Enzymes PDF

Enzymes  INTRODUCTION Enzymes are biocatalysts – the catalysts of life. Catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process. Enzymes may be defined...

Enzymes  INTRODUCTION Enzymes are biocatalysts – the catalysts of life. Catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process. Enzymes may be defined as biocatalysts synthesized by living cells, that have the property of accelerating specific chemical reactions without being consumed in the process. They are protein in nature (exception – RNA acting as ribozyme), thermolabile in character, and specific in their action.  Enzymes Enzymes are proteins that increase the rate of reaction by lowering the energy of activation. Present in all living cells. They catalyze nearly all the chemical reactions taking place in the cells of the body by converts substrates into products. Remain unchanged by chemical reaction (Not consumed). They are globular proteins. Highly selective, (usually specific in action). They can be denatured by physical and /or chemical agents and they loose their biological function. Rate of chemical reaction: It is the change in the amount (moles, grams) of starting materials (substrates) or products per unit time. Substrate: It is the substance upon which the enzyme acts and convert it to product.  Cellular distribution of enzymes Enzymes are sometimes considered under two broad categories (Distribution according to their site of action) (a) Intracellular enzymes: Synthesized and act inside the cells e.g. metabolic enzymes. (b) Extracellular enzymes: These enzymes are Produced inside the cells and act outside the cells e.g. digestive enzymes.  Nomenclature and Classification In most cases, enzyme names end in –ase, the suffix-ase. was added to the substrate for naming the enzymes e.g. lipase acts on lipids; nuclease on nucleic acids; lactase on lactose. Some names are historical. For example, the names pepsin, trypsin and chymotrypsin. These are known as trivial names of the enzymes which, however, fail to give complete information of enzyme function (type of reaction, or the nature of the substrate on which they act cofactor requirement etc.)  The International Union of Biochemistry (IUB) system of enzyme classification has been in force. Enzymes are divided into six major classes (Systematic name). 1. Oxidoreductases : Enzymes involved in oxidation- reduction reactions. 2. Transferases : Enzymes which catalyze transfer of functional groups other than hydrogen. 3. Hydrolases : These enzymes act by splitting (cleavage) of a certain bond by adding water. 4. Lyases : Addition or removal of a chemical group other than by hydrolysis. 5. Isomerases : They catalyze the interconversion of two isomers. 6. Ligases : These enzymes link two molecules using energy from ATP.  Enzyme structure and activity Enzymes are either simple or conjugated proteins. If the enzyme is a simple protein, only the native conformation of the protein is required for activity. If the enzyme is a conjugated protein, it is called: holoenzyme and its activity will depend upon: a) Conformation of the protein which is called apoenzyme. b) The availability of a non protein part which is called cofactor (Either organic(Coenzyme) or inorganic). Holoenzyme Apoenzyme + Cofactor (active enzyme) (protein part) (non-protein part)  The Important coenzymes 1· Hydrogen carriers: a) NAD and NADP. b) FAD and FMN. c) Lipoic acid. d) Coenzyme-Q. 2- Carriers of groups other than hydrogen: Most of them are derived from vitamins as vit B complex. Coenzyme A(Co-A) : acyl group carrier. Thiamine pyrophosphate (TPP): C02 and ketone group carrier. Biotin : C02 carrier. Pyridoxal phosphate (PLP) : (-NH2 ) group carrier. Folic acid : one carbon group carrier. Cobalamine : methyl group carrier.  CHEMICAL NATURE OF ENZYMES: 1. Monomeric enzyme: it is made up of a single polypeptide e.g. ribonuclease, trypsin. 2. Oligomeric enzymes: the enzymes which possess more than one polypeptide (subunit) chain e.g. lactate dehydrogenase, aspartate transcarbamoylase etc. 3. Multienzyme complexes: possessing specific sites to catalyze different reactions in a sequence.  Zymogens or Proenzymes Zymogens or proenzyme are Inactive (precursor) form of enzymes. Zymogens are inactive because their catalytic sites are masked by a polypeptide chain. Activation of zymogen, into active enzyme is done at the time of action, by removal of the polypeptide chain to open the catalytic site for its substrate. Examples of zymogens: are pepsinogen and trypsinogen.  Factors affecting enzyme activity The important factors that influence the velocity of the enzyme reaction are : 1. Concentration of enzyme As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. (There is a linear relationship between reaction rate and enzyme concentration ) Effect of enzyme concentration on enzyme velocity 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 hyperbolic curve is obtained when velocity is plotted against the substrate concentration. Effect of substrate concentration on enzyme velocity (A-linear; B-curve; C-almost unchanged)  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. The temperature at which the enzyme activity is maximum is called optimum temperature. Any strong change in the optimum temperature results in the loss of enzyme activity. Optimum temperature of enzymes in the human body is 37°C. The optimum temperature for most of the enzymes is between 35°C–40°C. 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. most enzymes are denatured and become permanently inactive at 55°C - 70°C. Effect of temperature on enzyme velocity 4. Effect of pH Increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained. The optimal pH for enzyme activity is that pH at which the enzyme acts maximally. Above or below this pH, the ionic state of both enzyme and substrate will be changed, and the rate of reaction will therefore decrease. Extremes of pH can also lead to denaturation of the enzyme. Effect of pH on enzyme velocity. Each enzyme has its own optimal pH e.g. - Salivary amylase 6.8. - Pepsin 2 - Trypsin 8 - Alkaline phosphatase 8.4 -  5. Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity. For certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity. 6. Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for their optimum activity. Rarely, anions are also needed for enzyme activity e.g. chloride ion (Cl–) for amylase. 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 (Mg2+ and Ca2+) Enolase (Mg2+) Metalloenzymes: These enzymes hold the metals rather tightly which are not readily exchanged. e.g. alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase, carboxypeptidase and aldolase contain zinc. Phenol oxidase (copper); Pyruvate oxidase (manganese); Xanthine oxidase (molybdenum); Cytochrome oxidase (iron and copper). 7. 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. ACTIVE SITE The active site (or active center) of an enzyme represents as the small region at which the substrate(s) binds and participates in the catalysis. A diagrammatic representation of an enzyme with active site  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. Two general classes of inhibitors are recognized according to whether the inhibitor action is: 1. Reversible inhibition. 2. Irreversible inhibition. 1. 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 I. Competitive inhibition ( Fig. A) II. Non-competitive inhibition ( Fig. B) A diagrammatic representation of (A) Competitive and (B) Non-competitive inhibition. I. Competitive inhibition : The inhibitor (I) 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 EI complexes are formed as shown below. The inhibition could be overcome by high substrate concentration. In competitive inhibition, the Km value increases whereas Vmax remains unchanged. Sulfanilamide and p-aminobenzoic acid (PABA): PABA is required for synthesis of folic acid in bacteria. Folic acid is essential for bacterial growth and multiplication. Sulfanilamide acts as competitive Inhibitor for enzyme system that uses PABA for synthesis of folic acid and acts as antibacterial drug II. 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 resemblance 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 relation in non-competitive inhibition is represented below For non-competitive inhibition, the Km value is unchanged while Vmax is decreased Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non- competitively inhibit the enzymes by binding with cysteinyl sulfhydryl groups. The general reaction for Hg2+ is shown below 2. Irreversible inhibition The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usually toxic substances that poison enzymes. Iodoacetate is an irreversible inhibitor of the enzymes like papain and glyceraldehyde-3-phosphate dehydrogenase. Iodoacetate combines with sulfhydryl ( SH) groups at the active site of these enzymes and makes them inactive. Diisopropyl fluorophosphate (DIFP) is a nerve gas developed by the Germans during Second World War. DIFP irreversibly binds with enzymes containing serine at the active site, e.g. 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 The penicillin antibiotics act as irreversible inhibitors of serine – containing enzymes, and block the bacterial cell wall synthesis. Cyanide inhibits cytochrome oxidase (binds to iron atom) of electron transport chain. Fluoride inhibits enolase (by removing manganese), and thus glycolysis. 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. Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthine, a more effective inhibitor of this enzyme.  Regulation of enzyme activity: 1. Regulation by allosteric enzyme: Allosteric enzyme is a regulatory enzyme. Like all enzymes, allosteric enzymes have active site for binding of the substrate but they also have one or more regulatory (or allosteric) sites for binding regulatory metabolites which is called modulator. Allosteric enzymes may be inhibited or stimulated by their modulators. Regulation of enzyme activity: 2. Regulation by covalent modification: Many enzymes may be regulated by covalent modification, mostly by phosphorylation or dephosphorylation. Regulation by covalent modification is slower than allosteric regulation. 3. Regulation by induction and repression of enzyme synthesis: The increased (induction) or decreased (repression) synthesis of the protein (enzyme) results in alteration of total number of active sites. The alteration of enzyme levels due to induction or repression in protein synthesis takes more time (hours to days) compared to that of allosteric regulation.  Isoenzymes: Isoenzymes or isozymes are multiple forms of the same enzyme that catalyze the same biochemical reaction. Isoenzymes differ in their physical and chemical properties which include the structure, electrophoretic and immunological properties, Km and Vmax values, pH optimum, relative susceptibility to inhibitors and degree of denaturation. For example: 1. Lactate dehydrogenase (LDH) 2. Creatine kinase (CK) (formerly called creatine phosphokinase (CPK)  ENZYME in Clinical Diagnosis For the right diagnosis of a particular disease, it is always better to estimate a few (three or more) serum enzymes, instead of a single enzyme. Examples of enzyme patterns in important diseases are given here 1. Enzymes in Myocardial Infarction (MI) The elevation of these enzymes in serum in relation to hours/days are important for the diagnosis of MI: 1. creatine phosphokinase (CPK). 2. aspartate transaminase (AST). 3. lactate dehydrogenase (LDH). Creatine phosphokinase (CK or CPK) (precisely isoenzyme) is the first enzyme to be released into circulation within 6-18 hours after the infarction. Therefore, CPK estimation is highly useful for the early diagnosis of MI. This enzyme reaches a peak value within 24-30 hours, and returns to normal level by the 2nd or 3rd day. Aspartate transaminase (AST or SGOT) rises sharply after CPK, and reaches a peak within 48 hours of the myocardial infarction. AST takes 4-5 days to return to normal level. Lactate dehydrogenase (LDH1) generally rises from the second day after infarction, attains a peak by the 3rd or 4th day and takes about 10-15 days to reach normal level. Thus, LDH is the last enzyme to rise and also the last enzyme to return to normal level in MI. Enzyme pattern in myocardial infarction (CPK-Creatine phosphokinase; SGOT- Serum glutamate oxaloacetate transaminase; LDH-Lactate dehydrogenase) Enzymes in liver diseases The following enzymes—when elevated in serum – are useful for the diagnosis of liver dysfunction due to viral hepatitis (jaundice), toxic hepatitis, cirrhosis and hepatic necrosis 1. Alanine transaminase(ALT or GPT) 2. Aspartate transaminase 3. Lactate dehydrogenase(LDH5). The enzymes that markedly increase in intrahepatic and extrahepatic cholestasis are : 1) Alkaline phosphatase, 2) 5’-Nucleotidase. Serum-γ-glutamyl transpeptidase is useful in the diagnosis of alcoholic liver diseases Enzymes in muscle diseases In the muscular dystrophies, serum levels of certain muscle enzymes are increased. These include: 1) Creatine phosphokinase, 2) Aldolase 3) Aspartate transaminase. Enzymes in cancers Increase in the serum acid phosphatase (tartarate labile) is specific for the detection of prostatic carcinoma. [Note : Prostate specific antigen (PSA) though not an enzyme, is a more reliable marker for the detection of prostate cancer. Normal serum concentration of PSA is 1-4 ng/ml] Neuron–specific enolase serves as a marker for lung cancer, neuroblastoma. Bone disease Alkaline phosphatase (ALP) Increased in rickets disease Pancreatic disease 1) Amylase Significantly elevated in acute pancreatitis. 2) Lipase Markedly increased in acute pancreatitis.