Enzymes Definition and History Enzymes are protein catalysts for biochemical reactions in living cells. The term enzyme, derived from Greek means “in yeast’ pecause the yeast cells were the first to reveal enzyme activity in living organisms. It was first introduced by W. Kuhne in 1878. Berzelius ; who was one of the first to define and recognize the nature of catalysts, proposed in 1827 that “ferments” were catalysts produced by living cells. In 1926 J.B. Sumner isolated the enzymes urease as a crystalline protein for the first time. He was awarded Nobel Prize in 1946 for this work. Since that time, several hundred enzymes have been obtained in highly purified form and more than 150 of these in the crystalline state. The study of enzymes, nowadays. is dealt with under a separate branch of Biochemistry, known as the enzymology. . Enzymic reactions were used by man long before written history. The discovery of fermentation to produce wines was attributed by the Greeks to Bacchus. The making of cheese, the leavening of bread, and the manufacture to vinegar are enzymic processes that stem from the ancient times. These practical activities have occupied an important place in the history of enzymology. The brewaries and their research laboratories have provided much information concerning the processes by which living cells utilize sugar because the fundamental reactions of fermentation in a yeast culture are much the same as in the tissues of mammals. Indeed, the name enzyme, coined by Kuhne means “in yeast”, but the word is now used to denote a biological catalyst, regardless of origin. A substantial part of the study of living cells is today devoted to enzymes inasmuch as all physiological functions ¢.g. muscular contraction, nerve conduction, excretion etc., as well as life itself are intimately linked to the activity of enzymes. 147os ee PHYSIOLOGY AND BIOCHEMsppy NOMENCLATURE, variety of ways B s are named in a varie’) ' aay Enzymes are 0 iy actice is to add the ae a hename a omary pre ayers vl DA we o., the substance on whic a by urease, arginine fa rene he enzyme that attacks ure caer by Urleea’ For at the e ase, tyrosine by tyrosinase, U a ine yros acted upon by arg o ete. starch) by amylase e a Bi anne may also be name ie chemical reactions such as ee 5 s ecarboxylases etc. . a ea . en used to denote en ee 3) s 1X ee : | ; : é sate such as proteolytic for protein sp) vere ae = th Stipes) = “splitting, and sucrolytic for enzy: e 0S) — S Sawa enzymes retain their old traditional nomenclature, efi s i i i sin ete. and we find such names as ea ee canted fps aa i and mi 5) Some proteinases of plan , ; eae from the species of origin, e.g., pepain from papaya, ficin from ficus, subtilisin from Bacillus substilis etc. CLASSIFICATION , According to International Union of Biochemistry (TUB), the present system of classification of enzymes is based on their reaction specificity. Six classes have been recognized : . . 1. Oxidoreductases (Catalyze oxidation-reduction-reactions) This class includés enzymes which are concerned with biological oxidation and reduction, and therefore, with respiration and fermentation processes, The important subclasses are : (i) Dehydrogenases, that catalyze reactions in which the electron transfer is accompanied by the transfer of hydrogen atoms ee, alcohol dehydrogenase and glutamic dehydrogenase of animal iver, (ii) Oxidases, in which molecular o: en is one of the reactants, eg, cytochrome oxidase, oe \ i Gerdases, which use H,0, as the oxidant 1V) Oxygenases, which int gen i a double bond inthe substrate, mute Taloeular Sraeeninplaceof 2. Hydrolases categories - (catalyze hydro d by groups that catalyze similar oxidases, proteinases, lytic Teactions). These fall into 3 (Proteolytic enzymes), These are enzymes : of Proteins and Peptides. It is customary exopeptidacesy Proteinase (endopeptidases) and the 0 distin, Peptidases (,— nays M9 (a) Peptidases (Exopeptidases) act on peptide bonds adjacent pafree amino or carboxyl group. Among the principal types are the followin . : () Carboxypeptidases require a free a-carboxyl group in the substrate and split the peptide bond adjacent to this group, liberating a free amino acid. (i) Aminopeptidases act on the peptide bond adjacent to the free amino group of the simple peptides. (iii) Dipeptidases specifically act on certain dipeptides. An example is glycylglycine dipeptidase which requires CO** or Mn? for its action. (b) Proteinases (Endopeptidases) act on the interior peptide bonds of proteins; they can, however, also split peptide bonds in suitable simple peptides and their derivatives. Examples are pepsin, rennin, trypsin, chymotrypsin and plasmin from animals. Cathepsins are intracellular proteinases found in most animal tissues. The richest sources are liver, kidney and spleen. Pant Proreinases. Papain is obtained from the unripe fruit of the papaya. Similar enzymes are bromelin, found in pineapples and ficin in the milky sap of the fig tree. (B) ESTERASES. These enzymes catalyze hydrolysis of ester linkages. Classification depends on both the type of acid and the type of alcohol constituting the ester. (a) Simple esterases such as esterase which catalyzes reversibly the scission and synthesis of esters of lower alcohols and fatty acids. (b) The true lipases hydrolyze fats into long-chain fatty acids and glycerol. Examples are pancreatic lipase and plant lipases. (c) Phosphatases hydrolyze esters of phosphoric acid. (d) Pyrophosphatases hydrolyze pyrophosphate linkages. ” (e) Nucleases cause hydrolysis. of nucleic acids, liberating aligo— or mononucleoitides. These enzymes are phosphodiesterases. (C) CARBOHYDRASES. These enzymes hydrolyze the glycosidic linkages of simple glycosides, oligosaccharides, and polysaccharides. (a) Glycosidases hydrolyze simple glycosides and oligosaccharides. An example is yeast invertase, which hydrolyzes sucrose to glucose and fructose. (b) Polysaccharidases act on complex polysaccharides. f- amylases hydrolyze starch and glycogen to maltose and to residual polysaccharides. The polysaccharides of wheat, barley, soyabeans and other plants are of this type. ‘ a-amylases can hydrolyze glycogen and starch and the residual polysaccharides of starch (amylodextrins) to give glucose, maltose,PHYSIOLOGY AND BIOCHEM spy, and product that no longer Rive 4 colour ay iodine. ao amylane Af this type have been obtained in crystalline form from pancreas of this type 0 algo found in blood and urine. a Phese are + wn var aneteraach (Catalyze group transfer reactions), Thege talyze the transfer of a group from one substance ty enzymes © Ikyl, glycosyl, acyl, amino, aldehyde, another, The groups may he a an OT TRAN SFERE ‘ASES, e.g., choline acyltransferase, i i YCOS) TRANSFERASES, ¢.g., phosphorylase. o Co TRANSFERASES or TRANSAMINASES, eg, glytamicaspartic aminotransferase. ferofaph (iv) KINASES. Enzymes catalyzing transfer of a pl osphate group, specifically from the ATP to particular substrates, .g., hexokinase. 4. Lyses. (Catalyze removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds). t C-C=X-Y+C=C These enzymes act on C—C, C—O, C—N, C—S and C—halide bonds. The important ones are as under : () HYDRASES (Carbon-oxygen lyases). These enzymes catalyze addition to or removal of water from their specific substrates. For examples, fumarase catalyzes the interconversion of malic and fumaric acids; HOOC—CH,—CHOH—COOH = HOOC—CH = CH—COOH + H,0 Malic acid (ii) ALDEHYDE-LYASES, e.g., Aldolase. ii) DECARBOXYLASES. These enzymes remove CO, from carboxylic acids. Keto acid decarboxylases are Important in the liberation of CO,, An example is the catalyzed decarboxylation of pyruvic acid to acetaldehyde and Co,. Fumaric acid Decarboxylase CH,COCOOH————» cH. CHO + CO, catal » Isomerases (Catalyze isomeration reactions). These enzymes of: ‘aldose and ket yeeular rearrangements, e.g., the interconversion ‘ose sugars. For example. i se catalyzes the following interconversion 2 phosphohexose isomera! rose 6-Phosphate = Frunctose 6-phosphate &g., uridine ie of enzymes are also included the epimerases, Phosphate galactose 4-epimerase, which catalyzespNzYMES the following reaction : Uridine diphosphate galactose == Uridine diphosphate glucose. Uridine diphosphate glucose plays an important role in several aspects of carbohydrate metabolism. Other examples of isomerases are alanine recemase and retinene isomerase. 6. Ligases or Synthetases. (Ligare = “to bind”. Catalyze synthesis by condensation of two groups requiring ATP or a similar triphosphate). These enzymes catalyze reactions forming C—O, C—S, C—N and C—C bonds. Some sub-classes are : (i) Enzymes, catalyzing formation of C—S bonds, e.g., succinic thiokinase. Gi) Enzymes, catalyzing formation of C—N bonds, e.g., glutamine synthetase. (iii) Enzymes, catalyzing formation of ‘C—C bonds, e.g., acetyl COA carboxylase. CHEMICAL NATURE OF ENZYMES Enzymes are the largest and most specialized class of protein molecules. They are of two types : simple and holoenzymes. 1. Simple Enzymes. Some enzymes are simple proteins, i.e., on hydrolysis, they yield amino acids only. Digestive enzymes such as pepsin, trypsin and chymotrypsin are of this nature. ~ 2. Conjugated Enzymes. Many enzymes possess chemical groups that are non-amino acid in nature. These conjugated proteins are called holoenzymes. A holoenzyme may be dissociated into a protein component, termed the apoenzyme and a non-protein moeity, the cofactor. Cofactors may be divided into 3 groups which include : (a) prosthetic groups, (b) coenzymes, and (c) metal activators. Conjugated protein enzyme == Apoenzyme (Protein part) or + Holoenzyme Non-protein cofactor (Prosthetic group/Coenzyme/ Metal activator) (a) Prosthetic Group. A prosthetic group is firmly bound tothe enzyme protein. Thus, for example, the parphyrin part of Enzyme the enzyme. Haemoprotein Ne peroxidase and FAD in succinic on RQ _ at) dehydrogenase are prosthetic groups — that are firmly Substrate Cofactor associated with their protein Fig. 11.1. Combination of an apoenzyme and a co-factor with the substrate._—_"_—— PHYSIOLOGY AND BlocHEy EN MISTRy 152 counter Cooney A coenzyme is a small, heat-stable, dialyzable ( ens) f ye te a es ; se molecule whieh readily dissociates off an enzyme protein ore AD, pyrophosphate and Tetrahydrofolic acig Thus NAD, NADP, Thiamine ¢ nzymes. ator. An enzyme may contain only aminoacids here are enzymes t hat are copper protein, s; c acid oxidase is one of them. In this enzyme Cu is tightly Seas rated readily from the protein apoenzyme, metal ions for their activation. The jonsof Ca, Co, Cu, Mg, Mn, Mo, Na, Kand Zn are known to participate enzymic reactions, In some cases these activators: function in combination with the protein, in others the metal ion forms a compounds with a substrate. ENDO-AND EXOENZYMES Enzymes which act only within’ the cells are called endoenzymes, hence they are metabolic enzymes ¢.g., cytochrome oxidase. On the contrary, certain enzymes are liberated by living cells and catalyze vital reactions in the cell’s environment. These are called exoenzymes, e.g., the digestive enzymes : amylase, lipase, proteases etc. : GENERAL PROPERTIES OF ENZYMES (ENZYME ACTIVITY) (1) Remain unaltered in the End. One of the primary characteristics of enzymes is their emergence from a reaction or set of reactions in an unaltered state. This enzyme may now enter into another reaction with another substrate of the same type. (2) Required in small Quantities. Enzymes are active in small quantities. Since the enzyme is reused, only a small quantity of an individual enzyme is required by biological systems. It has been estimated that a single enzyme can act upon 500,000 substrate molecules per minute. This value is referred to as the turn-over number. ee ve olen head The third general property of enzymes is sericea ae The protein structure provides for weak nn subatr , side Broups of the amino acids within it, The and substrate by We amplex involves the association of the enzyme Enzymes exhibit all > ae rather than covalent bondings. weight, colloidal hha caine of proteins viz.. High molecular most living mombrs , Slow diffusion, inability to pass through ane ines, and movement in response to an electric are examples of coe’ ic) Metal Activ anda metal. For example, tl ascorbi bound and is not separater Many other enzymes requireNf MES 153 f gyAccelerate the Rate of Reaction. An enzyme cha ofthe reaction, not the direction of the equilibrit es only proceed at a faster rate in the presence of an a fan piibrium between the substrase and the product will canals stant with or without the intervention of the enzyme. (5) Reversibility of Enzyme Action. Certain enzymes exhibi _cersibility of their action. This property is very halpfil ms jretabolism. For example, succinic dehydrogenase eatalieea vorsibly the dehydrogenation of succinic acid (in the presence ofa table hydrogen acceptor A) of hydrogenation of fumaric acid poral «i ef eon cooH HCCOOH CH, . + AS =HOOCCH + AH, CH, | cooH Succinic Acid + Acceptor —= Fumaric acid + Reduced acceptor (6) Enzyme Specificity. The speclficity of enzymes is one of their most fundamental and important properties. By contrast to non-protein catalysts (H', OH or metal ions), enzymes catalyze only one specific reaction or act upon only one kind of substrate. The specificity may be of any kind, e-g., reaction specificity, substrate specificity, group specificity or optical specificity. (i) Reaction specificity. Most enzymes can catalyze the same type of reaction (phosphate transfer, oxidation-reduction etc.) with several structurally related substrates. For example, some esterases can act upon the esters different fatty acids with a variety of alcohols. Nevertheless, the esterases are specific in their esterase actions, they do not catalyze other hydrolytic reactions, nor do they function as oxidises, decarboxylases etc. Specificity is evident in the type of reaction that is catalyzed. (ii) Substrate Specificity. Avery important feature of enzyme activity is that it is substrate specific, i.e., a particular enzyme will act only on a certain substrate. For example, urease is a specific enzyme, it acts on urea only but it would not exert any influence on the catabolism of protein or carbohydrate. (iii) Group Specificity. A particular enzyme acts only on Particular chemical groupings, ¢.g., glycosidase on glycosides, alcohol dehydrogenase on alcohols, pepsin and trypsin on peptide bonds and esterases on ester linkages. Chymotrypsin preferentially hydrolyzes Peptide bonds in which the carboxyl group is contr ibuted by the——_——s—assss—h i PHYSIOLOGY AND Bloenp eM Igy no acids phenylalanine, tyrosine, or ty; TRY d aminopeptida es split of amino. aeldae 8 one aromatic am! no terminal end of polypepti lypeptid an le chaing Carboxypeptidase atime from the car respectively tiv) Op! of epimerases show absolute optic molecule. Thus ma O choxyl or am ty (Stereospecificity). With th vert optical isomers, omayme ge btion city for at least a portion ofa a ohentlly yzes the hydrolysis of a- but cate ot fi. tical Speeifict e which intercom ral specific Itase catal Enzyme Fig. 11.2. Enzyme Specificity. Reactants B and C fit i zy partially i the enzyme, but reactant A does get eX glycosides, while enzymes of the ] oxidative pathways catalyze the picaaentre ti pn ee phosphosugars. With a few exception, such as the D. re mot Le itn ary opm ne enzymes is the ener ee example of stereospecificity ae Tha neacess can be n of pyruvic acid to lactic acid by reducti S accomplished by two different enzymes, cach producing but one of the optical isomers : Oh ' woken i Om COOH Cn, Pyruvie acid L-Lactic acid COOH Hoon or, D-Lactic acidSNZYMES a 155 PROENZYMES Some enzymes are produced in a catalytically inactive or proenzyme form which must undergo limited proteolysis, to become catalytically active. This phenomenon is best illustrated by certain digestive enzymes, as well as by enzymes of blood coagulati id blood clot dissolution. agulation andof Conversion of the proenzyme to the active enzyme is catalyzed either by proteolytic enzymes or by hydrogen ions : " Pepsinogen—1-o* Pepsin, Pepsin rae Trypsin ‘Tripsinogen vee oe enterokinase ay Chymotrypsinogen. psi, Chymotrypsin Procarboxypeptidase PSN, carboxypeptidase Since activation of proenzyme is catalyzed by the active form of the enzyme itself, the activation of pepsinogen and of trypsinogen proceeds with ever-increasing velocity and is said to be autocatalytic. Conversion of fibrinogen to fibrin involves limited proteolysis catalyzed by thrombin. Under normal physiologic conditions, thrombin exists as the inactive precursor, prothrombin, and its acti- vation requires a complex sequence of reactions. Significance of Proenzymes. Synthesis of inactive enzyme precursors provides a mechanism for rapidly increasing the availability of an enzyme in response to particular physiologic demand. It would be serious, for example, if the enzymes of blood coagulation had first to be provided by the somewhat slower process of protein synthesis. MECHANISM OF ENZYME ACTION t 8 i ion, but itself remains The enzyme promotes a given reaction, i unchanged at the end ofthe reaction. In 1913, Michaelis and Menten proposed that an intermediate enzyme-substrate complex Sian during enzymic activity. The following scheme may be written to illustrate concept : Enzyme (E) + Substrate (S) =Enzyme-Substrate Complex (Es) Enzymes + Products (P): i ides The enzyme itself remains almost passive. It merely provi— PHYSIOLOGY AND BIOCHEM spay 13 template on which certain molecules could react or templi “platform” ee ena ‘ i jyother, Such an enzyme-platform brings reacting molecules each 0 s ve collisions at that te ene net much faster (han chance collisions at that temperature. ey reactions are accelerated. The result is that the huce the Energy of Activation : of enzymes is to lower the activation-energy equirements, thereby promoting appreciable reaction rates at lower = peratures than would be possible otherwise. Energy of activation js that minimal amount of energy which is required ofa molecule to take part in a reaction. For example, decomposition of hydrogen peroxide without a catalyst has an energy activation of some 18,000 ca/mole. When the enzyme catalase is added, it is less than 2,000. Enzymes red The effec Transition state Activated A-B <— Uncatalyzed Energy of activation Free energy —». Reactants Progress of reaction —» Fig. 11.3. Enzymes accelerate catalysis by reducing the Energy of Activation. Enzymes accelerate reactions by reducing the free energy of activation. This may be interpreted in terms of the transition state theory illustrate in Fig. 11.3. According to this concept, reactants A and B, in order to react to form the product C, must pass through a transition state (A—B) in which the activated complex is formed and certain amount of activation energy (E,) is required. When the proper enzyme is present, less energy is required for activation (E,’). In other words, the combination of substrate with enzyme creates a new-reaction pathway that has a transition state of lower energy than in the absence of enzyme, The Catalytic Site The larger size of, i FI Protein- . rates led biochemists in the begi fer mymbes compared to thelr subst of the century to postulate thatnz MES 1a? some restricted region of the enzyme was concerned with the at eataly sis. This region was termed as the active site, Nowadays, wer to the catalytic sito, since it is now known that other sites arealso ‘active (e.g., allosteric sites), Although enzymes differ widely instracture, specificity, and mode of catalysis, a numbor of gonorali- zations concerning their catalytic sites can bo stated as follows : 1. The catalytic sites take up a relatively small portion of the total volume of an enzyme. It is interesting to note that most of the amino acid residues in an enzyme ave not in contact with the sub- strate. 2. The catalytic site is a three-dimensional entity : The catalytic site of an enzyme is not a point, a line, or even a plane. It consists of contact amino acid residues, which are fairly close to each other in the native structure of the enzyme because of the three-dimensional folding of the molecule, but may be actually far apart in the primary sequence. For example, in lysozymes the important groups in the catalytic site are contributed by residues numbered 35,52,62,68 and 101 in the linear sequence of 129 amino acids. 3. Catalytic sites are clefts or crevices. In all enzymes, substrate molecules are bound to a cleft or crevice from which water is largely excluded. 4. Substrates are bound to enzymes by relatively weak forces. The binding of the substrate to the catalytic site involve forces of a non-covalent nature (ionic and hydrogen bonds, Van der Waals forces), which are of very short range. process The interaction of Substrates and Enzymes The mechanism of enzyme action has been explained by two theories : (1) Lock and Key theory of Emil Fischer and (2) Induced fit theory of Koshland. (1) Lock and Key Theory (Rigid Model of the Catalytic Site) The original model of a catalytic site, proposed by Emil Fisher in 1890, visualized interaction between substrate and enzyme in terms of a “lock and key”. Just as only particularly shaped keys fit into particularly shaped locks, similarly, only certain types of molecules will establish a close fit with a given type of enzyme protein, This concept was developed to explain the great specificity of en- zymes. According to this concept, a structurally well defined catalytic site will accept only those substrate molecules which have a matching shape and will repell others that differ structurally. In other words, the catalytic site of the enzymes by itself is complementary in shape to that of the substrate.15s, PHYSIOLOGY AND BIOCHEMISTRY \A Substrate ~ ES Complex Enzyme Fig. 11.4. Lock-and-key model of the interaction of substrate and enzyme. (2) Induced Fit Theory (Flexible Model of the Catalytic Site). An essential feature of the “induced fit” model of Koshland is the flexibility of the region of catalytic site. In the Fischer model, the catalytic site is presumed to be pre-shaped to fit the substrate. In the induced fit model, the substrate induces a conformational change in the enzyme. According to this postulate, the catalytic sites of some enzymes are not rigid. In thesé enzymes, the shape of the catalytic site is modified by the binding of substrate. The catalytic site has a shape complementary to that of the substrate only after the substrate is bound. This process of dynamic recognition is called Substrate + ES Complex Enzyme Fig. 11.5. Induced-fit model of the interaction of substrate and enzyme.ny fit. It brings amino acid residy in the correct spatial orientati < or both. At the same time, othe ly a s i i ate purried in the interior of the en: eco , es or other groups on the on for substrate binding, T amino acid residues may zyme molecule, ind onnyme ont) — a Ww Y SH) ————+ FACTORS AFFECTING ENZYME ACTIVITY (1) Temperature. Between a Tange of 0°C and 45°C, the activity of the enzymes in increased when the temperature is raised, as in most chemical reactions. It has been. found that a rise of 10°C hastens the reaction 2 or 3 times. The increase is due to two factors (i) the influence of heat on the chemical reaction itself and (ii) the increased enzyme activity. 40 35 30 25 Relative activity 20 15 ete Optimal Comp! \ temperature inactivation ~~ 70 80 10 20,~—«30si«isC(iS OO Temperature in °C 1 tivity. Fig. 11.7. Effect of temperature on the rate of enzyme a¢PHYSIOLOGY AN) °HEMIS1 160 PHYSIOLOGY AND BIOCHEMISTRY The rate of enzyme catalyzed reactions do not, however, increase indefinitely as the temperature is increased. At some point, usually inthe range between 45° to 60°C, thermal denaturation will destroy the catalytic function of the enzyme protein. As the enzyme is inactivated, the reaction which it catalyzes, slows down and ultimately stops. There is thus, an optimum temperature for each enzyme. Most enzymes operate at their best in a temperature range of about 25° to 40°C (human body temperature is 37°C) however, certain enzymes are very resistant to high temperature. For example, crystalline trypsin is denatured reversibly even at 100°C and so is Taka Diastase. (2) Hydrogen Ion Concentration (pH). One of the most interesting environmental influences on enzymatic activity is the effect of pH. Each enzyme is catalytically active within a limited range of pH. Though each particular type of enzyme has its own specific optimum, most enzymes operate effectively in a pH range of about 5.0 to 9.0. For example, the optimal range for the activity of malt amylase is 5.2 ; for salivary amylase, between 6.7 and 6.8; for trypsin, between 8 and 11. However, a few enzymes, e.g., pepsin are active at pH values between 1.5 and 2.5. In all these cases activity declines gradually on either side of the optimum. In addition to the purely, ionic effects, extremes of pH values can cause considerable denaturation and hence inactivation of the enzyme protein. 75: 50 Relative rate-percent 25: Optimal pH. 9 12 pH Fig. 11.8. Influence of Acidity on Enzyme action. (3) Enzyme Concentration. The rate of a chemical reactionENZYMES 161 influcnced by a particular enzyme, in most cases, increases propotio- nally with increasing concentration of the enzyme, provided that the substrate is present abundantly and is therefore not rate limiting ~y 400 : 300 1 4 Relative concentration of cae (rityeost sacchexase) Fig. 11.9. Effect of Enzyme Concentration on the rate of 2 reaction when the substrate is available in excess. (4) Substrate Concentration. At a constant concentration of enzyme, the reaction rate increases with i increasing substrate concentration until a maximal velocity is reached. At a sufficiently high substrate concentration, the catalytic sites are filled and so the reaction rate reaches a maximum. Therefore, further increase in concentration of substrate has no effect on the rate of the reaction. In contrast, uncatalyzed reactions do not show this saturation effect. Rate of hydrolysis (1 moles/litre/sec) a 5 0.1 02 os Concentration of substrate (ATP, millimotesslitre) Fig. 11.10. Effect of Substrate Concentration on the rate of reaction when 3 constant amount of enzyme is present.162 PHYSIOLOGY AND BlocHEMsTRy (5) Cofactors. Some enzymes require small NON-prote; components called cofactors for their activity. For example, the cytochromes are conjugated proteins having tightly bound prosthetje groups. The cytochromes | ave a metalloporphyrin complex and are involved in electron-transfer reactions (6) Chemical Substances. It has been found that the activity of many hormones and certain drugs results from inhibition or exhancement of enzyme action. ENZYME KINETICS The kinetic characteristics of enzymes can best be illustrated by a graph (Fig. 11.11). Without having anything to react with, the enzyme will not show any activity. Therefore, assuming a ready supply of enzyme, the apparent activity will increase with the availability of the substrate. However, as the substrate supply in- creases, a point will be reached when all available enzyme molecules will be fully employed; this is the point of maximum velocity of reaction for a given enzyme concentration with a given substrate. Obviously, this point will vary according to the enzyme and the substrate, and is a measure of the detailed mechanism of enzyme action on the particular substrate. We can also note the halfway Reaction velocity (E +S 2 where k,, k, and k, are rate constants for the reactions. The rate (V) of formation of product is calculated by the Michaelis-Menten equation : (S} V= Vina (S]+ Ky where V,,,, is the rate when the enzyme is fully saturated with substrate, and K,,, the Michaelis Menten Constant, is the substrate concentration at which the reaction rate is half maximal. The maxi- mal rate, V _, ,,, is equal to the product of k, and the total concentration of enzyme. The kinetic constant k,, called the turnover number, is the number of substrate molecules converted into product per unit time by a single catalytic site when the enzyme is fully saturated with substrate. Turnover number for most enzymes are between 1 and 10¢ per second. ENZYME INHIBITION Enzymes can be inhibited by specific small molecules or ions. The inhibition of enzymes serves as a major control mechanism in biological systems. Certain enzyme inhibitors such as cyanide, carbon monoxide, oxalic acid etc. are poisonous for living organisms. Their toxicity is due to their ability to inhibit enzymic reactions. On the other hand, an inhibitor blocking a disease producing reaction represents a medicinal drug. It is thought that the action of sulphonamides in killing bacteria, or preventing their reproduction, may be due to a similar interference with enzyme system in the bacterial cell. Furthermore, enzyme inhibition can provide insight into the mechanism of enzyme action. Some of the strongest evidence for the localization of catalytic sites comes from studies with specific164 PHYSIOLOGY AND BIOCHEMISTRY inhibitors of enzymes. By specific inhibitors are meant compoundy that can combine with an enzyme in such a manner as to prevent the normal enzyme-substrate combination and the catalytic reaction, Enzyme inhibition may be reversible or Irreversible. 1. REVERSIBLE INHIBITION. This type of enzyme inhibition is characterized by a rapid equilibrium of the inhibitor and enzyme, There are two major types of reversible inhibition: (a) Competitive and (b) Non-competitive. (a) Competitive or Surface Analog Inhibition. Competitive inhibition involves a compound which resembles the substrate and binds to the catalytic site of the enzyme. The substrate is then prevented from binding to the same active site. The formation of an enzyme-inhibitor complex may be represented as E+] = #H where E is the enzyme, I the inhibitor, and El the enzymeinhibitor complex. In competitive Inhibition, the formation of EI is reversible and their is continuing competition between the substrate and the inhibitor for the same locus on the enzyme. This situation is, therefore, designated as “competitive Inhibition”. The actual rate of the catalyzed reaction is then strictly dependent on the relative con- centrations of substrate and inhibitor. The inhibition can be reversed by increasing the substrate concentration so that the substrate molecules outnumber those of the inhibitor. Subtrate Competitive inhibitor “_ 4 J Noncompetitive y; vy inhibitor CY) ey Fig. 11.12. Distinction between a competitive Inhibitor and a Non-competitive Inhibitor. A classic example of competitive inhibition is the action of H n-4 coon HOOC—C—H oe H—C—COOH H dehydrogenase HOO! H Sucecinic acid ic acidENZYMES 165 malonic acid on succinic dehydrogenase. An enzyme that dehydrogenates succinic acid. Many compounds that are structurally similar to succinic acid but which are not dehydrogenated can combine with the enzyme and inhibit catalysis of the normal reaction by blocking the “active site” of the enzyme. The inhibition of succinic dehydrogenase by malonic acid, whose molecular structure is very similar to that of succinic acid, serves as an example of competitive inhibition : (ooH COOH CH, CH, CH, cooH COOH Succinic acid Malonic acid (Substrate) (nhibitor) Enzyme = Succinic dehydrogenase. In this case, both the substrate (succinic acid) and the inhibitor (malonic acid) will compete for the active (catalytic) site, and the enzyme activity will be reduced. Compounds like oxalic acid, glutaric acid and phenyl propoionic acid also inhibit succinic dehydrogenase. This inhibition can be reversed by increasing the substrate concentration, so that the substrate molecules outnumber those of inhibitor. One more example of competitive inhibition is shown below: (Hi, COOH CH@).COOH HO—C_cOOH HO—C—COOH CH,.COOH CH,.COOH Citric acid Fluorocitric acid (Substrate) (Inhibitor) Enzyme = Aconitase hydratase A physiologically important example of competitive inhibition is found in the formation of 2,3-Diphosphoglycerate from 1,3 Di- phosphoglycerate. Diphosphoglycerate mutase, the enzyme cata- lyzing this isomerization, is competitively inhibited by low levels of 2, 3-Diphosphoglycerate. In such a case the product of an enzymatic reaction has a structural resemblance to the substrate and, therefore, it serves as a competitive inhibitor.PHYSIOLOGY AND BIOCHEMIstp 7 Y 166 0 i topo? ° n-_OH == = H-C_OPO; Hy¢—-0PO? H,C—OPO; 1, 3-Diphosphoglycerate * 9, 3.Diphosphoglycerate (b) Non-Competitive Inhibition. In this type, the inhibitor and the substrate are not structurally related; they can bind simultaneously to an enzyme molecule. This means that their binding sites do not overlap. Since these inhibitors bear no structural resemblance to the substrate, an increase in the substrate concentration generally is ineffective in relieving this inhibition. A non-competitive inhibitor acts by decreasing the turnover number of an enzyme rather than by diminishing the proportion of enzyme molecules that have a bound substrate. In contrast to the competitive type, it may be assumed that the formation of EI in non-competitive inhibition occurs at a locus on the enzyme that is not attacked by the substrate. E+I =— EI and ES+I =— ESI where EI and ESI are inactive. Reversible, non-competitive inhibition is, in any case, rare. _ _U. IRREVERSIBLE INHIBITION. In this type of inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly jas is dissociation from the enzyme is very low, An example of acetyicholinesterase ‘an 3 the action of nerve gas poisons on 7 nzyme that has an important role in the q H Hyl—-CHy HyC-4_cH ; Enzyme—CH,OH + mT O —+Enzyme —cuo-+ =0O+HF 5 | H ! i CH HyC—C—CH, HX k Diisopropylphospho- fluoridate (DIPF)ENZYMES 167 transmission of nerve impulse. Disoprophylophospho-fluoridate (DIPF), one of these agents, reacts with a critical serine residue at the active site on the enzyme to form an inactive diisopropylphosphoryl enzyme. Similarly, lodoacetic acid blocks the sulfhydral (—SH) groups by alkylation. thus inhibiting certain enzymes. I Enzyme —CH,SH + ICH,—C—NH;——> Todoacetamide I Enzyme —CH,—S—CH,—C—NH, + HI Other examples of irreversible inhibition are the inhibition of many enzymes by heavy metal ions such as Ag*, Hg”* and Pb* etc. Urease is extremely sensitive to traces of these ions. ALLOSTERIC ENZYMES The kinetic properties of many enzymes cannot be accounted for by the Michaelis-Menten model. An important group consists of the allosteric enzymes which usually exhibit sigmoidal rather than hyperbolic plots of the reaction velocity V, versus substrate concentration [S]. They are oligomers containing two (dimer), four (tetramer), or more subunits which are able to interact with one another. In allosteric enzymes, one active site in an enzyme molecule ES Fig. 11.13. The Sigmoidal Curve.yes PHYSIOL ogy e su plot ofy the ean ateet another active site in the same D Moctpy this interaction across subunits, the bindin nee Molec sty cooperative, Which would give a sigmoidal J Dstt Due to sigmoidal shape of the curve results from the fact t Verse res st substrate molecule increases the affine tt hing The send substrate molecule, and so forth, Allosteric for binding grit regulatory value, since large changes in activity ana es i he dy small change in substrate concentration [8], They Oban importance in the regulation of cell metabolism, ate OF utmost Wo models have been postulated to accoun properties of allosteric enzymes, They are (1) the concertey Some and (2) the sequential model. | modes (1) The Concerted Model for Allosteric Interactions According to this model proposed in 1965 by Monod, W, and Changeux, an allosteric enzyme is made up of two ‘dena subunits, each with one active site. A subunit can exist in citherat two conformational states, called R and T. R has a high affinity fo, the substrate whereas T has a low affinity. Forms R and T are interconvertible. An important supposition of this model is that bot subunits must be in the same conformational state, in order that the symmetry of the dimer is conserved. Thus RR and TT are permissible conformations, but RT is not allowed. Tense state Relaxed state Fig. 11.14. R and T forms of an allosteric enzyme. In the absence of substrate, most of the enzyme molecules are in the T form. Since the substrate binds preferentially to the R form, the addition of substrate shifts the conformational equilibrium in the direction of the R form when substrate binds to one site, the other site on the same enzyme molecule must also be in the R form, according to the basic concept of this model. It could be said, therefore, that the transition from T to Ror vice versa is concerted or arranged. In this way, the proportion of enzyme molecules in the R form increases progressively as more substrate is added, and so the binding of the substrate is cooperative, i.e., the binding of the first molecule of substrate enhances the binding of those that follow. When th active sites are fully saturated, all of the enzyme molecules the R form.ENZYMES me In the concerted model, an allosteric inhibitor (represented hy a hexagon in Fig. 11.15) binds preferentially to the T form whereas an allosteric activator (represented by a triangle) binds to the & form. Therefore, an allosteric inhibitor shifts the R « 1 conformational equilibrium toward T, whereas an allosteric activator shifts it toward R. Such modulators or modifiers do not bind to the active site, but to a different site—the allosteric one, Fig. 11.15. Concerted model for the cooperative binding of substrate in an allosteric enzyme. The low affinity form TT, changes to the high affinity form, RR, upon binding the first molecule of the substrate. Allosteric interactions between identical legands* are homotropic as in the cooperative binding of substrate to the enzyme. Homotropic interactions are necessarily positive in the concerted model. Interactions between different ligands are referred to as heterotropic. For example, the effect of an activator or inhibitor on the binding of substrate is heterotropic because it involves interactions between different kinds of molecules. Heterotropic effects can be either positive or negative. Allosteric Allosteric inhibitor activator “ » v Fig. 11.16. Allosterlc Modifiers in the concerted model. (2) The Sequential Model for Allosteric Interaction Allosteric interactions can also be explained by the sequential - ; - e433 = =] ql —_ = RT RA Fig. 11.17. Sequential model for the cooperative binding of substrate in an allosteric enzyme. *Ligand : Any small molecule bound to the enzyme by non-covalent forces. The term includes activators, substrates, and inhibitors. TT70 PHYSIOLOGY AND BIOCHEMISTRY model which has been developed by Koshland, Nemethy and Filmer, It is based on three assumptions : @) Any one subunit can exist in only two conformational states Rand T. (i) The binding of substrate to one of the subunits induces a change in its conformation, However, the conformation of the other subunits in this enzyme molecule does not change, (ii) The conformational change induced by the binding of substrate in one subunit can increase or decrease the substrate binding affinity of the other subunits in the same enzyme molecule. The sequential model differs from the concerted model in many ways : (1) An equilibrium between the R and T forms in the absence of substrate is not assumed. On the contrary, the conformational change-over from T to R is induced by the binding of substrate. (2) The conformational change from T to R in different subunits of an enzyme molecule is sequential, not concerted, i.e., arranged or adjusted. (3) The hybrid form RT is prominent in the sequential model but it does not exist in the concerted model (4) The concerted model assumes that symmetry is essential for the interaction of subunits in oligomeric proteins and, therefore Tequires that it should be conserved in allosteric interactions. (5) Homotropic interactions are always positive in the converted model but can be either positive or negative in the sequential model. The most studies allosteric enzyme is aspartate transcarbamy- Jase(ATCase) which catalyzes the conversion of carbamyl phosphate to carbamyl aspartate, the first of a sequence of eight reactions leading to the synthesis of Cytidine Triphosphate (CTP), which, in turn, is required for the synthesis of the nucleic acids, This is illustrated in the following equations : Carbamy Es Carhamyl--S8P » Crp----Nucleic phosphate aspartate acids When CTP begins to accumulate as a result of a decrease in the rate of nucleic acid synthesis within the cell, it reaches a concentration which makes it an inhibitor of ATCase, thereby inhibiting its own synthesis. This effect is termed negative feed back control. The enzyme ATCase consists of 12 subunits equally divided among two kinds : six subunits of A, and six of B. Subunits A contain binding sites for the substrate, carbamy] phosphate, but do not bind |ENZYMES 171 CTP; they contain the catalytic sites. Subunits, B bind CTP and lack catalytic activity. The binding site for CTP is termed the allosteric site (allo= Other) ; such an enzyme is called an allosteric enzyme, and the inhibition is called allosteric inhibition. COENZYMES “Coenzymes generally act as acceptors or donors of a functional group or of atoms that are removed from or contributed to the substrate.” Types ofreactions that frequently require the participation of coenzymes are oxidoreductions, group transfer, isomerization reactions and reactions resulting in the formation of covalent such as those catalyzed by the enzymes of the digestive tract, are not known to require coenzymes. The majority of coenzymes are chemical derivatives of nucleotides. In most coenzymes, the nitrogen-base portion of nucleotides is replaced by another chemical unit. This unit is usually a derivative ofa B vitamin. Thus many enzymes concerned with the metabolism of amino acids require enzymes containing vitamin B, or pyridoxine. The B vitamins nicotinamide, thiamin, riboflavin, cid, and lipoic acid are important constituents of biological redox reactions. Similarly folic acid and function in one-carbon metabolism. pantothenic a coenzymes for cobamide coenzymes of Coenzymes Based on Functional Characteristics Classification (A) Coenzymes for transfer of H TABLE 11.1 S.No, Coenzyme Function Essential nutri- tional factor of vitamin Hydrogen Carrier Nicotinic acid Nicotinamide adenine dinucleotide (NaD*) Nicotinamlde adenine di- Hydrogen Carrier nucleotide phosphate (NADP*) Flavin mononucleotide Hydrogen Carrier | Riboflavin (FMN) Nicotinic acid Flavin adinine dinucleotide | Hydrogen Carrier Riboflavin (FAD) Lipoic acid Hydrogen Carrier None Coenzymes Q (Ubiqui- Hydrogen Carrier | None none)—An important component of the ETS in the mitochondriaPHYSIOLOGY AND BIOCHEMISTRy "Yoo pue s8jsue.y Y 10 SouLAzue0g “BI'TT “SLT (S@ NIWVLIA) lOv SINSHLOLNYd 1 t d-0 t | ¥ (¥05) v SWAZN309 d-O0 J euiuepy—esogiy d- ct euluepy—ssoqid4—O—d—}-O—d —O—2S0g]tj—eplweunooIN, (SGVN) 31VHdSOHd 3CLLOFIONNIG 3NINIGV JGINVNILOSIN (AVN) 3a1L0370NNIG SNINSGV AGINYNILOOIN @pIWeUNOSIN S3WAZN3OO Ide SIUODIN (Fg ueyr) awy NWS upeN ees j—O-OSOF!!-UIWE)-} < 8SOG!I-UIAE] 4 (q ujweyta) (NW4) SCILLOSTONNONOW NIAW14 uIAeYoqly euluepy—esoqiy—o—g. d—O—980q)!-U/Ae] 4 (ava) ag1L0a1OnNIG aNINZGY NIAVT4ENZYMES ra In older literature, nicotinamide adenine dinudeotide(NAD*) is also called diphosphopyridine nucleotide (DPN*) or coenzyme 1. Similarly nicotinamide adenine dinucleotide phosphate ( NADP") is called triphosphopyridine nucleotide (TPN) or coenzyme II. Because DPN? is not a nucleotide of ‘diphosphopyridine’, because this name does not indicate the presence either of nicotinamide or adenine, and because this nomenclature is not in keeping with that used for many related compounds, an International Commission on Nomen- clature in 1961 suggested that these two coenzymes be designated as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Their reduced forms in this convention, are denoted as NADH, and NADPH, respectively. (i) NAD* consists of 2 nucleotide units : nicotinamide mononucleotide (NMN: nicotinamide-ribose—O—P), derived from the dietary supply of nicotinamide; and adenosine monophosphate (AMP: adenine—ribose—O—P). Gi) NADP* is essentially identical with that of NAD*, except that it possess in addition a phosphate ester group attached to the 2'-hydroxyl group of the ribose portion of the adenosine unit. (iii) FMI is a metabolically active form of the vitamin riboflavin (B,) to which in cells, a phosphate group becomes linked. (iv) FAD comprises two nucleotides : FMN and AMP. Functions. Vitamin B derived coenzymes as mentioned above are capable of being reversibly oxidised and reduced. They are associated with essentially all of the oxidation and reduction reactions occurring within living cells. They serve as carriers in many processes in which hydrogen is transferred from one compound to another. (B) Coenzymes for Group Transfer of Groups other than H. TABLE 11.2. Nutrient factor or Eseential vitamin ATP and its ‘Transphosphorylation None relatives Coenzyme A (CoA) Acetyl of other acyl group Pantothenic acid transfer: fatty acid synthesis and oxidation. Oxidative decarboxylation of a-keto acids; active aldehyde carrier. Reactions involving amino acids e.g.. decarboxylation transmination. Thiamin Thiamin pyrophosphate (Cocarboxylase) Pyridoxal phos- phate Pyridoxine174 PHYSIOLOGY AND BIOCHEMISTRY S.No| Coenzyme Function Nutrient factor oy Eseential vitamin 5. | Tetrahydrofolic | Reactions involving one'| Polic acid acid carbon compounds. 6. | Biotin CO, transfer. Biotin 7. | Cobamide Carbon chain isomerizations Cyanacobalamin coenzymes and certain —CH, group (B,y) transfers. 8. | Lipoic acid Oxidative decarboxylation acyl acceptor. Coenzymes A. A molecule of this compound consists of three parts : Aphosphate derivative + Pantothenic acid (a B vitamin) +A chain of N, C and S atoms to which H is attached. CoA serves as a carrier of a molecular group called acetyl, a2- carbon unit of importance in respiration and synthesis. An acetyl group gets attached to the S end of the CoA, and the product acety] CoA, is held together by an —S~C bond. Like the O~P bond, the —S-C bond is also a high energy bond. Thus CoA may be considéred to be both an acetyl carrier and an energy carrier. CoA was discovered by the American Biochemist Dr. Fritz Lipmann for which he got the Nobel Prize in 1955. Since coenzymes are frequently readily dissociable from the enzyme protein, they may properly be regarded as second substrates or cosubstrates. This is so for 2 reasons : 1. The chemical changes in the coenzymes exactly counter balance those taking place in the substrate, e.g., in transphosphorylation reactions involved in the metabolism of: sugars, for every molecule of sugar phosphorylated, one molecule of ATP is dephosphorylated and converted to ADP. ATP + Sugar ~ Sugar~phosphate + ADP Similarly in oxidoreduction (dehydrogenase) reactions, one molecule of substrate is oxided (dehydrogenated) and one molecule of coenzyme is reduced (hydrogenated). 2. Asecond reason to give equal importance to the reactions of the coenzyme is that this aspect of the reaction may actually be of greater fundamental physiological significance. For example, without NAD‘, glycolysis cannot continue and anaerobic ATP synthesis ceases.ENZYMES 17% ISOENZYMES. An enzyme which has multiple molecular forms in the same organism catalyzing the same reaction is called an Isoenzyme. Isoenzymes are physically distinct forms of the same catalytic activity. Thus they catalyze the same chemical reaction. Isoenzymes of a particular enzyme are analogous to 10-paisa coins minted at several different mints e.g., at Kolkata, Pune, Chennai etc. The monetary value (reaction catalyzed == biologic value) is identical in each instance, and each coin (isoenzyme) is physically quite similar. But just like “mint marks” on a coin, minute physical, chemical and immunological differences between isoenzymes become apparent on careful examination. The best studied isoenzyme is Lactic dehydrogenase (LDH) which can occur in 5 possible forms in the blood sera and tissues of : most vertebrates. Two basically different types of LDH occur : 1. Heart LDH. It occurs in the heart and consists of 4 identical H subunits, viz, HHHH. 2. Muscle LDH. It is present in the skeletal muscles and consists of 4 similar M subunits, viz., MMMM. Only the tetrameric molecule possesses catalytic activity. Synthesis of two subunits H and M have been shown to be controlled by distinct genetic loci. Combinations of H and M subunits will produce three additional types of hybrid enzymes. The possible combinations of the H and M subunits are as follows : Lactate Dehydrogenase Subunits Isoenzyme 1, HHHH L HHHM ly HHMM L, HMMM I, MMMM There are more than 100 enzymes which are known to exist as isoenzymes. Isoenzymes of numerous dehydrogenases, and of several oxidases, transminases, phosphatases, transphosphorylases and proteolytic enzymes have been reported. It is now well established that these isoenzymes are produced by genetic changes that determine differences in the amino acid sequence.z "yp aurdkzus0g [ay pue v aUIAZUa0Q ‘6 IT a i] n 3 dnoad g Vo9 IAjo0y woo we 8 0 g la. { : a -O Vvoo—s~0O—*HD <— V90—-SHtO—0—" HO a | ® eee (woo) S HO. OO v 3WAZN309 ° 5 (NIVHO uNJINS-NOBYVO) & H H LINN NIN x H oa LINN SAWNAHLOLNWd “VIAHISOLdvOUaW- °y H H *400d—0—d—0—*HO pte tp ttm ®HO O