C 34 Azymes are biocatalysts ~ the catalysts of life, A catalyst is defined as a substance that creases the velocity or rate of a chemical action without itself undergoing any change in overall process. The student-teacher relationship may be a ood example to understand how a catalyst ‘works. The students often find it difficult to learn from a text-book on their own, The teacher explains the subject to the students and increases their understanding capability. It is no wonder that certain difficult things which the students take days together to understand, and sometimes do not understand at all - are easily learnt under the guidance of the teacher. Here, the teacher acts like a catalyst in enhancing the understanding ability of students. A good teacher is always a good catalyst in students’ life! Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception- RNA acting as ribozyme), colloidal and thermolabile in character, and specific in their action. 85 The enzymes "We are the catalysts of the Huge in ed Highly exploited for In the laboratory, hydrolysis of proteins by a strong acid at 100°C takes at least a couple of days. The same protein is fully digested by the enzymes in gastrointestinal tract at body temperature (37°C) within a couple of hours. This remarkable difference in the chemical reactions taking place in the living system is exclusively due to enzymes. The very existence of life is unimaginable without the presence of enzymes. deltsdre]1oy Vit 7. Cel (ej -felh Te} Berzelius in 1836 coined the term catalysis (Greek ; to dissolve). In 1878, Kuhne used the word enzyme (Greek : in yeast) to indicate the catalysis taking place in the biological systems. Isolation of enzyme system from cell-free extract of yeast was achieved in 1883 by Buchner. He named the active principle as zymase (later found to contain a mixture of enzymes), which could convert sugar to alcohol. In 1926, James © scanned with OKEN Scanner86 Sumner first achieved the isolation ancl enstallization of the enzyme urease from jack bean anc ified it as a protein. Ainisnes er iseauve Crests Nt In the early days, the enzymes were given names by their discoverers in an arbitrary manner, For example, the names pepsin, trypsin and chymotrypsin convey no information about the function of the enzyme or the nature of the substrate on which they act. Sometimes, the suffiv-ase was added to the substrate for naming the enzymes e.g. lipase acts on lipids; nuclease on nucleic acids; lactase on lactose. These are known as trivial names of the enzymes which, however, fail to give complete information of Enzyme class with examples* |. Oxidoreductases oyfocivome oxidase, L- and D-amino acid oxidases Transferases ~ ~ _Mansaminases, transmethyiases, phosphorylase Hydrolases: yases lumarase, histidase somerases ‘Triose phosphate isomerase keloisomerase, E.C. 6.3.1.1) phosphohexose isomeras “6. Ligases Glutamine synthetase (L-gutamate ammonia ligase, acetyl CoA carboxylase, succinate thiokinase retinal isomerase, "Fr one enzyma in eth ass, soni rae ao enzyme in each class, systematic name along with EC 1 SS sptemalé name alongwith E.C, number is given in the brankon ‘Alcoho! dehydrogenase (alcohol : NADY oxidoreductase E.C. 1.1.1.1.), Hexokinase (ATP : D-hexose 6-phosphotransterase, E.C. 2.7.1.1.), Aldolase (ketose 1-phosphate aldehyde lyase, E.C, 4.1.2.7), (O-glyceraldehyde 3-phosphate EC. 63.1.2), BIOCHEMISTRY enzyme reaction (type of reaction, cofactor requirement etc.) Enzymes are sometimes considered under two broad categories ; (a) Intracellular enzymes ~ ‘They are functional within cells where they are eymthesized. (b) Extracellular enzymes — These enzymes are active outside the cell; all the digestive enzymes belong to this group. “The International Union of Biochemistry (IUB) appointed an Enzyme Commission in 1961. This Committee made a thorough study of the existing enzymes and devised some basic principles for the classification and nomenclature of enzymes. Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six major classes (in that order). Each class on its own represents the general type of reaction brought about by the enzymes of that class (Table 6.1). * Ura h CeEPe re uP ec ld Reaction catalysed Oxidation —+ Reduction AH, + B—> A+BH, Group transfer A-X+B—+ A+B-X Lipase (viacyiglycerl acyl hydrolase E.C. 3.1.1.3) choline é esterase, acid and alkaline phosphatases, pepsin, urease Hydrolysis A-B+H,0 ‘Addition —> Elimination . A-B+X-Y—> AX-BY Interconversion of isomers AN Condensation (usualy dependent on ATP A+B A-B Sl pein iat ATP ADP +P © scanned with OKEN Scanner aon -87 ductases Enzymes involved In ses ¢ Enzymes that catalyse the inctional groups. Enzymes that bring about sarious compounds. Enzymes specialised in the oval of water, ammonia, CO, etc. + Enzymes involved in all the Enzymes catalysing the synthetic -k : ligate—to bind) where two ich enzyme representing the class lass (second digit), sub-sub class action catalysed by the enzyme. UB names for the enzymes are ambiguous, they have not been neral use as they are complex fe to remember. Therefore, the long with the E.C. numbers as ded, are commonly used and Mera aL) =RTIES OF ENZYMES ymes are invariably proteins. In however, a few RNA molecules to function as enzymes. Each pwn tertiary structure and specific which is very essential for its ity. The functional unit of the as holoenzyme which is often made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part). Holoenzyme —+ Apoenzyme + Coenzyme {active enzyme) (pretein par (non-protein par The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme, The coenzyme can be separated by dialysis from the enzyme while the prosthetic group cannot be. The word monomeric enzyme is used if it is made up of a single polypeptide e.g. ribo- nuclease, trypsin. Some of the enzymes which Possess more than one polypeptide (subunit) chain are known as oligomeric enzymes e.g. lactate dehydrogenase, aspartate —trans- carbamoylase etc. There are certain multienzyme complexes possessing specific sites to catalyse different reactions in a sequence. Only the native intact multienzyme complex is functionally active and not the individual units, if they are separated e.g. pyruvate dehydrogenase, fatty acid synthase, prostaglandin synthase etc. The enzymes exhibit all the general properties of proteins (Chapter 4). Genetic engineering and modified enzymes Recent advances in biotechnology have made it possible to modify the enzymes with desirable characters-improved catalytic abilities, activities under unusual conditions. This approach is equired since enzymes possess enormous potential for their use in medicine and industry. Hybrid enzymes : It is possible to rearrange genes and produce fusion proteins. e.g. a hybrid enzyme (of glucanase and cellulase) that can more efficiently hydrolyse barley B-glucans in beer manufacture. Site-directed mutagenesis : This is a technique used to produce a specified mutation at a predetermined position in a DNA molecule. The result is incorporation of a desired amino acid (of one’s choice) in place of the specified amino acid in the enzyme, By this approach, it is possible to produce an enzyme with desirable characteristics. e.g. tissue plasminogen activator (used to lyse blood clots in myocardial © scanned with OKEN Scannera Enzyme velocity Enzyme concentration ——+ 2 Btfect of enzyme increased half-life. This is infarction) with achieved by replacing asparagine (at position 120) by glutamine. In recent years, it has also become possible to yoduce hybrid enzymes by rearrangement of nes. Another innovative approach is the juction of abzymes or catalytic antibodies, fe antibody enzymes. Potato e) ENZYME ACTIVITY The contact between the enzyme and substrate is the most essential pre-requisite for enzyme activity. The important factors that influence the velocity of the enzyme reaction are discussed hereunder 1. Concentration of enzyme As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases (Fig.6.1). In fact, this property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases, By using a known volume of serum, and keeping all the other factors (substrate, pH, temperature etc.) at the optimum level, the enzyme could be assayed in the laboratory. BIOCHEMISTRY 2. Concentration of substrate Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels. A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration (Fig.6.2). Three distinct phases of | the reaction are observed in the graph (A-linear, B.curve; C-almost unchanged). Order of reaction : When the velocity of the reaction is almost proportional to the substrate concentration (ie. (5) is less than Kr), the rate of the reaction is said to be first order with respect to substrate. When the (5] is much greater than Km the rate of reaction is independent of substrate concentration, and the reaction is said to be zero order. Enzyme kinetics and K,, value : The enzyme (® and substrate (5) combine with each other to form an unstable enzyme-substrate complex (ES) _ for the formation of product (P). Ky ky E+ aa ES—=+E+P 2 Here kj, kz and ky represent the velocity — constants for the respective reactions, as indicated by arrows. Km the Michaelis-Menten constant (or Brig’s and Haldane’s constand, is given by the formula The following equation is obtained after suitable algebraic manipulation. Naaxl8 equation (1) kK, +151 where v = Measured velocity, Vea = Maximum velocity, s = Substrate concentration, Kn = Michaelis - Menten constant. Let us assume that the measured velocity (Vv) is equal t0 Vinay. Then the equation (1) may be _ substituted as follows Vingx ($1 Kn tIS) Vax = 4 2 © scanned with OKEN Scannerchaptor 6 : ENZYMES ae) Ky tis) = 2m!) Vinax kK, +1] = 215} Ky = ISI K stands for a Michaelis (in Km). constant and m stands for Kp or the Michaelis-Menten constant is defined as the substrate concentration (expressed in moles/l) to produce half-maximum velocity in an enzyme catalysed reaction. It indicates that half of the enzyme molecules (ie. 50%) are bound with the substrate molecules when the substrate concentration equals the K, value. Km value is a constant and a characteristic feature of a given enzyme (comparable to a thumb impression or signature). It is @ representative for measuring the strength of ES complex. A low K,, value indicates a strong affinity between enzyme and substrate, whereas a high Km value reflects a weak affinity between them. For majority of enzymes, the Km values are in the range of 10°5 to 10°? moles. It may however, be noted that K,, is not dependent on the concentration of enzyme. Lineweaver-Burk double reciprocal plot : For the determination of Km value, the substrate saturation curve (Fig.6.2) Is not very accurate ry Vmax IS approached asymptotically. By ‘aking the reciprocals of the equation (1), a Straight line graphic representation is obtained. 1 Kna[s Vo Vinax {S| 1 Ka 1 x x View 1 K, 1.4, vo" Via [5] The above equation is similar to y = ax + b. Therefore, a plot of the reciprocal of the velocity 1 (2) vs, the reciprocal of the substrate concen- tration (4) gives a straight line. Here the slope is Kp/Vynax and whose y intercept is 1/Vmax- The Lineweaver-Burk plot is shown in Fig.6.3. It is much easier to calculate the Km from the intercept on x-axis which is ~(1/Ky). Further, the double reciprocal plot is useful in understanding the effect of various inhibitions (discussed later). Enzyme reactions with two or more substrates : The above discussion is based on the presumption of a single substrate-enzyme reaction. In fact, a majority of the enzyme- catalysed reactions involve two or more substrates. Even in case of multisubstrate a © scanned with OKEN ScannerBIOCHEMISTRY 90 SS ISTRY enzymes, despite the complex mathematical expressions, the fundamental principles conform to Michaelis-Menten Kinetics. 3. Effect of temperature Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. A bell-shaped curve is usually observed (Fig.6.4). Temperature coefficient or Qyo is defined as increase in enzyme velocity when the temperature is increased by 10°C. For a majority of enzymes, Qio is 2 between 0°C and 40°C. Increase in temperature results in higher activation energy of the molecules and more molecular (enzyme and substrate) collision and interaction for the reaction to proceed faster. The optimum temperature for most of the enzymes is between 40°C-45°C. However, a few zymes (e.g. venom phosphokinases, muscle lenylate kinase) are active even at 100°C. Some lant enzymes like urease have optimum activity und 60°C. This may be due to very stable icture and conformation of these enzymes. In general, when the enzymes are exposed to a temperature above 50°C, denaturation leading to derangement in the native (tertiary) structure Of the protein and active site are seen. Majority of the enzymes become inactive at higher temperature (above 70°C). — Enzyme volocity ——> © 10 20 30 40 so 60 70 80 ‘Temperatura (°C) | j i Optimum pH o pH —> a 1 is worth noting here that the enzymes have been assigned optimal temperatures based on the laboratory work. These temperatures, however, may have less relevance and biological significance in the living system. 4. Effect of pH Increase in the hydrogen ion concentration (PH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained (Fig.6.5). Each enzyme has an optimum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive. Most of the enzymes of higher organisms show optimum activity around neutral pH (6-8). ‘There are, however, many exceptions like pepsin (1-2), acid phosphatase (4-5) and alkaline Phosphatase (10-11). Enzymes from fungi and Plants are most active in acidic pH (4-6). Hydrogen ions influence the enzyme activity by altering the ionic charges on the amino acids (particularly at the active site), substrate, ES complex etc. 5. Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity: : © scanned with OKEN ScannerFor certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity, In the living system, this type of inhibition is generally prevented by a quick removal of products formed. The end product inhibition by feedback mechanism is discussed later, 6. Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg?+, Mn?*, ant, Ca%*, Co2*, Cu2*, Nat, K* etc, for thelr optimum activity. Rarely, anions are also needed for enzyme activity eg. chloride ion (Cr) for amylase. Metals function as activators of enzyme velocity through various mechanisms— combining with the substrate, formation of ES-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme. Two categories of enzymes requiring metals for their activity are distinguished + Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchanged easily with other ions e.g. ATPase (Mg?* and Ca?*) Enolase (Mg”*) + Metalloenzymes : These enzymes hold the metals rather tightly which are not readily exchanged. eg. alcohol dehydro- genase, carbonic anhydrase, alkaline phos- phatase, carboxypeptidase and aldolase contain zinc, Phenol oxidase (copper); Pyruvate oxidase (manganese); Xanthine oxidase (molybdenum); Cytochrome oxidase (iron and copper). 7. Effect of time Under ideal and optimal conditions (like pH, temperature etc,), the time required for an enzyme reaction is less, Variations in the time of the reaction are generally related to the alterations in PH and temperature. ‘Substrate 8. Effect of light and radiation Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides. e.g. UV rays inhibit salivary amylase activity. Pe Enzymes are big in size compared to substrates which are relatively smaller. Evidently, a small portion of the huge enzyme molecule is directly involved in the substrate binding and catalysis (Fig.6.6). The active site (or active centre) of an enzyme represents as the small region at which the substrate(s) binds and participates in the catalysis. Salient features of active site 1. The existence of active site is due to the tertiary structure of protein resulting in three- dimensional native conformation. 2, The active site is made up of amino acids which are far from each other in the linear sequence of amino acids (primary structure of protein). For instance, the enzyme lysozyme has 129 amino acids. The active site is formed by the contribution of amino acid residues numbered 35, 52, 62, 63 and 101. 3. Active sites are regarded as clefts or crevices or pockets occupying a small region in big enzyme molecule. © scanned with OKEN Scanner92. it igid in structure and active site is not rigid tare re ae nor Mexible to promote the specific substrate binding. | Generally, the active site possesses a subsite binding site and a catalytic site. The aur is for the catalysis of the specific reaction. 6. The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site. 7. The substrate(s) binds at the active site by weak noncovalent bonds, 8. Enzymes are specific in their function due to the existence of active sites. 9. The commonly found amino acids at the active sit serine, aspartate, histidine, Among ri a ‘amino acids, a 10. The substrate[S] binds the enzyme (E) at the active site to form enzyme-substrate complex (ES). The product (P) is released after the catalysis and the enzyme is available for reuse. E+ SS ES—E+P --! ENZYME INHIBITION Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. There are three broad categories of enzyme inhibition 1, Reversible inhibit 2. Irreversible inhibition. 3. Allosteric inhibition. ion. 4. Reversible inhibition The inhibitor binds non-covalently with enzyme and the enzyme inhibition can_be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into 1. Competitive inhibition (Fig.6.7A) 1, Non-competitive inhibition (Fig.6.78) Cc Substrate oa Competitive Inhibitor 'Non-competitve| inhibitor |, Competitive inhibition : The inhibitor ti) which closely resembles the real substrate (S) is regarded as a substrate analogue. The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis. As long as the competitive inhibitor holds the active site, the enzyme is not available. for the substrate to bind, During the reaction, ES and El complexes are formed as shown below The relative concentration of the substrate and inhibitor and their respective affinity with the enzyme determines the degree of competitive inhibition. The inhibition could be overcome by a high substrate concentration. In competitive inhibition, the Ky, value increases whereas Vmax temains unchanged (Fig.6.8). The enzyme succinate dehydrogenase (SDH) is a classical example of competitive inhibition with succinic acid as its substrate. The compounds, namely, malonic acid, glutaric acid and oxalic acid, have structural similarity with succinic acid and compete with the substrate for binding at the active site of SDH. © scanned with OKEN ScannerChopter 6 : ENZYMES Se Soon GH.COOH He CH,COOH Coon ‘Succinic acld Malonic acid Methanol is toxic to the body when it is converted to formaldehyde by the enzyme alcohol dehydrogenase (ADH). Ethanol can compete with methanol for ADH. Thus, ethanol can be used in the treatment of methanol poisoning. Some more examples of the enzymes with substrates and competitive inhibitors (of clinical and pharmacological significance) are given in Table 6.2. Antimetabolites : These are the chemical compounds that block the metabolic reactions by their inhibitory action on enzymes. Antimetabolites are usually structural analogues of substrates and thus are competitive inhibitors (Table 6.2). They are in use for cancer therapy, gout etc. The term antivitamins is used for the antimetabolites which block the biochemical actions of vitamins causing deficiencies, e.g. sulphonilamide, dicumarol. Il. Non-competitive inhibition : The inhibitor binds at a site other than the active site on the enzyme surface, This binding impairs the ‘enzyme function, The inhibitor has no structural Fesemblance with the substrate, However, there Usually exists a strong affinity for the inhibitor to bind at the second site, In fact, the inhibitor does Not interfere with the enzyme-substrate binding. But the catalysis is prevented, possibly due to a distortion in the enzyme conformation. The Inhibitor generally binds with the enzyme as well as the ES complex. The overall felation in non-competitive ini fepresented below E+ SSSES—tE+P fod For non-competitive inhibition, the Ky, value is unchanged while Vmax is lowered (Fig.6.9). Heavy metal ions (Agt, Pb?*, Hg?* etc.) can non-competitively inhibit the enzymes by binding with cysteinyl sulfhydry! groups. The general reaction for Hg?* is shown below. E-SH + Hg E-S- + Hg? Ht © scanned with OKEN ScannerSubstrate Hypoxanthine xanthine Catecholamines (epinephne, norepinephrine) "Dinydrofolate reductase Dihydrofolc acid Enzyme Xanthine oxidase Monoamine oxidase Ephedrine, amphetamine ‘Aminopterin, amethoptetn, Inhibitor(s) ‘Alopurinol Used In the contr of gout fo red production of uric acid from hypaxant Useful for elevating catecholamine Employed in the treatment of leukemi methotrexate ‘Acetylcholine esterase Acetyicholine Suocinyt choline ‘Sulfonilamide Heavy metals also lead to the formation of covalent bonds carboxyl groups and histidine, often resulting in irreversible inhibition. 2. Irreversible inhibition The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usually toxic poisonous substances. lodoacetate is an irreversible enzymes like papain and _ glyceraldehyde 3-phosphate dehydrogenase. lodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzymes and makes them inactive. © scanned with OKEN ScannerDiisopropy! fluorophosphate (DFP) |s a nerve ‘gas developed by the Germans during Second World War. DFP irreversibly binds with enzymes containing serine at the active site, eg. serine proteases, acetylcholine esterase, Many organophosphorus insecticides like melathion are toxic to animals (including man) as they block the activity of acetylcholine esterase (essential for nerve conduction), resulting in paralysis of vital body functions. Disulfiram (Antabuse®) is a drug used in the treatment of alcoholism. It irreversibly inhibits the enzyme aldehyde dehydrogenase. Alcohol addicts, when treated with disulfiram become sick due to the accumulation of acetaldehyde, leading to alcohol avoidance. (Note : Alcohol Is metabolized by two enzymes. It is first acted upon by alcohol dehydrogenase to yield acetaldehyde. The enzyme aldehyde dehydro- genase converts acetaldehyde to acetic acid.) The penicillin antibiotics act as irreversible inhibitors of serine~containing enzymes, and block the bacterial cell wall synthesis. Irreversible inhibitors are frequently used to identify amino acid residues at the active site of the enzymes, and also to understand the mechanism of enzyme action. Suicide inhibition Suicide inhibition is a specialized form of irreversible inhibition. In this case, the original inhibitor (the structural analogue/competitive inhibitor) is converted to a more potent form by the same enzyme that ought to be inhibited. The so formed inhibitor binds irreversibly with the enzyme. This is in contrast to the original inhibitor which binds reversibly. A good example of suicide inhibition is allopurinol (used in the treatment of gout, Refer Chapter 17). Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthine, a more effective inhibitor of this enzyme. The use of certain purine and pyrimidine analogues in cancer therapy is also explained on the basis suicide inhibition, For instance, S-fluorouracil gets converted to fluorodeoxy- urldylate which Inhibits the enzyme thymidylate synthase, and thus nucleotide synthesis. 3. Allosteric Inhibition The details of this type of inhibition are given under allosteric regulation as a part of the regulation of enzyme activity in the living system, traf) ele ae Enzymes are highly specific in their action when compared with the chemical catalysts. The occurrence of thousands of enzymes in the biological system might be due to the specific nature of enzymes. Three types of enzyme specificity are well-recognised 1. Stereospecificity, 2. Reaction specifi 3. Substrate specificity, Specificity is a characteristic property of the active site. 1, Stereospecificity or optical specificity : Stereoisomers are the compounds which have the same molecular formula, but differ in their structural configuration. The enzymes act only on one isomer and, therefore, exhibit stereospecificity. e.g. L-amino acid oxidase and D-amino acid oxidase act on L+ and D-amino acids respectively. Hexokinase acts on D-hexoses; Glucokinase on D-glucose: Amylase acts on ceglycosidic linkages; Cellulase cleaves glycosidic bonds. Stereospecificity is explained by considering three distinct regions of substrate molecule specifically binding with three complementary regions on the surface of the enzyme (Fig.6.10), The class of enzymes belonging to isomerases do not exhibit stereospecificity, since they are specialized in the interconversion of isomers. © scanned with OKEN Scanner4—— Substrate Fig. 6.10 : Diagrammatic representation of stereo speciicity (a’, b’, ’)—three point attachment of — substrate to the enzyme (a,’b, 2, Reaction specificity : The same substrate glycosidases acting on glycosidic bonds carbohydrates, lipases cleaving ester bonds lipids etc. Broad specificity : Some enzymes act on closely related substrates which is commonly known as broad substrate specificity, e, hexokinase acts on glucose, fructose, mannose and glucosamine and not on galactose, It is possible that some structural similarity among the first four compounds makes them a common substrate for the enzyme | hexokinase. The protein part of the enzyme, on its own, is not always adequate to bring about the catalytic | activity. Many enzymes require certain non- | protein small additional factors, collectively referred to as cofactors for catalysis. The cofactors may be organic or inorganic in can undergo different types of reactions, each catalysed by a separate enzyme and this is referred to as reaction specificity. An amino acid can undergo trensamination, oxidative deami- ation, decarboxylation, racemization etc. The s however, ate different for each of these (For details, refer Chapter 15). er absolute, relative or broad. substrate specificity : Certain es act only on one substrate e. kinase acts on glucose to give glucose 6- hate, urease cleaves urea to ammonia bon dioxide. substrate specificity : Some enzymes ‘on structurally related substances. This, in turn, may be dependent on the specific group a bond present, The action of trypsin is B00d example for group specificity (Refer Fig.8.7). Trypsin hydrolyses peptide linkage involving argi ‘or lysine. Chymotrypsin leaves peptide bonds attached to aromatic ino acids (phenylalanine, tyrosine and Fyptophan). Examples of bond specificity. rate specificity : The substrate fy varies (rom enzyme to enzyme. It may (eonayme) The tem prose yup sd when a non-protein moiety is tightly bound to the enzyme which is not easily separable by dialysis. The term activator is referred to the inorganic cofactor (like Ca2*, Mg?*, Mn?* etc.) necessary to enhance enzyme activity. It may, however, be noted that some authors make n0 | distinction between the terms cofactor, coenzyme and prosthetic group and use them, interchangeably. Coenzymes are second substrates Coenzymes are often regarded as the second | substrates or co-substrates, since they have | affinity with the enzyme comparable with that of - the substrates, Coenzymes undergo alterations during the enzymatic reactions, which are later, regenerated. This is in contrast to the substrate. which is converted to the product. © scanned with OKEN ScannerChapter 6 : ENZYMES ‘Coenzymé ‘Thiamine pyrophosphate (TPP) ‘Details for each coanzyme are given in Ghapler 7 on vitamins Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen, aldehyde, keto, amino, acyl, methyl, carbon dioxide etc. Coenzymes play a decisive role in enzyme function. Coenzymes from B-complex vitamins : Most of the coenzymes are the derivatives of water soluble B-complex vitamins. In fact, the biochemical functions of B-complex vitamins are exerted through their respective coenzymes. The chapter on vitamins gives the details of structure and function of the coenzymes (Chapter 7). In 97 Atom or Dependent enzyme Broup transferred example (example) Transkata Table. 6.3, 2 summary of the vitamin related coenzymes with their functions is given. Non-vitamin coenzymes : Not all coenzymes are vitamin derivatives. There are some other organic substances, which have no relation with vitamins but function as coenzymes. They may be considered as non-vitamin coenzymes eg. ATP, CDP, UDP etc. The important non-vitamin coenzymes along with their functions are given in Table 6.4, Nucleotide coenzymes : Some of the coenzymes possess nitrogenous base, sugar and Rae Rc ae halls Coenzyme Abbreviation Adenosine triphosphate ATP Cylidine diphosphate Biochemical functions Donates phosphate, adenosine and adenosine monophosphate (AMP) moieties, © scanned with OKEN Scanner. Such coenzymes are, therefore, eos nucleotides e.g. NAD*, NADP*, FMN, FAD, coenzyme A, UDPG etc. Coenzymes do not decide enzyme specificity + ‘Aparticular coenzyme may participate in catalytic reactions along with different enzymes. For instance, NAD* acts as a coenzyme for lactate dehydrogenase and alcohol dehydrogenase. In both the enzymatic reactions, NAD* is involved in hydrogen transfer. The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzyme. MECHANISM OF ENZYME ACTION Catalysis is the prime function of enzymes. The nature of catalysis taking place in the biological system is similar to that of non- biological catalysis. For any chemical reaction to occur, the reactants have to be in an activated state or transition state. Enzymes lower activation energy : The energy required by the reactants to undergo the reaction is known as activation energy. The reactants when heated attain the activation energy. The catalyst (or the enzyme in the biological system) reduces the activation energy and this causes the reaction to proceed at a lower temperature. Enzymes do not alter the uilibrium constants, they only enhance the locity of the reac The role of catalyst or enzyme is comparable ith a tunnel made in a mountain to reduce the barrier as illustrated in Fig.6.41. The enzyme lowers energy barrier of reactants, thereby making the reaction go faster. The enzymes reduce the activation energy of the reactants in such a way that all the biological systems occur at body temperature (below 40°C). Enzyme-substrate complex formatio! for enzyme catalysis is must combine with the 10 form enzyme- Itimately results The prime requisite that the substrate (S) must enzyme (E) at the active site te substrate complex (ES) which ul te the wenclunt farmation (P). BIOCHEMIs Activation ener with enzyme zyme on activation E+SES—+E+P A few theories have been put forth to explain mechanism of enzyme-substrate complex. formation. Lock and key model or Fischer's template theo This theory was proposed by a German biochemist, Emil Fischer, his is in fact the very first model proposed to explain an enzyme catalysed reaction, According to this model, the structure or conformation of the enzyme is rigid. The substrate fits to the binding site (now active site) just as a key fits into the proper lock or a hand into the | proper glove. Thus the active site of an enzyme is a rigid and pre-shaped template where only a specific substrate can bind. This model does not give any scope for the flexible nature of enzymes, hence the model totally fails to explain many facts ‘of enzymatic reactions, the most important being the effect of allosteric modulators. Induced fit theory 4 or Koshland’s model Proposed a more ic_model for enzyme- acceptable and realistic _m ; substrate complex formation. As per this model, © scanned with OKEN Scannerthe active site is not rigid and pre-shaped. The essential features of the substrate binding site are present at the nascent active site. The interaction of the substrate with the enzyme induces a fit or conformation change in the enzyme, resulting in the formation of a strong substrate binding site. Further, due to induced fit, the appropriate amino acids of the enzyme are repositioned to form the active site and bring about the catalysis (Fig.6.12). Induced fit model has sufficient experimental evidence from the X-ray diffraction studies. Koshland’s model also explains the action of allosteric modulators and competitive inhibition on enzymes. Substrate strain theory In this model, the substrate is strained due to the induced conformation change in the enzyme. Itis also possible that when a substrate binds to the preformed active site, the enzyme induces a strain to the substrate, The strained substrate leads to the formation of product. In fact, a combination of the induced fit model with the substrate strain is considered to be operative in the enzymatic action. 99 — MECHaNIsn OF ENZYME CATALYSIS cohen fermation of an enzyme-substrate ‘enue a very crucial for the catalysis to occur, for the product formation. It is ‘ated ‘that an enzyme catalysed reaction Proceeds 10° to 1012 times faster than a non- Catalysed reaction, The enhancement in the rate Of the reaction is mainly due to four processes : 1. Acid-base catalysis; 2. Substrate strain; 3. Covalent catalysis; 4, Entropy effects, 1, Acid-base catalysis : Role of acids and bases is quite important in enzymology. At the Physiological pH, histidine is the most important amino acid, the protonated form of which functions as an acid and its corresponding. conjugate as a base. The other acids are -OH Broup of tyrosine, ~SH group of cysteine, and e-amino group of lysine. The conjugates of these acids and carboxyl ions (COO-) function as bases, Ribonuclease which cleaves phosphodiester bonds in a pyrimidine loci in RNA is a classical example of the role of acid and base in the catalysis, 2, Substrate strain : Induction of a strain on the substrate for ES formation is discussed above. During the course of strain induction, the energy level of the substrate is raised, leading to a transition state. The mechanism of lysozyme (an enzyme of tears, that cleaves B+1,4 glycosidic bonds) action is believed to be due to a combination of substrate strain and acid-base catalysis. 3. Covalent catalysis : In the covalent catalysis, the negatively charged (nucleophilic) or positively charged (electrophilic) group is present at the active site of the enzyme. This group attacks the substrate that results in the covalent binding of the substrate to the enzyme. in the serine proteases (so named due to the e of serine at active site), covalent base catalysis occur, thrombin ete. presenc n catalysis along with acid-| e.g. chymotrypsin, trypsin, © scanned with OKEN Scanner100 4. Entropy effect : Entropy is a term used in thermodynamics. It is defined as the extent of disorder in a system. The enzymes bring about a decrease in the entropy of the reactants, This enables the reactants to come closer to the enzyme and thus increase the rate of reaction. In the actual catalysis of the enzymes, more than one of the processes ~ acid-base catalysis, substrate strain, covalent catalysis and entropy are simultaneously operative. This will help the substrate(s) to attain a transition state leading to the formation of products. THERMODYNAMICS OF ENZYMATIC REACTIONS The enzyme catalysed reactions may be broadly grouped into three types based on thermodynamic (energy) considerations. 1. Isothermic reactions. : The energy exchange between reactants and products is negligible e.g. glycogen phosphorylase Glycogen + Pi —+ Glucose 1-phosphate 2. Exothermic (exergonic) reactions ; Energy is liberated in these reactions e.g. urease Urea —+ NH; + CO, + energy 3. Endothermic (endergonic) reactions |; Energy is consumed in these reactions eg. glucokinase Glucose + ATP —> Glucose 6-phosphate + ADP REGULATION OF ENZYME CUA A eA epost In biological system, regulation of enzyme activities occurs at different stages in one or more of the following ways to achieve cellular economy. 1. Allosteric regulation 2. Activation of latent enzymes 3. Compartmentation of metabolic pathways 4. Control of enzyme synthesis 5. Enzyme degradation 6. Isoenzymes 1. Some of the enzymes possess additional sites, _ known as allosteric sites (Greek : allo-other), | The existence of life 1s unimaginable without the presence of ensymes—the biocatalysts. fajority of the coenzymes (TPP, NAD*, FAD, CoA) are derived from Beomplex vitamins tn which form the latter exert thelr biochemical functions. © Competitive inhibitors of certaln enzymes are of great blological significance. Allopurinol, © employed In the treatment of gout, inhibits xanthine oxidase to reduce the formation |) of uric aeld. The other competitive inhibitors include aminoptertn used tn the treatment of cancers, sulfantlamide as antlbactericidal agent and dicumarol as an anticoagulant. “| e The nerve gas (diisopropyl fluorophosphate), first developed by Germans during Second World War, inhibits acetylcholine esterase, the enzyme essential for nerve conduction and paralyses the vital body functions. Many organophosphorus insecticides (e.g. “ metathion) also block the actiuity of acetylcholine esterase. Icillin anttblotics trreversibly Inhibit serine containing enzymes of bacterial cell wall _ t ‘ synthesis. © scanned with OKEN ScannerENZYMES ©) i (c) (0) Se ei re Y Chapter besides the active site. mes are known as allosteric enzymes. The ites are unique places on the enzyme molec Allosteric effectors : Certain substances referred to as allosteric modulators (effectors or modifiers) bind at the allosteric site and regulate the enzyme activity. The enzyme activity is increased when a pasitive (+) allosteric effector js at the allosteric site known as activator site. On the other hand, a negative (-) allosteric effector binds at the allosteric site called inhibitor site and inhibits the enzyme activity. Classes of allosteric enzymes : Enzymes that are regulated by allosteric mechanism are referred to as allosteric enzymes, They are divided into two classes based on the influence of allosteric effector on Kr and Vinax- « Keclass of allosteric enzymes, the effector changes the K,, and not the V; Double plots, similar to competitive ion are obtained e.g. phospho- fructokinase. + Veclass of allosteric enzymes, the effector alters the Veg, and not the K,,, Double reciprocal plots “resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase. allost Conformational ch enzymes The subunits may be identical or different. The norecovalent reversible binding of the effector molecule at the allosteric site brings about a conformational 101 change In the active site of the enzyme, leading © ihe inhibition or activation of the catalytic activity (Fig.6.13). In the concerted model, alesteric enzymes exist in two conformational sees the T (tense or taut) and the R (relaxed). he T and R states are in equilibrium. -y Mette actvator (1) substrate Sa Allosteric inhibitor Allosteric inhibitors favour T state whereas activators and substrates favour R state, The substrate can bind only with the R form of the enzyme, The concentration of enzyme molecule in the R state increases as more substrate is added, therefore the binding of the substrate to the allosteric enzyme is said to be cooperative. Allosteric enzymes give a sigmoidal curve (instead of hyperbola) when the velocity (v) versus substrate(S) concentration are plotted (Fig.6.14). The term homotropic effect is used if the substrate influences the substrate binding through allosteric mechanism, their effect is always positive. Heterotropic effect is used when an allosteric modulator effects the binding of substrate to the enzyme. Heterotropic interactions are either positive or negative. Selected examples of allosteric enzymes responsible for rapid control of biochemical pathways are given in Table 6.5. a © scanned with OKEN ScannerUe eee ae ee Enzyme Hexokinase Glycolysis Phosphotructokinase Glycolysis Isocitrate dehydrogenase robs cycle Pyruvate carboxylase Gluconeogenesis Fructose 1, 6- bisphosphatase Gluconeogenesis Carbamoyl phosphate synthetase! Urea cycle ‘Tryptophan oxygenase ‘Tryptophan metabolism = — E Acetyl CoA carboxylase Fatty acid synthesis Palmitate Metabolic pathway Re matey Feedback regulation The process of inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation. Look at the series of reactions given below AStyp Scape A is the initial substrate, B, C, and D are the intermediates and E is the end product, in a pathway catalysed by four different enzymes (€;, €2, €3, 4). The very first step (A > B by the enzyme e;) is the most effective for regulating the pathway, by the final end product E. This type of control is often called negative feedback ation since increased levels of end product ill result in its (e;) decreased synthesis. This is real cellular economy to save the cell from wasteful expenditure of synthesizing a which is already available within the inhibition or end product inhibition type of allosteric inhibition ‘control metabolic pathways for inction. moylase (ATCase) is f an allosteric enzyme back mechanism. ATCase st reaction in pyrimidine Feedbacl Kk gedback Carbamoy! aspartate + Pi Cytidine triphosphate (CTP) Carbamoy! phosphate undergoes a sequence of reactions for synthesis of the end product, CTP. When CTP accumulates, it allostericall inhibits the enzyme aspartate transcarbamoylase by a feedback mechanism. Feedback regulation or feedback inhibition? Sometimes a distinction is made between these two usages. Feedback regulation represents 4 the mechanism of regulation. Thus, in a tue sense, they are not synonymous. For instance, dietary cholesterol decreases hepatic cholestero biosynthesis through feedback regulation. This does not involve feedback inhibition, since) dietary cholesterol does not directly inhibit t regulatory enzyme HMG CoA reductase, However, the activity of gene encoding this enzyme is reduced (repression) by cholesterol. 2. Activation of latent enzymes i enzymes are synthesized as proenzymes zymogens which undergo irreversible cov © scanned with OKEN Scanneractivation by the breakdown of one or more peotide bonds. For instance, proenzymes name chymotrypsinogen, pepsinogen and plasminogen, are respectively ~ converted tothe active ensyic, chymotrypsin, pepsin and plasmin. Certain enzymes exist in the active and inactive forms which are interconvertible, Jepending on the needs of the body. the nterconversion is brought about by the reversible covalent modifications, namely »hosphorylation and dephosphorylation, and oxidation and reduction of disulfide bonds, Glycogen phosphorylase is a muscle enzyme at breaks down glycogen to provide energy, is enzyme is a homodimer (two identical subunits) and exists in two interconvertible forms. Phosphorylase b (dephospho enzyme) is inactive which is converted by phosphorylation of serine residues to active form phosphorylase a, The ‘nactive enzyme phosphorylase b is produced on dephosphorylation as illustrated below. ea nos 103 F There are some enzymes which are active in lephosphorylated state and become inactive When phosphorylated e.g. glycogen synthase, acetyl CoA carboxylase, A few enzymes are active only with sulthydryl (SH) groups, e.g. succinate dehydrogenase, urease. Substances like glutathione bring about the stability of these enzymes. E-SH+E-SH Reduced GS-SG active ‘enzyme 3. Compartmentation There are certain substances in the body (e.g., fatty acids, glycogen) which are synthesized and also degraded. There is no point for simultaneous ‘occurrence of both the pathways. Generally, the synthetic (anabolic) and breakdown (catabolic) pathways are operative in different cellular organelles to achieve maximum economy. For instance, enzymes for fatty acid synthesis are found in the cytosol whereas enzymes for fatty acid oxidation are present in the mitochondria. Depending on the needs of the body — through the mediation of hormonal and other controls — fatty are either synthesized or oxidized. The intracellular location of certain enzymes and metabolic pathways is given in Table 6.6. Organelle Enzyme/metabolic pathway a ‘Aminotranserases; peptidases, glycolysis; hexose monophosphate shunt; Be purine and pyiicine catabolsm. ; : Fatly acid oxidation; amino acid oxidation; Krebs cyte; urea synthess Mitochondria fae chain ad oxidative phosphor, : Nucleus ae ramet Endoplasmic reticulum (microsomes) OO ————— Cl © scanned with OKEN Scanner104 4. Control of enzyme synthe: Most of the enzymes, particularly the rate limiting ones, are present in’ very low concentration. Nevertheless, the amount of the enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate limiting enzymes have short halflives. This helps in the efficient regulation of the enzyme levels. There are two types of enzymes—(a) Consti- tutive enzymes (house-keeping enzymes)—the levels of which are not controlled and remain fairly constant. (b) Adaptive enzymes—their concentrations increase or decrease as per body needs and are well-regulated. The synthesis of enzymes (proteins) is regulated by the genes (Refer Chapter 26). Induction and repression : The term induction is used to represent increased synthesis of enzyme while repression indicates its decreased synthesis. Induction or repression which ultimately determines the enzyme concentration at the gene level through the mediation of hormones or other substances. Examples of enzyme induction : The hormone insulin induces the synthesis of glycogen synthetase, glucokinase, phosphofructokinase and pyruvate kinase. All these enzymes are involved in the utilization of glucose, The hormone cortisol induces the synthesis of many enzymes e.g. pyruvate carboxylase, tryptophan. oxygenase and tyrosine aminotransferase. Examples of repression : In many instances, substrate can repress the synthesis of enzyme. Pyruvate carboxylase is a key enzyme in the synthesis of glucose from non-carbohydrate sources like pyruvate and amino acids. If there is sufficient glucose available, there is no necessity for its synthesis. This is achieved through repression of pyruvate carboxylase by glucose. 5. Enzyme degradation Enzymes are not immortal, since it will create series of problems. There is a lot of variability the half-lives of individual enzymes. For some, is in days while for others in hours or in e.g. LDH4—5 to 6 days; LDH; —8 to amylase — 3 to 5 hours. BIOCHEMIS In general, the key and regulatory er are most rapidly degraded. If not needed, immediately disappear and, as and required, they are quickly sysnthesized. not always true, an enzyme with long half-li usually sluggish in its catalytic activity, Isoenzymes Multiple forms of the same enzyme will al help in the regulation of enzyme activity, of the isoenzymes are tissue-specific. Al isoenzymes of a given enzyme catalyse the reaction, they differ in Km, Vmax or both. isoenzymes of LDH and CPK. Cee rade Enzymes are never expressed in terms of t concentration (as mg or pg etc.), but expressed only as activities. Various metho enzyme activities (particularly for the plas enzymes). In fact, the activities have expressed in many ways, like King-Armstro units, Somogyi units, Reitman-Frankel unit spectrophotometric units etc. In order to maintain uniformity in expression of enzyme activities (as worldover, the Enzyme Commission of 1UB ha suggested radical changes. A new unit —n. katal (abbreviated as kat) — was introduced. kat denotes the conversion of one substrate per second (mol/sec). Activity may alst be expressed as millikatals (mkat), micro (kat) and so on. International Units (IU) Some workers prefer to use standard units SI units (System International). One St unit 0 International Unit (WU) is defined as the of enzyme activity that catalyses the conversia of one micromol of substrate per minute. Si units and katal are interconvertible. © scanned with OKEN ScannerChapter 6 : ENZYMES 1 = (or) 1 nkatal = 60 pkatal 1.67 1U Laboratory use of enzyme units the clinical laboratories, however, the units— namely katal or SI units—are yet to find a place. Many investigators still use the old units like King-Armstrong units, Somogyi units etc. while expressing the enzyme activities, It is therefore, essential that the units of enzyme activity, along with the normal values, be variably stated while expressing the enzymes for comparison. iis) its ra gsc} Ribozymes Ribozymes are a group of ribonucleic acids that function as biological catalysts, and they are regarded as non-protein enzymes. Altman and his coworkers, in 1983, found that ribonuclease P— an enzyme till then known to cleave precursors of RNAS to give tRNAs — was functional due to RNA component present in the enzyme and not the protein part of the enzyme. The RNA part isolated from ribonuclease P exhibited a true enzyme activity and also obeyed Michaelis-Menten kinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis. RNA molecules are known to adapt a tertiary structure just as in the case of proteins (i.e. enzymes). The specific conformation of RNA may be responsible for its function as biocatalyst. It is believed that ribozymes (RNAs) were functioning as catalysts before the occurrence of protein enzymes during evolution. APPLICATIONS OF ENZYMES Certain enzymes are useful as therapeutic agents, analytical reagents, in _ genetic manipulations and for industrial applications (Table 6.7). 105 icra) COO Re etd of enzymes ‘Application To remove biood clots In cancer therapy Antiinflammatory Totreat emphysema. (breathing citficuity due 4 f a to distension of lungs) Analytical application reagents (for estimation) Glucose oxidase and peroxidase Glucose Urease Urea Cholesterol oxidase Cholesterol ‘Uricase ‘Uric acid Upase Triacyiglycerols Luciferase To detect bacterial contamination of foods ‘Alkaline phosphatase! In the analytical technique horse radish peroxidase ELISA ‘Applications in genetic engineering Restriction endonucleases Gene transfer, DNA finger printing Taq DNA polymerase Polymerase chain Enzymes as therapeutic agents 1. Streptokinase prepared from streptococcus is useful for clearing the blood clots. Streptokinase activates plasma plasminogen to plasmin which, in turn, attacks fibrin to. convert into soluble products. Plasminogen Plasmin Sis eae (ct) © scanned with OKEN Scanner106 Se is used in the 2. the enzyme Fare cels are dependent leukem™'he host's plasma for their tes By administering asparaginase, the an le of asparagine are drastically depression in the viability treatment of on asparagine of th multiplic host’s plasma level reduced. This leads to d Enzymes as analytical reagents Some enzymes are useful in the clinical laboratory for the measurement of substrates, drugs, and even the activities of other enzymes. The biochemical compounds (e.g. glucose, urea, uric acid, cholesterol) can be more accurately and specifically estimated by _ enzymatic procedures compared to the conventional chemical methods. A good example is the estimation of plasma glucose by glucose oxidase and peroxidase method Immobilized enzymes Enzymes can be used as catalytic agents in industrial and medical applications. Some of these enzymes are immobilized by binding them to a solid, insoluble matrix which will not affect the enzyme stability or its catalytic activity. Beaded gels and cyanogen bromide activated sepharose are commonly used for lization of enzymes. The bound enzymes preserved for long periods without loss of ‘oxidase and peroxidase, immobilized ‘on a strip of paper, are used in the tory for the detection of glucose in Noi | iuconic acid + HO, H,0 Oxidized toluidine (blue colour) of the blue colour depends on of glucose. Hence, the strip for semi-quantitative estimation BIOCHEMIS PTET Cela em aL OF ENZYMES Estimation of enzyme activities in biologica fluids (particularly plasma/serum) is of gr clinical importance. Enzymes in the circulati are divided into two groups ~ plasma functi and plasma non-functional. 1. Plasma specific or plasma functional enzymes Certain enzymes are normally present in perform. Generally, these enzyme ac higher in plasma than in the tissues. They are mostly synthesized in the liver and enter t circulation e.g. lipoprotein lipase, plasmin, thrombin, choline esterase, ceruloplasmin etc, Impairment in liver function or ger disorders often leads to a fall in the activi plasma functional enzymes e.g. deficiency ceruloplasmin in Wilson's disease. 2. Non-plasma specific or plasma non-functional enzymes These enzymes are either totally absent present at a low concentration in pl compared to their levels found in the tissues The digestive enzymes of the gastrointest tract (e.g. amylase, pepsin, trypsin, lipase et present in the plasma are known as enzymes. All the other plasma et associated with metabolism of the cell collectively referred to as constitutive (eg. lactate dehydrogenase, transaminases, and alkaline phosphatases, creatine phospl kinase). Estimation of the activities of non-ph specific enzymes is very important for diagnosis and prognosis of several diseases. The normal serum level of an ena) indicates the balance between its synthesis and release in the routine cell turnover, The raisé enzyme levels could be due to cellular dami increased rate of cell turnover, proliferation 0 cells, increased synthesis of enzymes etc, © scanned with OKEN ScannerENZYMES > eee rae) Serum enzyme (elevated) Amylase ‘Serum glutamate pyruvate transaminase (SGPT) ‘Serum glutamate oxaloacetate transaminase (SGOT) Alkaline phosphatase ‘Acid phosphatase Lactate dehydrogenase (LDH) Creatine phosphokinase (CPK) Aldolase ‘5’-Nucleotidase +y-Glutamy/! transpeptidase (GGT) enzymes are conveniently used as markers to detect the cellular damage which ultimately helps in the diagnosis of diseases. ‘A summary of the important enzymes useful for the diagnosis of specific diseases is given in Table 6.8. Detailed information on the diagnostic enzymes including reference values is provided in Table 6.9. A brief account of selected diagnostic enzymes is discussed Amylase : The activity of serum amylase is increased in acute pancreatitis (normal 80-180 Somogyi units/dl). The peak value is observed within 8-12 hours after the onset of disease which returns to normal by 3° or 4'% day. Elevated activity of amylase is also found in urine of the patients of acute pancreatitis. Serum amylase is also important for the diagnosis of chronic pancreatitis, acute parotitis (mumps) and obstruction of pancreatic duct. Alanine transaminase (ALT/SGPT) : SGPT is elevated in acute hepatitis of viral or toxic origin, jaundice and cirrhosis of liver (normal 3- 40 IU/). Aspartate transaminase (AST/SGOT) + SGOT activity in serum is increased in myocal infarction and. also in liver diseases (normal 4-45 IU), ont Disease (most important) ‘cite pancreatitis Uver diseases (hepatitis) Hear attacks (myocardial infarction) Rickets, obstructive jaundice Canoet of prostate gland Heart attacks, Iver diseases ‘Myocardial infarction (early marker) Muscular dystrophy Hepatitis Alcoholism It may be noted that SGPT is more specific for the diagnosis of liver diseases while SGOT is for heart diseases. This is mainly because of their cellular distribution-SGPT is a cytosomal enzyme while SGOT is found in cytosol and mitochondria. Alkaline phosphatase (ALP) : It is elevated in certain bone and liver diseases (normal 3-13 KA units/dl). ALP is useful for the diagnosis of rickets, hyperparathyroidism, carcinoma of bone, and obstructive jaundice. ‘Acid phosphatase (ACP) : It is increased in the cancer of prostate gland (normal 0.5-4 KA units/dl). The tartarate labile ACP (normal <1 KA units/dl) is useful for the diagnosis and prognosis of prostate cancers ie. ACP is a good tumor marker. Lactate dehydrogenase (LDH) : LOH is useful for the diagnosis of myocardial infarction, infective hepatitis, leukemia and muscular dystrophy (serum LDH normal 50-200 IU/). LDH hus five isoenzymes, the details of which are described later. Creatine kinase (CK) + It is elevated in m) infarction (early detection) and muscular dystrophy (normal 10-50 1U/). CK has three isoenzymes (described laten. © scanned with OKEN Scanner