enzyme.pptx

enzyme

1. Factors Affect Rate of Enzyme Action DR VISESH KUMAR

2. Factors Affect Rate of Enzyme Action  Enzyme concentration  Substrate concentration  Temperature  pH  Concentration of coenzymes  Concentration of ion activators  Time  Inhibitors

3. Factors affecting the rate of enzyme action 1- Effect of enzyme concentration The rate of enzyme action is directly proportional to the concentration of enzyme provided that there are sufficient supply of substrate & constant conditions .

4. 2- Effect of substrate concentration - The rate of reaction increases as the substrate concentration increases up to certain point at which the reaction rate is maximal ( Vmax .) At Vmax , the enzyme is completely saturated with the substrate any increase in substrate concentration doesn't affect the reaction rate .

5. A plot of reaction velocity versus substrate concentration Here varying amounts of substrate are added to a fixed amount of enzyme. The reaction velocity is measured for each substrate concentration and plotted. The resulting curve takes the form of a hyperbola (a mathematical function in which the values initially increase steeply but eventually approach a maximum level).

6. Initial velocity(V ) Measured at the very beginning of a reaction when very little P has been made Enzyme present in nanomolar quantities, whereas [S] may be five or six orders of magnitude higher

7. At relatively low concentrations of substrate , V increases almost linearly with an increase in [S] At higher substrate concentrations ,V increases by smaller and smaller amounts in response to increases in [S] A point is reached beyond which increases in V are vanishingly small as [S] increases This plateau-like V region is close to the maximum velocity, Vmax . The kinetic pattern led Victor Henri, to propose in 1903 that the combination of an enzyme with its substrate molecule to form an ES complex is a necessary step in enzymatic catalysis This idea was expanded into a general theory of enzyme action, particularly by Leonor Michaelis and Maud Menten in 1913 .

8. They postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step The ES complex then breaks down in a slower second step to yield the free enzyme and the reaction product P: Because the slower second reaction must limit the rate of the overall reaction, the overall rate must be proportional to the concentration of the species that reacts in the second step, that is, ES the pre–steady state , during which the concentration of ES builds up The reaction quickly achieves a steady state in which [ES] remains approximately constant over time. The concept of a steady state was introduced by G. E. Briggs and Haldane in 1925

9. Steady State The more ES present, the faster ES will dissociate into E + P or E + S. Therefore, when the reaction is started by mixing enzymes and substrates, the [ES] builds up at first, but quickly reaches a STEADY STATE, in which [ES] remains constant. This steady state will persist until almost all of the substrate has been consumed.

10. Michaelis Menten equation: describe the rate of  steady state enzymatic reactions with relation to the  concentration of a substrate Where, S = substrate E = enzyme ES = enzyme-substrate concentration P = product k 1 , k -1 and k 2 are rate constants 

11. Where, v o = initial reaction velocity V max = maximal velocity Km = Michaelis constant = (k 2 + k -1 )/ k 1 [S] = substrate concentration Criteria for Michaelis kinetics: Steady state reaction: rate of formation of [ES] equal rate of breakdown of [ES] i.e. concentration of [ES] does not change Concentration of substrate is much greater than the concentration of enzyme Initial velocity, v o measured as soon as enzyme and substrate are mixed i.e. v o is dependent on [S] and Km.

12. Michaelis – Menten Kinetic theory M & M model accounts for the kinetic properties of some enzymes. It helps to describe many enzymatic reactions under the following assumptions: The reaction has only one substrate The substrate concentration is much higher than that of the enzyme Only the initial velocity is measured.

13. Michaelis constant (Km) - It is the substrate concentration that produces half maximum velocity of enzyme

14. v o dependent on [S] and Km When [S] <<< Km , Km + [S] equivalent to Km, v o = Vmax [S] Km since Vmax and Km are constant, v o α [S] 2. When [S] >>> Km , Km + [S] equivalent to [S] v o = Vmax 3. When [S] = Km , v o = Vmax/ 2

15. Importance of Michaelis Menten Kinetics 1. Characteristics of Km: Km is characteristic of an enzyme and its particular substrate , reflects the enzyme affinity Numerically Km equal to substrate concentration at which reaction velocity is equal to ½ Vmax . Km does not vary with enzyme concentration Small Km reflects high affinity between enzyme and substrate High Km reflects low affinity between enzyme and substrate

16. Effect of substrate concentration on reaction velocities for two enzymes: enzyme 1 with a small K m , and enzyme 2 with a large K m

17. Lineweaver Burk Plot In Michaelis Menten equation, when v o is plotted against [S] , as the curve is hyperbolic it is hard to calculate Vmax . If equation is reciprocated and the graph is plotted, a straight line is obtained . This plot is called Lineweaver Burk plot or Double reciprocal plot. Can be used for calculating Km and Vmax , as well as to determine the mechanism of action of enzyme inhibitors .

18. Measuring Km and V max Curve-fitting algorithms can be used to determine Km and Vmax from v vs. [S] plots Michaelis-Menton equation can be rearranged to the “double reciprocal” plot and Km and Vmax can be graphically determined

19. Temperature The effect of temperature on reaction rate is due to: 1- Increase of temperature increase the initial energy of substrate and thus decrease the activation energy 2- Increase of collision of molecules: more molecules become in the bond forming or bond breaking distance.

20. After the optimum temperature, the rate of reaction decrease due to denaturation of the enzyme (60-65 C).

21. 4- Effect of PH - Each enzyme has an optimum PH at which its activity is maximal • E.g. Optimum PH of pepsin = 1.5 - 2 • Optimum PH of pancreatic lipase = 7.5 - 8 • Optimum PH of salivary amylase = 6.8

22. Change of PH above or below optimum PH decrease rate of enzyme action due to: 1- The enzyme activity depends on the ionization state of both enzyme and substrate which is affected by PH. 2- Marked change in PH will cause denaturation of enzyme.

23. 5- Concentration of coenzymes : In the conjugated enzymes that need coenzymes, the increase in the coenzyme concentration will increase the reaction rate

24. 6- Concentration of ion activators : The increase in metal ion activator increase the reaction rate Enzymes are activated by ions: 1- Chloride ion activate salivary amylase 2- Calcium ion activate thromobokinase enzyme

25. 7- Effect of time: • In an enzymatic reaction, the rate of reaction is decreased by time. • This is due to: 1- The decrease in substrate concentration. 2- The accumulation of the end products. 3- The change in PH than optimum PH.

26. 8- Presence of enzymes inhibitor: Presence of enzyme inhibitor decreases or stops the enzyme activity.

27. Enzyme Inhibiton Any substance that can diminish the velocity of an enzyme catalyzed These include drugs, antibiotics, poisons, and anti-metabolites. Useful in understanding the sequence of enzyme catalyzed reactions, metabolic regulation, studying the mechanism of cell toxicity produced by toxicants. Forms the basis of drug designing.

28. Types of Enzyme Inhibiton Reversible inhibitors Irreversible inhibitors

29. Reversible inhibitors can be classified into : Competitive Non-competitive Un-competitive

30. Classes of Inhibition Two real, one hypothetical Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition

31. Competitive inhibitor: Inhibitor binds reversibly at same site of substrate; compete with substrate Inhibition is reversed by increasing [S] Inhibitor increases the apparent Km , but Vmax is not affected E.g.: Statin drugs inhibit HMG CoA Reductase Inhibitor malonate competes with succinate for succinate dehydrogenase

32. Competitive Inhibition

33. A. Effect of a competitive inhibitor on the reaction velocity ( v o ) versus substrate ([S]) plot. B. Lineweaver -Burk plot of competitive inhibition of an enzyme.

34. Lovastatin competes with HMG- CoA for the active site of HMG- CoA reductase .

35. Succinate dehydrogenase Reaction Malonate is a competitive inhibitor

36. A competitive inhibitor

37. Non competitive inhibition: Inhibitor and substrate binds at different sites Inhibitor binds either free enzyme or [ES] complex Vmax decreases as inhibition cannot be overcome by increasing [S] Km is not affected as inhibitor does not interfere substrate binding to enzyme Eg .: Ferrochelatase catalyses Fe 2+ insertion into protoporphyrin during Heme synthesis Lead is the non competitive inhibitor

38. Non-Competitive Inhibition

39. A. Effect of a noncompetitive inhibitor on the reaction velocity ( vo ) versus substrate ([S]) plot. B. Lineweaver -Burk plot of noncompetitive inhibition of an enzyme.

40. Un-competitive Inhibiton Binds only to the enzyme-substrate complex. Does not have the capacity to bind to the free enzyme. Not overcome by increasing substrate concentration. Both the K m and V max are reduced.  

41. Uncompetitive inhibition Uncompetitive inhibitors bind only the ES complex , not free enzyme (E). Both Vmax and Km are affected by inhibitor, [I]. Km is affected because the ESI complex prevents E and S from dissociating. Vmax is affected because the ESI complex can’t form product (ES is trapped as ESI at saturating substrate concentration).

42. Un-competitive Inhibiton Enzyme ES Complex + Inhibitor ESI complex

43. Irreversible inhibition Inhibitor act irreversibly by chemically modifying the enzyme involve formation of covalent bonds between inhibitor and aminoacyl residues on enzymes , Covalent changes are relatively stable ; and enzyme remains inactive even after removal of inhibitor from surrounding medium E.g.: Heavy metal, etc.

44. Irreversible inhibition Inhibition of Cyclooxygenase (COX)by aspirin. Iodoacetate , heavy metal ions, oxidising agents form covalent bonds with functional group of enzymes. Suicide Inhibition is a type of irreversible inhibition.

45. Mechanism based/ Suicide inhibition Suicide inhibitors, specialized substrate analogs , that are transformed into a more highly reactive group by the target enzyme Forms covalent bond and blocks the function of a catalytically active residue leading to irreversible inactivation of the enzyme. Basis for development of enzyme specific drugs Eg : Allopurinol used for gout/ hyperuricemia Competes with hypoxanthine for xanthine oxidase Allopurinol converted into more potent inhibitor, alloxanthine

46. Irreversible Inhibitor: Allopurinol