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Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------Enzyme: protein catalysts that increase the rate of reactions without being changed in the overall process. Occurrence: Enzymes are produced by all living organisms including humans and present only in small amounts. Medical and biological importance of enzymes 1. Enzymes are the chemical work horses of the body. Enzymes are biological catalysts that speed up the pace of chemical reactions. 2. A chemical reaction without an enzyme is like a drive over a mountain. The enzyme bores a tunnel through it so that passage is far quicker and takes much less energy. 3. Enzymes make life on earth possible, all biology from conception to the dissolution that follows death depends on enzymes. 4. Enzymes regulates rate of physiological process. So, defects in enzyme function cause diseases. 5. When cells are injured enzymes leak into plasma. Measurement of activity of such enzymes in plasma is an integral part of modern day medical diagnosis. 6. Enzymes are used as drugs. 7. Immobilized enzymes, which are enzymes attached to solid supports are used in clinical chemistry laboratories and in industry. For example glucose in blood or urine is detected by using immobilized glucose oxidase. In pharmaceutical industry, glucose isomerase is used to produce fructose from glucose. 8. Enzymes are used as biosensors. 9. AIDS detection involves use of enzyme dependent ELISA technique. 10. Enzymes are used as cleansing agents in detergent industry. Classification of enzymes 1. Oxidoreductases: Transfer of hydrogen or addition of oxygen; e.g. Lactate dehydrogenase (NAD); Glucose-6-phosphate dehydrogenase (NADP); Succinate dehydrogenase (FAD); di-oxygenases. 2. Transferases: Transfer of groups other than hydrogen. Example, Aminotransferase. (Subclass: Kinase, transfer of phosphoryl group from ATP; e.g. Hexokinase) 3. Hydrolases: Cleave bond and add water; e.g. Acetyl choline esterase; Trypsin 4. Lyases: Cleave without adding water, e.g. Aldolase; HMG CoA lyase; ATP Citrate lyase. (Subclass: Hydratase; add water to a double bond) Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------5. Isomerases: Intramolecular transfers. The include racemases and epimerases. Example, Triose phosphate isomerase. 6. Ligases: ATP dependent condensation of two molecules, e.g. Acetyl CoA carboxylase; Glutamine synthetase; PRPP synthetase Enzymes Lowering of Activation Energy i. Enzymes lower the energy of activation. ii. Activation energy is defined as the energy required to convert all molecules of a reacting substance from the ground state to the transition state. Active site 1. Two parts: (a) Catalytic site. It is the portion (part) of the enzyme that is responsible for catalysis. It determines reaction specificity. Occasionally, catalytic site and active site are used synonomously. (b) Binding site. It is the part of the enzyme that binds with substrate. It determines substrate specificity. (Fig). 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. E+S E–S Complex E + P Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------2. The active sites of enzyme are clefts within the enzyme molecule. For example, the active site of ribonuclease lies within cleft 3. Active site consists of few amino acid residues only. 4. Active site is three dimensional. 5. The active site is contributed by amino acid residues that are far apart in the enzyme molecule. During catalysis, they are brought together. 6. The amino acids at the active site are arranged in a very precise manner so that only specific substrate can bind at the active site. 7. Usually serine, histidine, cysteine, aspartate or glutamate residues make up active site. Enzymes are named according to the active site amino acid. For example, trypsin is a serine protease and papain is cysteine protease. Factors effect on enzyme activity 1. concentration of enzyme As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. 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. Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------Enzyme kinetics and Km value : The enzyme(E) and substrate( S) combine with each other to form an unstable enzyme-substrate complex (ES) for the formation of product (P). K1 K3 E + S --------------> E--S ---------------> E + P K2 If concentration of substrate is increased, the forward reaction K1 is increased, and so K3 as well as total velocity is correspondingly enhanced. The three different constants may be made into one equation, Km = K2 + K3 K1 Km is called as Michaelis Constant. It is further shown that Mechaelis-Menten equation: Velocity (v) = Vmax [S] Km + [S] When concentration of substrate is made equal to Km, i.e. When [S] = Km Velocity (v) = Vmax [S] = Vmax [S] = Vmax [S] + [S] 2 [S] 2 or v = ½ Vmax 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 Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------4. Effect of pH increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained. 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. Enzyme inhibitors 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 inhibition. 2. Irreversible inhibition. 3. Allosteric inhibition 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 l. Competitive inhibition (Fig.) ll. Non-competitive inhibition Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------- Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------- CO-ENZYMES i. Enzymes may be simple proteins, or complex enzymes, containing a non-protein part, called the prosthetic group. The prosthetic group is called the co-enzyme. It is heat stable. ii. The protein part of the enzyme is then named the apo-enzyme. It is heat labile. iii. These two portions combined together is called the holo-enzyme. Enzymes 4th lecture 2ndclass biochemistry Vet.Med.Collage --------------------------------------------v. Co-enzymes may be divided into two groups v-a. Those taking part in reactions catalyzed by oxidoreductases by donating or accepting hydrogen atoms or electrons. v-b. Those co-enzymes taking part in reactions transferring groups other than hydrogen Examples of co-enzymes Co-enzyme Thiamine pyrophosphate (TPP) Pyridoxal phosphate (PLP) Biotin Coenzyme-A (Co-A) Tetra hydrofolate (FH4) Adenosine triphosphate (ATP) Group transferred Hydroxy ethyl Amino group Carbon dioxide Acyl groups One carbon groups Phosphate Co-factors The term co-factor is used as a collective term to include co-enzymes and metal ions. Co-enzyme is an organic and co-factors is non-organic. Metal Enzyme containing the metal Zinc Carbonic anhydrase, carboxy peptidase, alcohol dehydrogenase Magnesium Hexokinase, phospho fructo kinase, enolase, glucose-6-phosphatase Manganese Phospho gluco mutase, hexokinase, enolase, glycosyl transferases Copper Tyrosinase, cytochrome oxidase, lysyl oxidase, superoxide dismutase Iron Cytochrome oxidase, catalase, peroxidase, xanthine oxidase Calcium Lecithinase, lipase