WEL-COME

1. WEL-COME

2. DHANAJI NANA MAHAVIDYALAYA,FAIZPURDEPARTMENT OF MICROBIOLOGYEnzyme InhibitionAssi.Prof.Rupali B . Sali

3. Enzyme Inhibition There are several motivations to study enzyme inhibition: • Distinguish among the different potential mechanisms in multisubstrate reactions • Relative binding affinity of competitive inhibitors  active site structure research in the absence of 3-D structure info • Control mechanisms in biology: balance of protease NZs and their inhibitors in a tissue  help to achieve homeostasis • Commercial applications: • Insecticides, weed killers • Drugs

4. Enzyme Inhibition • Since most clinical drug therapy is based on inhibiting the activity of enzymes, analysis of enzyme inhibition kinetics is fundamental to the modern design of pharmaceuticals • Well-known examples of such therapy include the use of methotrexate in cancer chemotherapy to semi-selectively inhibit DNA synthesis of malignant cells • the use of aspirin to inhibit the synthesis of prostaglandins which are at least partly responsible for the aches and pains of arthritis • the use of sulfa drugs to inhibit the folic acid synthesis that is essential for the metabolism and growth of disease-causing bacteria .

5. Enzyme Inhibition • Reversible inhibition: the effect of an inhibitor can be reversed by decreasing the concentration of inhibitor • Irreversible inhibition: there is no reversal of inhibition on decreasing the inhibitor concentration: an example of enzyme inactivation • e.g. Cyanide: by covalently binding mitochondrial cytochrome oxidase, it inhibits all the reactions associated with electron transport • Penicillin for bacterial peptidase • The distinction between reversible and irreversible inhibition is not absolute and may be difficult to make if the inhibitor binds very tightly to the enzyme and is released very slowly (tight-binding inhibitors)

6. Mixed Inhibition • Always possible • Not possible with uncompetitive inhibitors • Not possible with competitive inhibitors • Not possible with competitive or uncompetitive inhibitors A noncompetitive inhibitor is capable of all four reactions, but the classical noncompetitive inhibitor, as opposed to a mixed one, is a special case. With these inhibitors Ks and Ks' are equal to each other, as are Ki and Ki'

7. When [E]0<< [I]0: Mixed Inhibition

8. Simplified cases: Competitive inhibition • Both the substrate and inhibitor compete for binding to the same form of the enzyme: free form • ESI complex is not formed • The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] • Vmax remains unaffected • Attaining Vmax requires higher [S] in the presence of competitive • inhibitor • Apparent Km is increased

9. Kapp Simplified cases: Competitive inhibition To obtain accurate data: • At [S] = Km  check broad range of [I] • Choose [I] that yield between 30 % to 75 % inhibition • Measure vo values as a function of [S] at several fixed [I]

10. Simplified cases: Competitive inhibition • Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the reversal of enzyme inhibition • like all kinds of reversible inhibitors, a decreasing concentration of the inhibitor reverses the equilibrium • since substrate and competitive inhibitors both bind at the same site, raising [S], while holding [I] constant, provides the second route for reversal of competitive inhibition Examples of competitive inhibitors 2,3-biphosphoglycerate • Inhibits its own formation by inhibiting biphosphoglycerate mutase • Metabolic regulation by product inhibition

11. Examples of competitiveinhibitors Sulphonamides: widely used in medicine to limit bacterial growth Antifreeze: ethylene glycol competes for active site of alcohol dehydrogenase • Ethylene glycol is metabolized to oxalic acid, which crystallizes in kidneys and causes renal failure • Can be treated by alcohol infusion • A new treatment, approved by the FDA in December 1997, uses the alcohol dehydrogenase inhibitor 4-methylpyrazole (trade name = Antizol). Unlike ethanol, 4-methylpyrazole is not a substrate for the enzyme, is therefore not metabolized

12. Simplified cases: Noncompetitive inhibition • Since noncompetitive inhibitors do not interfere in the binding of the substrate (the dissociation constant of ES and ESI have the same value Ks) • Km is not affected • However, increasing [S] can not abolish the inhibition  (ESI) complex are formed and these are incapable of progressing to reaction products • The effect of a noncompetitive inhibitor is to reduce [ES] that can advance to product • Since Vmax = k2[Et], and the concentration of competent Et is diminished by the amount of ESI formed • Vmax is decreased

13. Examples of noncompetitive inhibitors • Heavy metals like lead, mercury (breaks disulfide bonds), chromium will act as non-competitive inhibitors • Mono-amine oxidase (MAO) inhibitors that are used as anti-depressants: They covalently react with the enzyme in the liver and effectively remove it. • There are many potent drug interactions with MAO inhibitors. One of these is tyramine, a compound that is present in red wine and aged cheeses • MAO inhibitors and tyramine (blocks neurotransmitter reuptake in the brain)  hypertensive crisis • Patients are still subject to hypertension for as long as two weeks after discontinuing the drug

14. THANK YOU….