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Enzymology Enzymes are biological catalysts which bring about chemical are present in very small amounts in various cells . Almost all the functions of the body such as digestion , breathing , synthesis and breakdown of carbohydrates m 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 catalysed by enzymes . The substance upon which an enzymes acts is called the substrate . The enzyme will convert the substrate into the product or products . The enzymes are generally named by adding the suffix-ase to the name the substrate . Thus the enzyme lactase acts on the substrate lactase and products glucose and galactose are formed . All enzymes are proteins . Enzymes follows the physical and chemical reaction of proteins . They are heat labile , soluble in water , precipitated by protein precipitating reagents ( Ammonium sulphate or trichloroacertic acid ) and contain 16 % weight nitrogen . General Properties of Enzymes 1.All enzymes are proteins . 2. They accelerate the reaction , but a. do not alter the reaction equilibrium b. not consumed in overall reaction c. required only in very small quantities. 3. They have enormous power for catalysis . 4. Enzymes are highly specific for their substrate . 5. Enzymes possess active sites at which interaction with substrate takes place . 6. Enzymes lower activation energy . 7. They form substrate complex as intermediates during their action 8. Some enzymes are regulatory in function . Classification of Enzymes As per the International Union of Biochemists , enzymes are divided into six major classes . Oxidoreductases One compound oxidized , another reduced , e.g. tyrosinase , urease , lactic hydrogenase , catalase and peroxidase . Transferase This class of enzymes transfers group containing C , N, or S from the substrate to another substrate substrate . They are important in is , biological synthesis , e.g. transaminases , hexokinases transcylases , transaldolases . Hydrolases They catalyse hydrolysis of esters , ether , peptide or glycosidic bond by addition of water molecules across the bond which is split , e.g. esterases , peptidases . Lyases They catalyse the addition or removal pf groups without hydrolysis , oxidation or reduction m producing double bonds at times , e.g. decarboxylases , carboxylases , carbonic anhydrase . Isomerases These can produce optical , geometric or position isomers of substrates by intermolecular rearrangement , e.g. reacemases, epimerases , isomerases . Ligases These enzymes also called synthetases link two substrates together usually with the linking of pyrophosphate bound in ATP ( Ligare = to bind ) , e.g. glutamine synthetase . ENZYME SPECIFITY 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. Sterospecificity : Some enzymes show specificities only with a specific group of a substrate , e.g. urease catalyses the hydrolysis of urea . O ║ Urease H 2N –C-NH2 + 2H2O (NH4)2 CO3 The catalysis dose not take place when structure of urea is altered , e.g. N methhyyl urea thiourea are not hydrolysed by urrase . O S ║ ║ H2N –C-NH-CH3 H2N-C-NH2 N-Methyl Urea Thiourea D – amino acid – oxidase acts only on the D- form of amino acid and not on L- form . 2. Substrate specificity : Some enzymes catalyes similar type of reaction but differ in their action due to substrate specificity , e.g. (a) Pepsin hydrolyses peptide bonds involving aromatic amino acids like phenyl alanine and tyrosine . (b) Trypsin hydrolyses peptide bonds involving carboxyl group of basic amino acids like arginine or lysine . 3. Reaction specificity : A substrate can undergo many reactions but in reaction specificity , one enzyme can catalyse only one of the various reaction . For example , oxalic acid can undergo different reactions , but each of these reactions is catalysed by separate enzyme . Mechanism of Enzyme Acids According to Michaelis and Menten , the enzyme molecule (E) first combines with a substrate molecule (S) to form an enzyme 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 if free to combine with another molecule of the substrate . The site at with a substrate can meet with the enzyme molecule is extremely specific and is called active site or catalytic site . Normally the molecule size and shape of the substrate molecule is extremely small compared to the enzymes molecules . The active site is made up of several amino acid residues that come together as a result of the folding 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 Enzymes Activity Substrate Concentration At a low substrate concentration the initial velocity of an enzymes 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 assumes a constant rate as a result of enzymes 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 the reaction is not proceeding at maximum velocity whereas when the enzymes is saturated with substrate , maximum velocity is observed . The enzymes 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 substrate proportional to the enzyme – substrate complex . The velocity of the reaction at this high substrate concentration is termed as maximum velocity (Vmax) . The velocity is called the Michael's constant (Km) . Km indicates the affinity of the substrate towards the enzymes and is inversely proportional to the affinity . 1 Km = α Affinity Higher the affinity the smaller will be the Km and lower the affinity , the higher will be the Km ( Fig .7.1) . The Michaelis – Menten equation is given by the expression Vmax [S] V= Km + [S] Where V = Initial velocity Vm= Maximum velocity Km = Michaela's constant [S] = Substrate concentration When the initial velocity is exactly half the maximum velocity , Vmax [S] Km + [S] 2 Km + [S] = 2[S] 1 Vmax = Km = [S] VMAX V E L O C I T Y 1/ 2 vmax Substrate concentration Fig . 7.1 : Enzyme activity and substrate concentration Therefore , Km is equal to substrate concentration at which the velocity is half the maximum . Effect of Enzyme concentration The rate of an enzyme catalysed reactioly is direct proportional to the concentration of the enzyme. The greater the concentration of enzyme , the faster will be reaction taking places( Fig 7.2). Effect of pH The enzyme activity is maximum at a particular pH which is called the optimum pH . This id due to the change in the net Reaction rate Substrate concentration Fig . 7.2: Enzyme activity and enzyme concentration Charge on enzymes resulting from change in pH. Excessive changes in pH brought on by addition of strong acids or bases may completely and inactivate enzymes ( Fig. 7.3) . Optimum pH Reaction rate 0 7 14 pH Fig .7.3. : Enzyme activity and pH Effect of Temperature The velocity of enzyme reaction increase when temperature of the medium is increased ; reaches a maximum and then falls . The temperature at which maximum amount of substrate is converted to the product per unit time is called the optimum temperature ( Fig . 7.4). Reaction rate Denaturation Optimum Temperature 0 20 40 60 Fig.7.4: Enzyme activity and temperature As temperature is increased, more molecules get activation energy, or molecules are at increased rate of motion, and so their collision probabilities along with the reaction rate is increased. Above this temperature the reaction rate decreases as enzymes being protein in nature are denatured by heat and becomes inactive. Effect of Time The time required for completion of an enzyme reaction increases with decrease in temperature from its optimum. Under the opti-mum conditions of pH and temperature, time required for enzyme reaction is less. Enzyme Inhibition Enzyme are proteins and they can be inactivated by the agents that denature them. The chemical substances which inactivate the enzymes are called as inhibitors and the process is called enzyme inhibition. Most of the substances commonly referred to as poisons are harmful in 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 The inhibitor molecule is a structural analog of the normal substrate of the enzyme, i.e. 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 bound to the inhibitor, the enzyme is not a catalyst. Since both are competing for combination with same active site, the term competitive inhibition is often used. An important example is provided by the sulfa drugs. These are structural analongs of paraaminobenzoic acid, a compound required by many bacteria for certain metabolic processes but which is not used by higher 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. NH2 NH2 O=S=O C=O OH p-aminobenzoic Acid NH2 Sulfanilimide Non-Specific Inhibition Every protein molecule has a number of reactive groups present on side chains of the constituent amino acids, groups such as –CO2H, -SH, -NH2, etc. 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 to a number of protein groups in particular –SH and –CO2H at the active site inhibiting the normal catalytic. 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-compe-titive in nature. CO-ENZYMES Many enzymes require 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, function as cosubstances. Usually, it binds loosely and can be easily separated from its enzyme by dialysis, but when 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. The term apo-enzyme refers to the protein part of the enzyme. The apo-enzyme with its prosthetic group (or co-enzyme) consti-tute a complete enzyme or holoenzyme. Conjugated protein enzyme protein + prosthetic group Or Holoenzyme Apo-enzyme + co+enzyme =Protein part + non-protein part (prosthetic group) Classification of Co-enzyme Co-enzymes for group transfer Co-enzymes for transfer of H 1.ATP and its relatives 1. NAD+ , NADP+ 2. Sugar phosphates 2. FMN, FAD 3. CoA 3. Lipoic acid 4. Thiamine pyrophosphate 4. Co-enzyme Q 5. B6 phosphate 6. Folate co-enzyme 7. Biotin 8. Cobamide (B12) Co-enzyme 9. Lipoic acid 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 Vitamins Thiamine (B1) Co- enzymes Thiamine pyrophosphate TPP Riboflavin (B2) Flavin mononucleotide ( FMN) Flavin adenine dinucleotide (FAD) Pyridoxal phosphate Pyridoxine(B3) Niacin (B3) Din Pantothenic acid Biotin E Types of reaction Decarboxylation of α - keto acid certain reaction of ketosugars Several kind of Oxidation – reduction reactions. Several kinds of Reactions involving Amino acids , e.g. decarboxylation transamination. Nicotinamide adenine Numerous oxidation dinucleotide ( NAD+) - reduction reaction Nicotinamide adenine dinucleotide phosphate (NADP+) co-enzyme 'A' Enzyme bound biotin Many reaction of fatty Acids , particularly those involving transfer of acetyl groups . Certain carbon dioxide Folic acid Tetrahydrofolic acid Cyanoco-balamine Several " Cobamide " co-enzymes fixation reactions. Various reactions involving single carbon compounds Carbon chain Isomerizations , certain methyl group transfers. 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 . ( See Table 7.2) . Table 7.2: Increase of Different Enzymes in Diseases Enzymes Amylase Acid Phosphatase(Optimum pH = 5 Alkaline phosphatase ( Optimum pH=10 Aspartate transaminase AST ( previously GOT ) Alanine transaminase ALT ( previously GPT) Lactase dehydrogenase LDH Creatine kinase (CK) Γ Glutamyl transferase (γGT) Urinary Elevation Increased in Acute pancreatitis Prostatic carcinoma. Liver disease , rickets . Myocardial infarction. Liver disease especially with liver cell damage Myocardial infarction but also increased in liver disease and some blood disease . Myocardial infarction and skeletal muscle disease ( muscular dystrophy , dermatomytomyositis ) . Diagnosis of liver diseases , particularly biliary obstruction and alcoholism . N acetyl glucosaminidase in the urine can be used to indicate renal transplant rejection .