‎⁨انزايم ميد أول⁩.pdf

CHAPTER 1 Structure And Function Of Enzymes 1.1 What Are Enzymes? Enzymes are, mostly proteins, a biological catalysts. They speed up or increase the rate of biochemical reactions taking place within living cells without themselves undergoing any overall change (i....

CHAPTER 1 Structure And Function Of Enzymes 1.1 What Are Enzymes? Enzymes are, mostly proteins, a biological catalysts. They speed up or increase the rate of biochemical reactions taking place within living cells without themselves undergoing any overall change (i.e. are not consumed or changed during the reaction they catalyze). The rates of the reactions they catalyze are generally increased by the order of at least 102-108 time. A very large number of chemical reactions are involved in an organism’s metabolic processes, catalyzed by many different enzymes. An example of a biochemical pathway is glycolysis, in which glucose is converted to pyruvate. Most of the chemical reactions which occur in biological systems are catalyzed by enzymes. Some relatively simple reactions such as hydration of carbon dioxide and the modification of small organic molecules are enzyme-catalyzed. Enzymes are involved in the reading of genetic infor- mation stored in DNA, the first step in the synthesis of proteins them- selves. In addition, the non-protein components of cells are synthesized by enzymes. Thus, enzymes are a central component of “cellular machin- ery” and they are very important classes of compounds in living systems. There are a huge number of enzymes that we cannot count it. It found in the cell of bacteria (Escherichia coli) there is about 2000, where in animal cell (Eukaryotic cell) there is about 10000 of enzymes. The fol- lowing examples of some enzyme structures are shown in Figure 1.1 by the methods of: v NMR or v X Ray Diffraction 12 available, their structure and properties could be determined. Today, enzymes still form a major subject for academic research. Because of their specificity of action they are used in hospitals (as an aid to diagnosis), in manufacture of cheeses, breads, alcoholic beverage, and for the tenderizing of meats. 1.3 Structure Of Enzymes Chemically every known enzyme is either a protein alone or a protein associated with either an organic molecule or a metal cation, or both. In a conjugated system of this sort, the protein part of the enzyme is called an apoenzyme, while the nonprotein part is called a cofactor. If the cofactor is an organic molecule, it is called a coenzyme or prosthetic group when the organ- ic molecule binds tightly to the apoenzyme. A complete active enzyme system containing one or more cofactors is called a holoenzyme (holo---, meaning whole). Apoenzyme + Cofactor Holoenzyme (globular protein) metal ion (inorganic) or coenzyme (organic) The following scheme represents the structural classification of enzymes as simple and conjugated protein. 16 1.4 Structure Of Proteins Since most enzymes are proteins, a knowledge of protein structure is clearly a prerequisite to any understanding of enzymes. Proteins are macromolecules (i.e. large molecules) with molecular weights of at least several thousand Daltons (The molecular mass of enzyme is expressed in units of Daltons which are defined as 1/12th the mass of a 12C atom). They are found in abundance in living organisms, making up more than half the dry weight of cells. Two distinct types are known: fibrous and globular proteins. Fibrous proteins are insoluble in water and are physically tough, which enables them to play a structural role. Examples include α-keratin (a compo- nent of hair, nails and feathers) and collagen (the main fibrous element of skin, bone and tendon) (Figure 1.2). In contrast, globular proteins are generally soluble in water, the poly- peptide chain or chains are tightly folded into spherical or globular shapes, and may be crystallized from solution. They have a functional role in living organ- isms, most enzymes being globular proteins (see Figure 1.12). Figure 1.2: The structure of α-keratin (a component of hair, nails and feathers) and collagen (the main fibrous element of skin, bone and tendon). The protein structures are taken from Protein Data Bank (PDB). Unlike polysaccharides and lipids, which may store by cells solely as a store of fuel, each protein in a cell has some precise purpose which is related to its shape and structure. Nevertheless, should the need arise, proteins may be bro- ken down, either to provide energy or to supply raw materials for the synthesis of other macromolecules. Every protein molecule can be considered as a polymer of amino acids. The most are 22 common amino acids found in proteins. In protein, the α-carbox- 17 Figure 1.4: Levels of structure in proteins. The primary structure consists of a sequence of amino acids linked together by covalent peptide bonds, and includes any disulfide bonds. The resulting polypeptide can be coiled into an α helix, one form of secondary structure. The helix is a part of the tertiary structure of the folded polypeptide, which is itself one of the subunits that make up the quaternary structure of the multimeric protein, in this case glutamine synthetase. All structures are taken from Protein Data Bank (PDB). There are four levels of protein structures found in polypeptides and proteins. Primary Structure Secondary Structure Tertiary Structure Quaternary Structure (Figure 1.4). 1.4.1 Primary Structure The primary structure of polypeptides and proteins is the sequence of amino acids in the polypeptide chain with reference to the locations of any disulfide bonds. Disulfide bonds are formed between the side chains of cysteine by oxi- dation of two thiol groups (SH) to form a disulfide bond (S-S), also sometimes called a disulfide bridge. The primary structure may be thought of as a complete description of all of the covalent bonding in a polypeptide chain or protein. The most common way to denote a primary structure is to write the amino acid sequence using the stan- 19 This structure is stabilized by hydrogen bonds, hydrophobic interactions, ionic interactions, and disulfide bonds between nearby amino acids in the protein (Figure 1.7). The two main secondary structures are the α -helix and the β-pleated sheet (Figure 1.8). There are other periodic conformations (as anti-parallel β-pleated sheet (Figure 1.9), but the α-helix and β-pleated sheet (Figure 1.10) are the most stable. A single polypeptide or protein may contain multiple secondary structures. Figure 1.7: The bonds (ionic bond, hydrogen bond, and disulfide bond) that stabilize the structure of proteins. 21  Figure 1.10: The α-helix and β-pleated sheet. These are the two most important regular sec- ondary structures of proteins. In the α-helix the hydrogen bonds are within a single chain, whereas in the β pleated sheet hydrogen bonds are between chains that run side by side. 1.4.3 Tertiary Structure The tertiary structure of a polypeptide or protein is the three-dimensional arrangement of the atoms within a single polypeptide chain. During and after synthesis, a protein folds into α-helices and β- sheets. These areas of second- ary structure are connected by bridging sequences that will cause the protein to fold in specific ways. At the completion of this process, the protein takes on its final shape. The mature stable structure of a single peptide sequence is termed its tertiary structure (Figure 1.11). Also, for a protein composed of a single polypeptide molecule, tertiary structure is the highest level of structure that is attained and is largely maintained by disulfide bonds. Bonds : Covalent bonds ( disulfide bonds ) , Hydrogen bonds , 25 Salt bridge , Hydrophobic interactions , Metal ion coordination Figure 1.11: Three-dimensional folding of the protein myoglobin. Each amino acid is indi- cated by a circle corresponding to its α-carbon atom. Side chains are not shown, in order to emphasize the way in which the polypeptide backbone is wrapped into helices and folded. This protein folds about a heme group (dark blue color), a planar heterocyclic structure that chelates iron and serves as the oxygen binding site. The enzyme structures from Protein Data Bank (PDB) site (PDB ID = 3HC9). So, the mature stable structure of a single peptide sequence is the tertia- ry structure which is globular and compact. For example, in the structure of myoglobin (is a protein with 70% α-helix), the helix is bent and fold to form a compact or globular molecule. Within that folding is formed a pocket to hold a prosthetic group, the hem (Figure 1.11). 1.4.4 Quaternary Structure Quaternary structure is used to describe proteins composed of multiple subunits (multiple polypeptide molecules, each called a ‘monomer’). Most proteins with a molecular weight greater than 50,000 Daltons consist of two or more noncovalently linked monomers. The arrangement of the monomers in the three-dimensional protein is the quaternary structure. The most com- mon example used to illustrate quaternary structure is the hemoglobin protein (Figure 1.12). Hemoglobin’s quaternary structure is the package of its mono- meric subunits. Hemoglobin is composed of four monomers. There are two α-chains, each with 141 amino acids, and two β-chains, each with 146 amino acids. Because there are two different subunits, hemoglobin exhibits hetero- quaternary structure. If all of the monomers in a protein are identical, there is homoquaternary structure. Hydrophobic interaction is the main stabilizing force for subunits in quater- nary structure. When a single monomer folds into a three-dimensional shape 26 to expose its polar side chains to an aqueous environment and to shield its non-polar side chains, there are still some hydrophobic sections on the exposed surface. Two or more monomers will assemble so that their exposed hydropho- bic sections are in contact. Literally, quaternary is subsequent to tertiary. In the context of biology, qua- ternary structures, such as enzymes, are assemblies of tertiary structural units, such as proteins. The assembly of biomolecules into quaternary structures pro- vides multiple or novel functional roles. These assemblies may contain as few as two units, as in an enzyme complex, or hundreds, as in a virus. Often qua- ternary structure is organized symmetrically. This allows the formation of large complexes with only a few different tertiary units. In multiple unit (multimeric) complexes the single units (monomers) form contacts between each other. In some cases, the monomer must change its conformation in order to make these contacts. Finally, large assemblies play not only functional but structural roles on the cellular level. Figure 1.12: The structure of hemoglobin revealed by x-ray diffraction analysis, from Protein Data Bank (PDB) site. The α subunits are shown in pink; β subunits are shown in yellow; and the heme groups are shown in blue. 1.5 Enzyme Are Monomeric Or Oligomeric Proteins 1.5.1 Monomeric Enzyme This kind of enzyme consist of only a single polypeptide chain, therefore they cannot be dissociated into smaller units. Monomeric enzymes are very few that possible to find in all catalyze hydrolytic reactions (digestion processes). Their molecular weights in the range of 13,000-35,000 Daltons and consist between 100 and 300 amino-acid residues. The example of monomeric en- 27 zymes are proteases or proteolytic enzymes (as serine proteases, e.g. trypsin, chymotrypsin, and elastase) (Figure 1.13) which catalyze the hydrolysis of peptide bonds in other proteins. So, to prevent them doing generalized damage to all cellular proteins, they are often produced in an inactive form known as a proenzyme or zymogen. Example of the proenzyme is chymotrypsinogen which is in the present of trypsin can be convert to the active form called chy- motrypsin, so they be activated as required (Figure 1.14). Carboxypeptidase A (Figure 1.15) is a rare one of monomeric enzymes because it is associated with a metal ion, since most react without the help of any cofactor. Figure 1.13: Some example of monomeric enzymes as trypsin, chymotrypsin, and elastase. The enzyme structures revealed by x-ray diffraction analysis, from Protein Data Bank (PDB) site. Figure 1.14: Chymotrypsinogen is a proenzyme, in the present of trypsin is converted to the active form called chymotrypsin. The enzyme structures revealed by x-ray diffraction analy- sis, from Protein Data Bank (PDB) site. 28 Figure 1.15: Carboxypeptidase A is a rare one of monomeric enzymes associated with a metal ion. The enzyme structure revealed by x-ray diffraction analysis, from Protein Data Bank (PDB) site. 1.5.2 Oligomeric Enzymes Oligomeric enzyme are those which contain two or more polypeptide chains, attached to each other by noncovalent bonds. These polypeptide chains are termed subunits or monomers, which they may be identical to or different from each other. When the subunits are identical, they are called protomers. Oligomeric enzymes consist two, four or any even subunits (Figure 1.16), they are called dimeric, and tetrameric enzymes, respectively. Oligomeric enzymes have a molecular weight of more than 35,000 Daltons. Most majority of known enzymes are oligomeric such as those involved in glycolysis pathway, citric acid cycle, β- oxidation of lipids and other processes in living cells, such as enolase (Figure 1.17) and glyceraldehyde 3-phosphate dehydrogenase (Figure 1.18). Oligomeric enzymes are usually two or four subunits gain properties in association that they do not have in isolation. Such enzymes are not synthesized as inactive zymogens, but their activities can be regulated by feed-back inhibition. 29 1.6 Cofactors Sometime biochemical reaction cannot be catalyzed by the function groups in the amino acid side-chain alone of the enzyme with the substrate. In these cases the protein enzyme act in a cooperation with other smaller molecules or ions which called cofactors. 1.6.1 Metal Ions Although many enzymes require metal ion cofactors, one can subdivide the list between what are called metalloenzymes and metal-activated enzymes on the basis of the strength of metal binding. Metalloenzymes generally engage stoichiometric amounts of the metal cofactor quit tightly. Metal-activated enzymes retain their metal in an equilib- rium with binding groups on the surface. Metal ions are essential cofactors in many enzyme system as of carboxy- peptidase A (Figure 1.19). Sometimes these cations are referred to as enzyme activators. Some metal ions (such as calcium, magnesium, and iron) are needed in relatively large quantities, while others only are needed for trace amount; in many cases, such as cobalt or chromium, an overdose can be lethal. We do not entirely understand how metal ions activate enzymes. In other cases, the metal ion is only loosely associated with the protein or polypeptide. In either type of enzyme system, the metal ion may be instrumental in attract- ing the substrate to the enzyme or in holding the substrate to the enzyme. The cation of the metal may also participate in catalysis of a biochemical reaction by interaction with a negatively charged portion of the substrate (Figure 1-19). 31 Table 1.1: Some enzymes with its metals. Cofactor Enzyme (Metal) Zn2+ Carbonic anhydrase Zn2+ Carboxypeptidase Mg2+ EcoRV Mg2+ Hexokinase Ni2+ Urease Mo+ Nitrate reductase K+ Propionyl CoA carboxylase Se2+ Glutathione peroxidase Mn2+ Superoxide dismutase 1.6.2 Coenzymes Coenzymes are non-protein organic molecules consist of vitamin B or other as lipoic acid. It works as part of an enzyme system in catalyzing bio- chemical reactions. In some instances, the metal ion such as the cobalt ion in vitamin B12 (as a coenzyme), is an integral part of a coenzyme structure. The B vitamins are necessary in order to: Support and increase the rate of metabolism. Maintain healthy skin and muscle tone. Enhance immune and nervous system function. Promote cell growth and division, including that of the red blood cells that help prevent anemia. Reduce the risk of certain cancer. 33  The B vitamins are eight water-soluble vitamins that play important roles in cell metabolism. They are: 1) Vitamin B1 (thiamine) 2) Vitamin B2 (riboflavin) 3) Vitamin B3 (niacin) 4) Vitamin B5 (pantothenic acid) 5) Vitamin B6 (pyridoxine) 6) Vitamin B7 (biotin) 7) Vitamin B9 (folic acid) 8) Vitamin B12 (cobalamins). 1. Thiamine (B1) Is a water-soluble vitamin of the B complex (Figure 1.20). All living organisms use thiamine in their biochemistry, but it is synthesized in bacteria, fungi and plants. Animals must obtain it from their diet. Thiamine pyrophosphate coenzyme derived from Thiamine. Often plays a role in the removal of carboxyl (-COOH) groups from organic acids. This coenzyme, for example, helps to remove a carboxyl group from pyruvic acid (Krebs cycle) in the present of pyruvate dehydrogenase enzyme. Figure 1.20: Thiamine (B1) water-soluble vitamin of the B complex. 34 2. Riboflavin (B2) The two flavin, coenzymes, flavin mononucleotide (FMN) and flavin ade- nine dinucleotide (FAD), are derived from riboflavin (vitamin B2). Flavin base with the ribose molecule form riboflavin which in the present of phosphate group can assemble the coenzyme flavin mononucleotide (FMN). Adenine mononucleotide (AMN) with flavin mononucleotide (FMN) form flavin ade- nine dinucleotide (FAD) the other coenzyme of riboflavin (vitamin B2) (Figure 1.21). 35 Figure 1.21: Flavin base with the ribose molecule form riboflavin which in the present of phosphate group can form the coenzyme riboflavin mononucleotide (FMN). Adenine mono- nucleotide (AMN) with riboflavin mononucleotide (FMN) form flavin adenine dinucleotide (FAD). FAD is a prosthetic group in the enzyme complex succinate dehydrogenase (Figure 1.22) that oxidizes succinate to fumarate in the eighth step of the citric acid cycle. 36 Figure 1.22: FAD is a prosthetic group in succinate dehydrogenase that oxidizes succinate to fumarate. 3. Niacin (B3) The two flavin, coenzymes, flavin mononucleotide (FMN) and flavin ade- nine dinucleotide (FAD), are derived from riboflavin (vitamin B2). Flavin base with the ribose molecule form riboflavin which in the present of phosphate group can assemble the coenzyme flavin mononucleotide (FMN). Adenine mononucleotide (AMN) with flavin mononucleotide (FMN) form flavin ade- nine dinucleotide (FAD) the other coenzyme of riboflavin (vitamin B2) (Figure 1.21). 37 Figure 1.23: Niacin can synthesize from the essential amino acid tryptophan. 4. Pantothenic Acid (B5) Pantothenic acid, is called vitamin B5. It is a water-soluble vitamin required to sustain life (essential nutrient). Pantothenic acid is needed to form coenzyme A (CoA), and it is critical in the metabolism and synthesis of carbohydrates, proteins, and fats. Pantothenic acid (vitamin B5) is used in the synthesis of coenzyme A (CoA) as it is shown in Figure 1.24. 38 Figure 1.24: Pantothenic acid (vitamin B5) is used in the synthesis of coenzyme A (CoA). 5. Pyridoxine (B6) Pyridoxine is one of the compounds that can be called vitamin B6 (Figure 1.25), along with pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) as a derivatives of vitamin B6. Pyridoxine assists in the balancing of sodium and potassium as well as promoting red blood cell production. 39 Figure 1.25: The structure of pyridoxine (vitamin B6) and its derivatives pyridoxal phosphate pyridoxamine phosphate (PMP). 6. Biotin (B7) Biotin like lipoic acid in that it performs a highly specialized biochemical function. Biotin acts as a cofactor for enzymes that add a carboxyl group to substrates (Figure 1.26). 40 Figure 1.26: This show the structure of biotin and the reaction that biotin acts as a cofactor hold carboxyl group to provide it to the substrate of the enzyme. 7. Folic Acid (B9) Folic acid (or folate) is one of the most important vitamins, both because of its key biological functions and its medicinal importance. The pterin ring system of folate is reduced by the enzyme dihydrofolate reductase (DHFR) to give tetrahydrofolate as the active cofactor (Figure 1.27). 41 Figure 1.27: This show the structure of folic acid as inactive form, but when the nitrogen atoms of pterin rings hydrated it will become tetrahydrofolate as the active cofactor. 8. Cobalamin (B12) Vitamin B12, called cobalamin, is a water soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood. 42 Figure 1.28: The structure of Cobalamin (B12) as a coenzyme. 9. Lipoic Acid Lipoic acid (LA), also known as α-lipoic acid (ALA) and thioctic acid is an organosulfur compound derived from octanoic acid (Figure 1.29). ALA is made in animals normally, and is essential for aerobic metabolism. It is marketed as an antioxidant, and is available as a pharmaceutical drug. Lipoic acid is cofac- tor for at least five enzymes systems (as dehydrogenase enzymes). Two of these are in the citric acid cycle. 43 Figure 1.29: The structure of lipoic acid (ALA) and its function with the enzyme as cofac- tor to remove the hydrogen ions and it converted to dihydrolipoic acid. Coenzymes is classified on the bases of their functional characteristics: A. Coenzymes for transfer of H. B. Coenzymes for transfer group other than H. A. Coenzymes for transfer of H such as: 1) NAD, NADP 2) Lipoic acid 3) FMN, FAD 4) Coenzyme Q B. Coenzymes for transfer group other than H such as: 1. ATP and its relative 2. Sugar phosphates 3. Thiamine pyrophosphate(B1). 4. Co A(B5) 5. Pyridoxal phosphate (PLP) (B6) 6. Biotin (B7) 7. Folic Acid (B9). 8. Cobamide coenzyme (B12) 9. Lipoic acid 44 Table 1.2: Some coenzymes with its enzymes. Cofactor (Coenzyme) Enzyme Thiamine pyrophosphate Pyruvate dehydrogenase Flavin adenine nucleotide Monoamine oxidase Nicotinamide adenine denucleotide Lactate dehydrogenase Pyridoxal phosphate Glycogen phosphorylase Coenzyme A (CoA) Acetyl CoA carboxylase Biotin Pyruvate carboxylase 5′- Deoxyadenosyl cobalamin Methylmalonyl mutase Tetrahydrofolate Thymidylate synthase 1.7 Non-Protein Biocatalysts-Ribozymes It was assumed that all biochemical catalysis (enzymes) was carried out by proteins. In fact, some RNA molecules, called ‘ribozymes’ have been shown to have catalytic activity. Therefore, not all enzymes are proteins. Example, Ribonuclease P, an enzyme that cleaves the precursors of tRNAs (pro-tRNA) to yield the functional tRNAs (Figure 1.30). It had been known for some time that active ribonuclease P (RNA-protein) contained both a protein portion and an RNA (as cofactor), but it was generally assumed that the active site resided on the protein portion. Altman S. A. and co-workers in 1983 revealed an astonishing fact: where- as the protein component alone was wholly inactive, the RNA by itself, with magnesium plus the small basic molecule spermine, was capable of catalyzing the specific cleavage of pre-tRNAs. RNA acted like a true enzyme, being unchanged in the process and obeying Michaelis-Menten kinetics. 45  CHAPTER 2 How Enzyme Works As Catalyst-Principles And Examples 2.1 Biological Importance Without catalysis by enzymes, most of the reactions which take place in living cells would occur very slowly, and life would not be possible. Indeed, as Willstatter has said “Life is a system of co-operating enzyme reaction”. It is well known that digestion, the various reactions involved in the intermediary metabolism of carbohydrates, fats and proteins, cellular respiration, calcifica- tion of bones, blood clotting and detoxification, are examples of physiological activities of great importance, all dependent on enzyme action. 2.2 Active Site The active site of an enzyme is the region that binds the substrate and contributes the amino acid residues that directly participate in the making and breaking of chemical bonds. The amino acid residues (12 amino acids) are called the catalytic groups (of enzyme). Enzymes differ widely in structure, function and mode of catalysis so active sites vary, but possible to make some generalizations. 1. Enzymes are usually very large in comparison with substrate (Figure 2.1), so only small portion of amino acid residues (12 amino acids) are near or in direct contact with substrate. Evidence for this includes the observation that large portions of certain enzymes may be removed without loss of catalytic activity. 2. Active site is a 3-dimensional entity (3-D). Not a point or a plane, usually an intricate pocket or cleft structurally designed to accept the structure of the substrate in 3-D terms. The residues which constitute the active site are often not close to each other in the primary sequence, but the tertiary fold brings them close together in three-dimensional space. Active sites often involve res- idues on connecting loops between helices and sheets, rather than those which are part of these regular secondary structures. In many instances, active sites occur at the junction between two domains making tertiary contacts (Figure 2.1). 48  3. Substrate is bound by relatively weak forces usually electrostatic and hydrogen bonds, and van der Waals interactions (between atoms or nonpo- lar molecules) all are important when there is steric complementary between the substrate and the active site of the enzyme). The free energy of interaction between E and S ranges -12 to -36 kJ/mole comparing this with the strength of a covalent bond which reach up to -450 kJ/mole (Figure 2.2). Figure 2.2: A is the structure of the enzyme and the substrate before binding to each other, B is after the binding which is showing the weak bonds between the enzyme and the substrare. 50  4. Two identified sites within enzymes are: a. The catalytic site, which is a region within the enzyme involved with catalysis, and b. The substrate binding site which is the specific area on the enzyme to which reactants called substrates bind to (Figure 2.3). The catalytic site and substrate binding site are often close or overlapping and collectively they are called the active site (Figure 2.3). If the catalytic site is not near the substrate binding site it can move into position once the enzyme is bound to a substrate. Enzymes do two important things at the active site: They recognize very specific substrates, and They perform specific chemical reactions on them at fast speeds. 5. Most are clefts or crevices designed to exclude water from the active site and are surrounded with non-polar amino acid residues (not have N,S,O molecule in their residues), which give the active site a non-polar environment. This appears essential for both binding and catalysis. Essential to exclude water (unless water involved in the reaction) because water disrupts bond breaking and making processes (Figure 2.4). 51  Active site is a buried pocket with lower energy of activation to reach tran- sition state by: Stabilizes transition Expels water Specificity (Reactive groups) Coenzyme helps 6. Specificity of the active site: Many enzymes are highly specific in terms of the substrate (S) which they bind, and this is determined by the arrangement of the atoms in the cleft or the pocket of the active site. Such enzymes include serine proteases, and zinc proteases. Proteases are enzymes which produce by pancreas. The active site differs from one protease to another. They are produced in the form of their respective pro-enzymes. They hydrolize the middle polypeptide chain as serine proteases called endopeptidases. The other type, is zinc proteases which hydrolize the end carboxyl group of the polypeptide chain of specific kinds of amino acids as shown below. Figure 2.4: The active site, avoids the influence of water, allowing to stabilize ionic bonds. 53  The serine proteases, so called because of the presence in the active site of an essential serine residue. This important class of enzymes includes trypsin, chymotrypsin, and elastase. Each of the serine proteases preferen- tially cuts a polypeptide chain just to the carboxyl side of specific kinds of amino acids. For example, trypsin cuts preferentially to the carboxylate side of basic amino acid residues like lysine or arginine, whereas chymotrypsin acts most strongly if there is a hydrophobic residue (like phenylalanine or leucine) in this position (Figure 2.5). Most of the serine proteases have simi- lar three-dimensional structures and are obviously evolutionarily related. The active site regions of all of the serine proteases (see Figure 2.6) have a number of common factors. In particular, there are always an aspartic acid, a histidine, and a serine residue clustered about the active site depression. These are Asp 102, His 57, and Ser 195 in the structure of chymotrypsin shown in Figure 2.7. A fourth feature of the active site differs from one serine protease to another. This is a “pocket,” always located close to the active site serine (Figure 2.7). In trypsin the pocket is deep and narrow, with a negatively charged carboxylate at its bottom- just the thing to catch and hold a long, positively charged side chain like lysine or arginine (of the substrate). In chymotrypsin it is wider and lined with hydrophobic residues, to accommodate a hydrophobic side chain of the substrate (tyrosin, tryptophan, methonine, phenalalanine and leucine). Where in elastase, the pocket is shallow and non-polar. Elastase just cut the polypep- tide chain at the amino acids of alanine, serine and glycine (Figure 2.8). It is the nature of this pocket that gives each of the serine proteases its specificity. 54 Figure 2.5: Serine proteases, and zinc proteases produced in the form of their respective pro-enzymes. Each of the serine and zinc proteases preferentially cuts a polypeptide chain just to the carboxyl side of specific kinds of amino acids. Figure 2.6: The differnces between serine protease pockets. 55  Zinc Proteases, such as carboxypeptidase A and B (Figure 2.9), hydrolyze the end of the polypeptide chain at a certain amino acid (see Figure 2.5). Figure 2.9: The structure of zinc proteases carboxypeptidase A and B from protein data bank. Binding the substrate to the active site of the enzyme can be explained by two hypotheses: - Lock and key hypothesis (Emil Fisher hypothesis,1890) and - Induced fit hypothesis (Koshland hypothesis, 1958). 2.3 Lock And Key Hypothesis This hypothesis was proposed by Fischer in 1890. In this hypothesis the substrate had a matching shape to fit into the active site of the enzyme which reveals the specificity of the enzyme to that substrate. Substrate and enzyme behaved like key in lock i.e. substrate (key) had a matching shape (as the lock and the key below) to fit into the active site of enzyme (lock) (Figure 2.10). 57  In this hypothesis there are no conformational changes upon the enzyme when substrate binds to it. The protein enzyme is viewed as a rigid structure (lock). It describes the active site as a rigid that is complementary to the substrate (the “key“ ) also it is a rigid. Figure 2.10: The lock and key hypothesis. In this early model, the shape relationship between the substrate and the active site in the enzyme is identical to the relationship between the key and lock. 58 2.4 Induced Fit Hypothesis Observation from different kind of experiment have revealed differences in structure between free and substrate-bound enzymes. Thus binding of substrate to an enzyme may induce conformational change in the three dimensional structure of the active site of the enzyme. This binding process which allows the enzyme to accommodate for the substrate, is called induced fit hypothesis, set up by Koshland in 1958. Koshland suggested that the structure of a substrate may be complemen- tary to that of the active site in the enzyme-substrate complex, but not in the free enzyme: a conformational change takes place in the enzyme during the binding of substrate which results in the required matching of structures. Such a mechanism could help to achieve a high degree of specificity for the enzyme (Figure 2.11). Figure 2.11: The induced fit hypothesis, the substrate binds to the enzyme inducing confor- mational change in the three dimensional structure of the active site of the enzyme. The induced-fit hypothesis essentially requires the active site to be floppy and the substrate to be rigid, allowing the enzyme to wrap itself around the substrate, in this way bringing together the corresponding catalytic sites (enzyme) and reacting groups (substrate). In some respects, the relationship between a substrate and an active site is similar to that between a hand and a wooden glove: in each interaction the structure of one compo- nent (substrate or hand) remains fixed and the shape of the second component (active site or glove) changes to become complementary to that of the first. 59  The lock and key hypotheses explains many features of enzyme specificity, but takes no account of the known flexibility of proteins. X-ray diffrac- tion analysis and data from several forms of spectroscopy, including nuclear magnetic resonance (nmr), have revealed differences in structure between free and substrate-bound enzymes. Thus, the binding of a substrate to an enzyme may bring about a conformational change, i.e. a change in three-dimensional structure (in tertiary structure) but not in primary structure. 2.5 Enzyme-substrate (ES) Complex Enzymes convert substrates to products by breaking and making chemical bonds. This process occurs at the active site and is producing ES complex. Evidence for existence of an ES complex is: a) Suggested by observation of maximum velocity in enzyme catalyzed reaction. At constant of the concentration of enzyme [E], increasing the concentration of substrate [S] will cause increase in reaction rate until a maximum velocity reached then further increases in [S] produce no fur- ther increase in activity. Maximum velocity is due to ES complex. At sufficiently high [S] all active sites are filled and working flat out so no more activity possible as showen in Figure 2.12. b) It is possible to isolate ES enzyme-substrate complex. Figure 2.12: Substrate concentration increases reaction rate until reach maximum velocity. 60