activity when frozen. Effects of pH • Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. • Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. Enzyme pH Optimum Lipase (pancreas) 8.0 Lipase (stomach) 4.0 - 5.0 Lipase (castor oil) 4.7 Pepsin 1.5 - 1.6 Trypsin 7.8 - 8.7 Urease 7.0 Invertase 4.5 Maltase 6.1 - 6.8 Amylase (pancreas) 6.7 - 7.0 Amylase (malt) 4.6 - 5.2 Catalase 7.0 • In addition to temperature and pH there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible. Enzyme Function • In simple terms, an enzyme functions by binding to one or more of the reactants in a reaction. • The reactants that bind to the enzyme are known as the substrates of the enzyme. • The exact location on the enzyme where substrate binding takes place is called the active site of the enzyme. • The shape of the active site just fits the shape of the substrate, somewhat like a lock fits a key. • In this way only the correct substrate binds to the enzyme. • Once the substrate or substrates are bound to the enzyme, the enzyme can promote the desired reaction in some particular way. • What that way is depends on the nature of the reaction and the nature of the enzyme. An enzyme may hold two substrate molecules in precisely the orientation needed for the reaction to occur. • Or binding to the enzyme may weaken a bond in a substrate molecule that must be broken in the course of the reaction, thus increasing the rate at which the reaction can occur. • An enzyme may also couple two different reactions. • Coupling an exothermic reaction with an endothermic one allows the enzyme to use the energy released by the exothermic reaction to drive the endothermic reaction. • In fact, a large variety of enzymes couple many different endothermic reactions to the exothermic reaction in which ATP is converted by hydrolysis to ADP. • In this way, ATP serves as the molecular fuel that powers most of the energy-requiring processes of living things.