Enzymes

1. Enzymes Dr. s.chakravarty MD

2. Learning objectives Define and classify enzymes based on the IUPAC agreement. Give examples to each class Classify cofactors and give examples for various types Discuss the general properties of enzymes and list the important functions Define KM, Vmax, Tranistion state and activation energy of enzymes Discusss the factors affecting enzyme activity Explain the MichaelisMenten reaction and differentiate it from lineweaverburk plot. Classify various types of enzyme inhibitions Mention the diagnostic importance of enzymes

3. Definition Thermolabile biocatalysts which enhances a chemical reaction without undergoing any chemical change. Properties : Specific for a reaction Does not dictate the direction of a reaction Increases the rate of a reaction by several thousand times Are proteins – except ribozymes (RNA) Lowers the activation energy of a reaction

4. Enzyme terminology Simple enzyme – made only of proteins Complex enzyme – also called Holoenzyme Apoenzyme – protein part Non-protein part - Prosthetic group –with covalent interaction ( usually metals) co-enzyme – without covalent interaction (vitamins )

5. Co-enzymes • Group I: take part in reactions transferring hydrogen atoms or electrons

6. Co-enzymes Group II: take part in reactions transferring groups other than hydrogen

7. Enzymes with metals Metalloenzyme – metal is the indegenous part of the enzyme itself. Seperation causes disruption Metal activated enzymes – required for activation but not indegenous to the enzyme.

8. Metalloenzymes

9. Classification of enzymes: based on function Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases

10. Catalysis occurs at the active site

11. Features of active site Occupies only a small portion of the whole enzyme Situated in a crevice or cleft of the enzyme Possesses a substrate binding site & a Catalytic site The substrate binds at the active site by weak noncovalent bonds The amino acids usually found at the active site are serine, lysine, histidine, arginine, cysteine

12. Theories explaining the binding of substrate to the enzyme Fischer’s template theory (lock and key model of enzyme attachment : According to this theory the active site of the enzyme is rigid. only a specific substrate complementary to the active site fits to it just as a key fits to its proper lock

13. Koshland’s induced fit theory: According to this theory the active site is not rigid & pre- shaped. The interaction of the substrate with the enzyme induces a conformational change at the active site so that proper alignment of the catalytic residues occur & the substrate fits in the active site

14. + ES Complex + E P + ES Complex + + E P E S E S Fischer’s template theory + E S E S Koshland’s Induced Fit Theory

15. Mechanism of action • Lowering of activation energy • Activation energy is defined as the energy required to convert all molecules of a reacting substance from ground state to transition state • Higher the activation energy ,slower the reaction & vice versa

16. Activation energy

17. Mechanism of action A A* B [Transition state] Reactant [ground state] Product Activation energy (Ea)

18. Factors affecting rate of a reaction Transition state /activation energy: Collision theory : for two molecules to react they must come as close as bond forming distances and collide with sufficient kinetic energy. Kinetic energy can be increased by: Increasing temperature Probability of bond formation can be increased by increasing substrate concentration

19. Factors affecting the rate of enzyme catalyzed reaction • Enzyme concentration • Substrate concentration • Temperature • PH • Product concentration • Presence of activators & Inhibitors

20. 1. Effect of enzyme concentration [E] on velocity of reaction [v]        Enzymeconcentration

21. 2. Substrate concentartion As the substrate concentration is increased the velocity also correspondingly increased in the initial phases but the curve flattens afterwards. • A rectangular hyperbola is obtained

22. Vmax Km ½ Vmax Substrate concentration

23. Substrate concentration on the reaction rate : First order reaction At low substrate concentration the velocity of the reaction is directly proportional to the substrate concentration. This is first order reaction Zero order reactions At high concentration the velocity of the reaction is independent of substrate concentration. This is zero order reaction

24. 3. Effect of temperature The velocity of enzyme reaction increases when the temperature is increased reaches a maximum & then falls The temperature at which maximum amount of substrate is converted to the product per unit time is called the optimum temperature

25. Effect of temperature Increase in velocity of the reaction Increase in temperature results in high activation energy of the molecules & more molecular collision & interaction for the reaction to proceed faster Decrease in velocity of the reaction When the temperature is increased more than the optimum temperature Denaturation of the enzyme occurs

26. 4. Effect of pH The pH decides the charge on the amino acid residues at the active site. The net charge on the enzyme would influence the substrate binding & catalytic activity

27. 5. Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity. The products combine with the active site of the enzyme & form a loose complex & thus inhibit the enzyme activity.

28. 6.Factors affecting enzyme activity Activators: Substances that increases the enzyme activity. Eg : Zinc activates carbonic anhydrase NAD+ activates LDH Inhibitors: substances that decreases the enzyme activity Eg : Fluoride inhibits Enolase. Cyanide inhibits cytochromeoxidase.

29. Specificity of enzyme activity Sterospecificity Reaction specificity Substarte specificity – Absolute Relative Broad

30. Reaction specificity The same substrate can undergo different type of reaction. Each reaction is catalysed by a separate enzyme PDH LDH Pyruvate Acetyl coA Lactate Carboxylase Transaminase Malic enzyme Oxalo acetate Alanine Malate

31. Substrate specificity Absolute specificity : Enzymes that can act only on one substrate & can catalyse only one reaction Eg : Glucose Glucose-6-phosphate Group specificity : Carboxy peptidase & amino peptidase are exopeptidase which hydrolyse peptide bond in the vicinity of free COOH or NH2 groups respectively Broad specificity :Hexokinase acts on Glucose, Fructose, Mannose etc Glucokinase

32. Michaelis –menten graph Enzyme concentration is kept constant. As the substrate concentration increases the velocity increases but after some conc.. The graph flattens due to enzyme saturation. The highest point on the graph is called Vmax. X- axis – substrate concentration Y-axis – velocity of the reaction (V) Vmax= maximum velocity a reaction can reach. Directly proportional to amount of the enzyme and substrate Km = substrate conc at the half maximal velocity.

33. Representation of enzymes at various substrate concentration

34. Michaelismenten equation Vmax [S] = Vi Km + [S] When substrate conc < Km value (point A), Vi will be directly proportional to substrate conc When substrate conc = Km value(point B), vi wil be half the maximal velocity When substrate conc > Km value(point C), Vi wil be equal to Vmax and independent of substarteconc on further increases.

35. What does the Km value say about a reaction? High Km value means – the enzyme has low affinity for the substrate . We have to load a large amount of substrate for the reaction to attain Velocity. Low Km value means - the enzyme has very high affinity for substrate. Even if small amounts of substrates are there the reaction will attain velocity Affinity / binding is inversely proportional to Km value. Binding is good when shapes of both enzyme and substrate match

36. Hill’s equation; Y=ax+b Conversion of michaelismentan equation which is hyperbolic to hill’s equation which is linear.

37. Lineweaver –Burk Double reciprocal plot Inverse plot of michaelismenten – both S conc and velocity plotted by taking 1/S and 1/V on x and y axis. This was done to make the equation linear. X axis = 1/S Point of intersection on x axis = (-1/Km) value Y axis = 1/V Point of intersection on Y-axis(slope) = 1/Vmax

38. Enzyme inhibition A substance that can inhibit enzyme activity is called enzyme inhibitor: Types : Competitive inhibition Non-competitive inhibition Uncompetitive inhibition Mixed inhibition

39. Competitive inhibition So in competitive inhibition: Km is increased Vmax remains unaltered because the number of enzymes remain the same. Inhibitor resembles substrate in shape. Substrate competes with catalytic site. Inhibition is reversible because the inhibitor forms non-covalent bonds Inhibition can be reversed by increasing substrate conc..

40. Michaelismenten plot : Competitive inhibition: Lineweaver plot:

41. Methanol Poisoning • “Wood” alcohol and antifreeze contain high methanol concentrations • Methanol poisoning causes decreased blood pressure and body temperature, and an increase in respiratory rate Methanol + NAD+ Formaldehyde + NADH + H+ Ethanol + NAD+Acetaldehyde + NADH + H+ • Alcohol dehydrogenase has a slightly lower Km for ethanol compared to methanol • The additional benefit of the ethanol reaction is that acetaldehyde is less physiologically damaging than formaldehyde, which is toxic to the retina and can cause permanent blindness if doses are high for prolonged periods of time • Displaced methanol can be safely excreted by the kidneys Alcohol Dehydrogenase Alcohol Dehydrogenase

42. MethotrexateStructural analogue of folate  inhibits folatereductase • Dicoumarol Structurally similar to VitKanticoagulant • Sulfonamide antibiotics structural analogues of PABA  inhibits folate synthesis in bacteria • Non-toxic to humans humans don’t synthesize PABA

43. Non-competitive inhibition: Inhibitors bind to site other than substrate binding site. Covalent bond formation – occurs Loss of enzyme activity. So Vmax decrease but Km unaltered.

44. Lineweaver plot: Non-competitive inhibition Michaelismenten plot:

45. Uncompetitive inhibition Inhibitor binds to the enzyme substrate complex. Increased Substrate affinity apparently decreases the Km but Vmax is decreased because the enzyme will take a long time to separate from the complex.

46. Uncompetitive inhibition Lineweaver plot: Km value decreases Vmax also decreases

47. Summary of inhibition:

48. Enzyme regulation The process by which cells can turn on, turn off, modulate the activities of various metabolic pathways For coordinating various processes as per the physiological needs of the body. Every pathway has enzymes which are the rate limiting/ or key enzymes

49. Types of enzyme regulation Coarse regulation – Regulating enzyme activity Fine regulation – Regulating enzyme concentration.

50. Types of enzyme regulation Coarse Regulation: • Covalent modification • Allosteric regulation • Feed back inhibition • Compartmentalization Fine Regulation: • Induction • Repression