CHAPTER Enzvmology ) Enzymes are biological catalysts which bring about chemical reactions in living cells. They are produced by: the. living organism and dre present in very small amounts in various cells. * Almost all the functions of the body such as digestion, breathing, synthesis and breakdown of carbohydrates, fats and proteins are catalysed and controlled by specific enzymes. Most chemical reactions of the living cells would have occurred very slowly had it not been catalysed by enzymes. The substance upon which an enzyme acts is called the si. strate. The enzyme will convert the substrate into the product ‘or products, The enzymes are generally named by adding the suffix-ase to the name of the substrate. Thus, the enzyme lactase acts on the substrate lactose and the products glucose and galactose are formed. All enzymes are proteins. Enzymes follow the physical and chemnical reaction of proteins. ‘They are heat labile, soluble in water, precipitated by protein _ precipitating reagents (Ammonium sulphate or trichloroacetic acid) and contain 16% weight nitrogen. es ees Cene : | Properties of Enzymes ss 1, All enzymesare proteins. 2. They accelerat the reaction, but a. Donotalter the'reaction equilibrium > b. Notconst md in ovérall reaction c. R quired only in very small quantities, + 3. They have enormous power for catalysis. © 4 -Epzymes are highly specific for their substrate. 8, Enzymes possess active sites at which interaction w trate takes place. 6. Eanes Jower activation energy, ‘7, They form $ substrate complex as intermediates during their action. “8, Some enzymes 47 regulatory in function, usEnzymology 95 Classification of Enzymes As per the International Union of Biochemists, enzymes are divided into six major classes. ‘Lactate dehydrogenase ee ey, NAO NADH#H* Oxidoreductases: One compound oxidised, another reduced, e.g., tyrosinase, urease, lactic dehydrogenase, catalasé and peroxidase. Lactate Pyruvate Transferase: This class of enzymes transfer group containing C,N or from the substrate to another substrate. They are important in biological synthesis, e.g.,’ transaminases, hexokinases transcy- Jases, transaldolases. Serine dehydrogenase Methyl transferase Hydrolases: They catalyse hydrolysis of esters, ether, peptide or -glycosidic bond by addition of water molecules across the bond which is split, é.g., esterases, peptidases. Urea + H,O Urease CO, + 2NH. _ Urea + H,0 Urease CO;+2NH, Serine. Glycine Lyases: They catalyse the addition or removal of groups without hydrolysis, oxidation or’ reduction, producing double bonds at times, €.g., decarboxylases, carboxylases, carbonic anhydrase. Pyruvi ate Pyruvate ———— Acetaldehyde + CO, Dehydrogenase Isomerases: These can produce optical, geometric or position isomers of substrates by intermolecular rearrangement, e.g,, reace- mages, epimerases, isomerases, Methyl Malonyl Coa M85 succinyl CoA Ligases: These enzymes also called synthetases link two Substrates together usually with the linking of pyrophosphate Pound in ATP (Ligare = to bind), e.g, glutamine synthetase.96 Nutrition and Biochemistry for Nurses Pyruvate Carboxylase —_—_— Pyruvate Oxaloacetate ATP ADP#+Pi ENZYME SPECIFICITY Some enzymes are very specific and show activity with only one substrate. Some others are much less particular. Generally three types of enzymatic specificities are observed. 1. Stereospecificity: Some enzymes show specificities only with 2 specific group of a substrate, e.g., urease catalyses the hydrolysis of urea. . ° H,N-C—NH, + 2H,0-———> (NH), COs The catalysis does not take place when structure of urea is altered, eg, N-methyl urea and thiourea are not hydrolysed by urease. ° : s Ml ‘ i H.N-C-NH-CH, . H.N=C-NH, N-Methyi Urea Thiourea D-amino acid-oxidase acts only on the D-form of amino acid. and not on L-forni. 2. Substrate specificity: Some enzymes catalyse similar type of reaction but differ in their action due to substrate specificity, eg. ~ (a) Pepsin hydrolyses peptide bonds involving aromatic amine acids like phenyl alanine: and tyrosine. (b) Trypsin hydrolyses peptide bonds involving carboxyl groups of basic aming acids like arginine or lysine. 3, Reaction specificity: A substrate can undergo many reactions but # reaction specificity, one enzyme can catalyse only one of the various reactions, For example, oxalic acid can undergo different reactions ut each of these reactions ee separate enzymes. Mechanism of Enzyme Action \_ _ According to Michaelis and Menten, the enzyme molecule (8) Gist combines with a substrate molecule ($) to forny an ensyeneEnzymology 97 7 substrate (ES) complex which further dissociates to form product (P) and enzyme (E) back, " E+S @ES—E + products Enzymes once dissociated from the complex is free to combine with another molecule of the substrate. The site at which a substrate can meet with the enzyme molecule is extremely specific and is called active site or catalytic site. Normally the molecular size and shape of the substrate molecule is extremely small compared to the enzyme molecules. The active site is made up of several amino acid residues that come together as a result of the foldings of secondary and tertiary structures of the enzymes. So the active site possesses a complex three dimensional form and shape, provides a, predominantly non-polar cleft or crevice to accept and bind the” substrate. Factors Affecting Enzyme Activity oo Substrate Concentration Ata low substrate concentration the initial velocity of an enzyme catalysed reaction is proportional to the substrate concentration. However, as the substrate concentration is. increased, the initial velocity increases less as it is no‘longer proportional to the substrate concentration. With a further increase in the substrate concentration and the velocity «ssumes a constant rate as a result of enzyme being saturated with its substrate. ‘It was Michaelis and Menten who suggested an explanation for these findings “by. postulating that at low substrate “concentrations; the enzyme is not saturated with the substrate and. thé ‘reaction is not proceéding at maximum velocity, whereas, when the enzyme is saturated with substrate, maximum velocity is observed. The enzyme combines with the substrate to. form an enzyme-substrate complex and the rate of decomposition of the substrate is proportional to the enzyme- substrate complex. The velocity of the reaction ‘at this high substrate concentration is termed as maximum velocity (Vinax) * The substrate concentration at which the velocity is half of the maximum velocity ‘is called the Michaelis’ constant (Km)-,Km indicates the affinity of the substrate towards the enzyme and is inversely proportional to the affinity.98 Nutrition and Biochemistry for Nurses x4-00rm< ‘Substrate concentration Fig: 7.1: Enzyme activity and substrate concentration Kye ciaaiy * Higher the affinity the smaller svi be the Ky anid dower the + affinity, the higher will be the K,, (Fiz. 7.1). The Michaelis - Menten equatior: is, en by the eprsion VmnaxlS] Vo +15) Where Ve Vo . = Initial velocity : = Maximum velocity e = Michaelis constant '[5] = Substrate coricentration When the initial velocity is exactly half the maximum velocity, Vous [S] Me = OHS Kp + [5] = 2 [5} K, = [5] ‘Therefore, K,, is equal to substrate concentration at which the velocity is half the maximum.knsymnoloyy 9% Enzyme concentration | __ Fig. 7.2: Enzyme activity and enzyme concentration Effect of Enzyme Concentration The rate of an enzyme catalysed reaction is directly proportional to the concentration of the enzyme. The greater the concentratlon of enzyme, the faster will be reaction taking place (Fig.7.2). Effect of pH The enzyme activity is maximum at a particular pH which is called the optimum pH. This is due to the changes in the net Fig. 7.3: Enzymé activity and pH4100. Nui ton and Biochemistry for Nurses charge on enzymes resulting from changes in pH. Excessive changes in p4 broughi on by addition of strong acids or bases may ‘ompletely denature and inactivate enzymes (Fig. 7.3). Sect of Temperature The velocity of enzy:ae reaction increases when temperature of the medium is increased; reaches a maximum and then falls. The temperature at which maximum amount of substrate is converted tu she product per-unit time’is called the optimum temperature we T@. As tensperature is increased, more molecules get activation eaergy, or molecules are at increased rate of motion, and so their probabilities along with the reaction rate is increased. aAdeve this temperature the reaction rate decreases as enzymes 7 protein in nature are denatured by heat and becomes inactive. . . "Effect of Time ‘The tine required for completion of an enzyme reaction increases with decrease i * -aperature from its optimum. Under the opti- mun. conditions of »H «nd temperature, time required for enzyme reaction 1s less Eazyme Irh biton Enzymes 7*j cms nd they can be inactivated by the agents thetdenature [Link] al substances which inactivate the LT Fi,. 7.4: Enzyme activity and temperaturerymology 101 | enzymes are called as inhibitors and the process is called enzyme inhibition. Most of the substances commonly referred to-as poisons are harmf«[Link] that they inhibit one or more essential enzymes. The inhibitors may be classified in two broad groups. First, compounds or ions which are specific in their effect, inhibiting only one enzyme or several closely related enzymes. And second, substances which are nonspecific, inhibiting many enzymes. Specific Inhibition Th. inhibitor molecule is a structural’ analog of the normal substrate of the enzyme, ie., it is chemically similar to the substrate. The inhibitor is capable of combining with the active _ site by virtue of its similar structure. ‘As long as the active site is” ‘. hound to the inhibitor, the enzyme is nota catalyst. Since, both are * competing for combination with the same active site, the term competitive inhibition (Fig. 7.5) is often used. i ‘An important example is provided by the sulfa drugs. These are structural analogs of para-aminobenzoic acid, a compound | required by many bacteria for synthesising their food (Vitamin) _ | tetrahydrofolic’acid (THFA) but which is not used by higher E NH NH, | Geo O=8=0 i OH NH, *e ee ‘paminobenzolc Acid. Sulfaniimide a i Fig. 7.5: Competive Inhibition 4 organisms such as man. The sulfa drugs inhibit growth of the |. bacteria in man by competing with the paraaminobenzoic acid for |. the active site of some bacterial enzyme. Non-Specific inhibition : : Every protein molecule ha3 a number of reactive groups present on side chains of the constituent amino acids, groups such402 ‘Nutrition and Biochemistry for Nurses as - CO,H, ~ SH, - NH;, ete. Any substance capable of combining with a common group of this type is a potential inhibitor of all. Heavy metal ions are non-specific inhibitors. They can bind toa number of protein groups in particular -SH and -CO,H at the active site inhibiting the normal catalytic activity. In sufficient concentration, heavy metal ions will inhibit most enzymes and are therefore poisonous to all living things. However, some of these heavy metal ions Cu**, Zn**, Co** are absolute requirements in low concentrations for cells as cofactors for a variety of enzymes. Thus, classification of a metal ion as poison depends _ on its concentration. Mostly non-specific inhibition is non- competitive in nature. a CO-ENZYMES Many enzymes tequire the presence of small non-protein organic molecules for their efficient performances. Only when both enzy- me and co-enzyme are present catalysis will occur. Co-enzymes are low molecular weight, non-protein organic compounds that are heat resistant, and function as cosubstances. Usually, it binds loosely [Link]‘be a easily separated from its enzyme by dialysis, but wheri it binds tightly, it is considered as a prosthetic group of the enzyme. Co-factor differs from a co-enzyme only because it usually is a metallic ion (Fe**, Mg**, Zn**, Cu**) rather than an organic molecule. Water soluble vitamins (Vit B complex and Vit C) from Co-enzymes. . The term apo-enzyme refers to the protein part of the enzyzhe. The'apo-enzyme with its prosthetic group (or co-enzyme) consti- tute a complete enzyme or holoenzyme. Conjugated Protein enzyme —_—_— Protein + Proathetic group or. Holoenzymez=—=—== Apo-enzyme + coenzyme = Protein part + non-protein part (prosthetic group) Classification of Co-enzyme Co-enzymes for group transfer" Co-enzymes for transfer of H _ of groups other than HYoeNourenn ATP and its relatives Sugar phosphates CoA Thiamine pyrophosphate Bg phosphate Folate co-enzyme Biotin. Cobamide (B,) Co-enzyme Lipoic acid. Enzymology 103 1, NAD*, NADPt 2. FMN, FAD 3. Lipoic acid 4, Co-enzymeQ Water Soluble Vitamins as Co-Enzymes _ Vitamins of B complex and vitamin C (Ascorbic acid) act as co-enzymes as shown in Table 7.1 Table 7.1: Relationship along water-soluble vitamins, their co-enzyme forms and metabolic reactions in which they participate Vitamins (Co-enzymes Types of reactions Thiamine (B,) Thiamine pyrophosphate TPP Decarboxylation of.a-Keto acid, certain reactions of ketosugars. Riboflavin (B,) Flavin mononucleotide (FMN) Several kinds of oxidation- Flavin adanine dinucleotide (FAD) " Ryridéxine (B,) Pyridoxal: phosphate " Niacin (B) Nicotinamide adenine E ~ dinucleotide. (NAD*), ‘Nicotinamide adenine, dinucleotide phorphate (NADP*) Pantothenic co-enzyme' ‘A’ acid Biotin Enzyme bound biotin Folic ad _—‘Tetrahydrofolic add Cyanoco-.’ Several “Cobamide” eduction reactions. Several kinds of reactions involving amino adds, og. decarboxylation, traneamination Numerous axidationrabatinn reactioris. Many reactions of fatty tS particularly those RWONR transfer of acetyl grays Certain carbon dhukte ROARS reactions, \ Various reactions OAR ‘single carbon aaynnnets Cabo chains iawieesien eens balamine coeazy! cortaln invetty! gone RAYSREI 404 Nutntion and Biochemistry for Nurses —————— TT Diagnostic Value of Plasma Enzymes When a tissue is injured some cells of that tissue are destroyed and their contents including enzymes are released into the blood stream. The increase of enzymes in blood stream will indicate the disease (Table 7.2). Table 7.2: Increase of Different Enzymes in Diseases Enzymes ‘ Increased in Amylase Acute pancreatitis. Acid Phosphatase Prostatic carcinoma. (Optimum pH = 5 : Alkaline Phosphatase Liver disease, rickets. (Optimum pH = 10) Aspartate transaminase - Myocardial infarction. AST (previously GOT) Alanine transaminase Liver disease especially with liver cell damage. ALT (previously GPT) Z Lactate dehydrogenase LDH Myocardial infarction but also increased in liver disease and some blood disease. Creatine kinase (CK) Myocardial infarction and skeletal muscle diseases (muscular dystrophy, dermatemyesits;- 1 1 Ghtamy transferase (7 GT) Diagnosis of liver disease, particularly biliary : obstruction and alcoholism. Urinary Elevation’ N-acetyl glucosaminidase in the urine can be used to indicate renal transpiast rejection. :