Chapter 1 Structure and Function of Enzymes 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 13 14 Figure 1.1: Shows crystal structure of some enzymes by using the methods of NMR and X Ray Diffraction. The enzyme structures are taken from Protein Data Bank (PDB). 1.2 History Of Enzymes It was considered, until the nineteenth century, that processes such as the souring of milk and the fermentation of sugar to alcohol could only take place through the action of a living organism. Berzelius (1835) published that the active agent breaking down the starch was better than sulfuric acid and given the name diastase of malt (now known as amylase). A little later, a substance which digested dietary protein was extracted from gastric juice and called pepsin. All these with other active substances were given the general name ferments. Liebig recognized that these ferments could be non-living materials obtained from living cells, but Pasteur and others still maintained that ferments must contain living material. Finally, the term ferment was gradually replaced by the name enzyme. This was first proposed by Kühne in 1878, and comes from the Greek, enzume (éνζυuη) meaning “in yeast”. Büchners, in 1897, succeeded in extracting from yeast cells the enzymes catalyzing alcoholic fermentation which can function independently of cell structure. In 1926, Sumner crystallized urease from Jack-bean extracts, and in the next few years many other enzymes were purified and crystallized such as ribonuclease, trypsin, chymotrypsin, and lysozyme. Once pure enzymes were 15 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 yl group of one amino acid is joined to the α-amino group of another amino acid by peptide bond. Any number of amino acids can be joined in this fashion (Figure 1.3) to form a polypeptide chain. Short chains up to a length of about 22 amino acids are called peptides. Proteins consist of one or more polypep- tide chains. Since amino acids in proteins can be arranged in an almost infinite number of sequence, there also possible infinite number of different proteins. Figure 1.3: The amino acid sequence of the peptide chain forming the primary structure of protein. The protein is composed of one polypeptide chain. A amino acids can be classified into three groups; polar, non-polar and charged. Polar and charged amino acids will most often be found on the sur- face of a protein, interacting with the surrounding water, while the non-polar (or hydrophobic) amino acids will bury themselves in the interior. The number and position of these types of amino acids in protein can greatly influence its function. Peptides and proteins are formed when 10 - 10,000 amino acids link together in a long polymer. This long chain is termed the primary sequence. The properties of the protein are determined, for the most part, by this primary sequence. The side group (R) of each amino acid gives its distinctive properties and helps to dictate the folding of the protein. The primary structure of a poly- peptide of protein determines its secondary, tertiary, and quaternary structures (Figure 1.4). 18 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 dard three-letter abbreviations for the amino acids. For example: gly- Ile-val…..-ala is the primary structure for a peptide composed of glycine, Isoleucine, valine,….. and alanine, in that order, from the N-terminal amino acid (glycine) to the C-terminal amino acid (alanine) (Figure 1.5). The example of this type is the structure of bovine insulin {PDB ID (313Z)} as is shown in Figure 1.6. Figure 1.5: Primary structure shows the hydrogen, disulfide bonds, and the N-terminal amino acid (serine) to the C-terminal amino acid (alanine). Figure 1.6: The structure of bovine insulin from Protein Data Bank {PDB ID (313Z)} as an example of primary structure. 1.4.2 Secondary Structure During and after synthesis, the primary sequence will associate in a fash- ion that leads to the most stable, “comfortable” structure for the protein. How a protein folds is largely dictated by the primary sequence of amino acids. 20 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 1.8: The two main secondary structures which are the α -helix and the β-pleated sheet. The enzyme structures from Protein Data Bank (PDB). 22 Figure 1.9: The β pleated sheet of polypeptide chains. Showing hydrogen-bond cross-links between adjacent chains. Parallel β sheets is inverse to Antiparallel β sheets, in which the amino-terminal to carboxyl-terminal orientation of adjacent chains. 23  The helix is stabilized by hydrogen bonding between amine and carbonyl groups of the same polypeptide chain. The β pleated sheet (Figure 1.10) is stabilized by hydrogen bonds are between two chains that run side by side. 24 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. 25 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 Figure 1.16: Glutamine synthetase composed of twelve identical subunits, each of which has an active site for the production of glutamine. The ligands (Mn2+ and ATP) are shown in a red color (Protein data Bank). Figure 1.17: Enolase plays a role in gluconeogenesis as well as glycolysis. Mg2+ or Mercury or Zinc ions are required for activity (red color). Two different forms of the monomer of enolase, α and β. It has ID: 12CA in the Protein data Bank (PDB). Figure 1.18: Glyceraldehydes 3-phosphate dehydrogenase is a tetrameric enzyme, respon- sible for the conversion of glyceraldehydes-3-phosphate to 1,3-bisphospho-glycerate in the presence of NAD and phosphate ion as aligands. It is showing the enzyme with ligands (red color) and with ID: 1A7K in the Protein data Bank (PDB). 30 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 Figure 1.19: The zinc atom in the red color of carboxypeptidase A serves as a metal ion cat- alyst to activate hydrolysis. The bond cleaved in the peptide bond of the phenylalanine. The white color is the substrate polypeptide in which the negative charge of the hydroxyl group of phenylalanine in the polypeptide is attracted by the metal ion in the active site of the enzyme carboxypeptidase A. 32 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 Figure 1.30: A typical pre-tRNA is shown. Production of tRNAs from pre-tRNAs (in the red and blue color) catalyzed by ribonuclease P. The RNA portion of the RNA-protein complex called ribonuclease P is by itself capable of catalyzing the hydrolysis of the specific phospho- diester bond indicated by the wedge. 46  CHAPTER 2 How Enzymes Work As Catalysts - Principles And Examples Page C ONTEN TS 47 2.1 Biological Importance 48 2.2 Active Site 48 2.3 Lock And Key Hypothesis 57 2.4 Induced Fit Hypothesis 59 2.5 Enzyme-Substrate (ES) Complex 60 2.6 Enzyme Specificity 61 2.6.1 Absolute Specificity 63 2.6.2 Relative Specificity 63 2.6.3 Stereo Specificity 64 2.6.4 Structural Specificity 65 2.7 Naming And Classification Of Enzyme 65 2.8 Location Within The Cell 69 2.9 How Enzymes Work 70 2.10 Factors Affecting Enzyme Activity 72 2.10.1 Effect Of Enzyme Concentration [E] 73 2.10.2 Effect Of Substrate Concentration [S] 73 2.10.3 Effect Of Temperature (T) 74 2.10.4 Effect Of pH 76 2.10.5 Contact Between Enzyme And Substrate 78 2.10.6 Products Of Reaction [P] 78 2.10.7 Oxidation 79 2.10.8 Effect Of Physical Factors 79 2.10.9 Concentration Of Cofactors 80 2.11 General Characteristics Of Enzymes 80 2.12 Allosteric Enzymes 81 2.12.1 General Characteristics Of Regulatory Enzymes 83 2.12.2 Mechanism Of Allosteric Enzymes 88 2.13 Regulation Of Enzyme Activity 91 2.13.1 Effect Of Substrate Concentration [S] 92 2.13.2 The Feedback Inhibition (Product Inhibition) 92 2.13.3 Noncovalent Modification 93 2.13.4 Covalent Modification 93 2.13.5 Induction And Repression Of Enzyme Synthesis 95 2.14 Isozymes 95 2.15 Autolysis 98 47  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 Figure 2.1: The two kinds of enzymes (glucokinase and ligase) are showing the binding of the substrate to the enzyme, and the size of both them. 49  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 Figure 2.3: A is shown the enzyme and the substrate before the binding between them. B shows the binding between the enzyme and the substrate with the binding site and the catalytic site. The active site of the enzyme has both the catalytic and the binding site. The enzyme is chymotrpsin that cut the polypeptide to tow smallor polypeptide. 52  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 Figure 2.7: Three-dimensional model of the polypeptide chain backbone of chymotrypsin as determined by x-ray analysis. Residues His 57,Asp 102, and Ser 195 function in the active site of chymotrypsin. Figure 2.8: Different active site pockets account for the different specificities of serine pro- teases, trypsin, chymotrypsin and elastase. 56  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  Here are some multiple-choice questions based on the document provided: 1. What is the primary function of enzymes in biochemical reactions? a) They consume substrates b) They act as catalysts without undergoing change c) They slow down chemical reactions d) They alter the chemical structure of substrates 2. Which enzyme was first crystallized in 1926? a) Pepsin b) Urease c) Chymotrypsin d) Ribonuclease 3. What is the term used for the non-protein part of an enzyme? a) Apoenzyme b) Holoenzyme c) Cofactor d) Substrate 4. Which of the following is NOT a fibrous protein? a) α-keratin b) Collagen c) Hemoglobin d) Elastin 5. What stabilizes the secondary structure of proteins like α-helix and β-pleated sheet? a) Hydrogen bonds b) Disulfide bridges c) Covalent bonds d) Ionic bonds 6. Which cofactor is essential for the activity of carboxypeptidase A? a) Zn2+ b) Mg2+ c) Ni2+ d) Se2+ 7. According to the lock and key hypothesis, how does a substrate interact with an enzyme? a) It induces a conformational change in the enzyme b) It binds to the active site because they have complementary shapes c) It forms a covalent bond with the enzyme d) It alters the tertiary structure of the enzyme 8. What is the term for an inactive form of an enzyme? a) Cofactor b) Holoenzyme c) Proenzyme d) Isozyme 9. Which vitamin is a precursor for the coenzyme thiamine pyrophosphate? a) Vitamin B2 b) Vitamin B5 c) Vitamin B1 d) Vitamin B6 10. Which enzyme requires Mg2+ as a cofactor? a) Urease b) Hexokinase c) Carboxypeptidase d) Glutathione peroxidase These questions focus on enzyme structure, function, and examples discussed in the document. Let me know if you'd like more!  c) It is possible to see ES complex using x-ray crystallography and see sub- strate at the active site (Figure 2.13). d) The spectrophotometric characteristics of enzymes and substrate change when ES complex is formed e.g. absorption spectrum of enzyme changes when it binds substrate. e) Existence of specificity for stereoisomers indicates an extremely precise interaction between E and S. Figure 2.13: The conformational change in glucokinase induced by glucose binding. (a) Before glucose binding. (b) After glucose binding. 2.6 Enzyme Specificity The most remarkable and significant property of enzymes is their high degree of specificity for their substrates. This property distinguishes them from the usual non-protein catalysts in non-living systems. This specificity is due to the fact that the enzyme attacks a compound at a definite of linkage. Enzyme specificity depends on the particular atomic structure and conforma- tion of both the substrate and the enzyme which it is explained by two hypothe- sis (Figure 2.14 and 2.15). Firstly, Emil Fisher in 1890 stated that substrate and enzyme behaved like key in lock. Secondary, Koshland in 1958 revealed that the substrate induce a change in conformation of the active site of the enzyme in order to accommodate it for the substrate. These two hypothesis show the great specificity of the enzymes to their substrate. However, the range of spec- ificity varies as follows: 61