Chapter 3 Enzyme Mode of Action How enzyme works.pptx

This chapter covers the way how enzyme works and the enzyme kinetics

1. Chapter 3 Enzyme Mode of Action

2. Important Terms to Understand Biochemical Nature and Activity of Enzymes Active site: The area on the enzyme where the substrate or substrates attach to is called the active site. Enzymes are usually very large proteins and the active site is just a small region of the enzyme molecule.

3. Enzyme molecules contain a special pocket or cleft called the active sites .

4. In enzymatic reactions, the substance at the beginning of the process, on which an enzyme begins it’s action is called substrate .

5. Apoenzyme and Holoenzyme The enzyme without its non protein moiety is termed as apoenzyme and it is inactive . Holoenzyme is an active enzyme with its non protein component.

6. Cofactor A cofactor is a non-protein chemical compound that is bound (either tightly or loosely) to an enzyme and is required for catalysis. Types of Cofactors: Coenzymes. Prosthetic groups. Coenzyme: The non-protein component, loosely bound to apoenzyme by non-covalent bond . Examples : vitamins or compound derived from vitamins . Prosthetic group The non-protein component, tightly bound to the apoenzyme by covalent bonds is called a Prosthetic group.

7. Enzyme Specificity Enzymes have varying degrees of specificity for substrates Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond

8. Activation energy or Energy of Activation All chemical reactions require some amount of energy to get them started. OR It is First push to start reaction. This energy is called activation energy .

9. Structure of enzymes Enzymes Complex or holoenzymes (protein part and nonprotein part – cofactor ) Simple (only protein ) Apoenzyme (protein part ) Cofactor Prosthetic groups usually small inorganic molecule or atom; usually tightly bound to apoenzyme Coenzyme -large organic molecule -loosely bound to apoenzyme

10. Rate Enhancements

11. Examples of Enzymatic Activity. Proteolytic Activity A protease Catalyzes cleavage of a peptide bond

12. Esterase Activity Many proteases also manifest esterase activity and catalyze cleavage of an ester bond.

13. Cleavage of a Peptide Bond Enzymatic cleavage occurs on the carboxyl side of the recognized sidechain. Trypsin

14. 3.1. Mode of Action of Enzymes Enzymes increase reaction rates by decreasing the Activation energy: Enzyme-Substrate Interactions: Formation of Enzyme substrate complex by : Lock-and-Key Model Induced Fit Model

15. Lock-and-Key Model In the lock-and-key model of enzyme action: - the active site has a rigid shape - only substrates with the matching shape can fit - the substrate is a key that fits the lock of the active site This is an older model, however, and does not work for all enzymes

16. Induced Fit Model In the induced-fit model of enzyme action: - the active site is flexible, not rigid - the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis - there is a greater range of substrate specificity This model is more consistent with a wider range of enzymes

17. Enzymes Lower a Reaction’s Activation Energy

18. 3.2. The Science- How do Enzymes Work? Enzymatic Catalysis Activation Energy (AE) – The energy require to reach transition state from ground state. AE barrier must be exceeded for rxn to proceed. Lower AE barrier, the more stable the transition state (TS) The higher [TS], the move likely the rxn will proceed.

19. Transition (TS) State Intermediate Transition state = unstable high-energy intermediate Rate of rxn depends on the frequency at which reactants collide and form the TS Reactants must be in the correct orientation and collide with sufficient energy to form TS Bonds are in the process of being formed and broken in TS Short lived (10 –14 to 10 -13 secs )

20. Intermediate s Intermediates are stable. In rxns w/ intermediates, 2 TS’s are involved. The slowest step (rate determining) has the highest AE barrier. Formation of intermediate is the slowest step.

21. Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation, decrease entropy) Need to stabilize TS to lower activation energy barrier.

22. ES complex must not be too stable Raising the energy of ES will increase the catalyzed rate This is accomplished by loss of entropy due to formation of ES and destabilization of ES by strain distortion desolvation

23. Transition State Stabilization Transition state analog Equilibrium between ES <-> TS, enzyme drives equilibrium towards TS Enzyme binds more tightly to TS than substrate

24. Mechanistic Strategies Polar AA Residues in Active Sites

25. Common types of enzymatic mechanisms Substitutions rxns Bond cleavage rxns Redox rxns Acid base catalysis Covalent catalysis Metal Ion Catalysis

26. A) Substitution Rxns Nucleophillic Substitution– Direct Substitution transition state Nucleophillic = e - rich Electrophillic = e - poor

27. B) Oxidation reduction (Redox) Rxns Loose e - = oxidation (LEO) Gain e - = reduction (GER) Central to energy production If something oxidized something must be reduced (reducing agent donates e - to oxidizing agent) Oxidations = removal of hydrogen or addition of oxygen or removal of e - In biological systems reducing agent is usually a co-factor (NADH of NADPH)

28. C) Cleavage Rxns Heterolytic vs homolytic cleavage Carbanion formation (retains both e - ) R 3 -C-H  R 3 -C: - + H + Carbocation formation (lose both e - ) R 3 -C-H  R 3 -C + + H: - Free radical formation (lose single e - ) R 1 -O-O-R 2  R 1 -O* + *O-R 2 Hydride ion

29. D) Acid-Base Catalysis Accelerates rxn by catalytic transfer of a proton Involves AA residues that can accept a proton Can remove proton from –OH, -NH, -CH, or –XH Creates a strong nucleophillic reactant (i.e. X: - )

30. : : Acid-Base Catalysis carbanion intermediate

31. E) Covalent Catalysis 20% of all enzymes employ covalent catalysis A-X + B + E <-> BX + E + A A group from a substrate binds covalently to enzyme (A-X + E <-> A + X-E) The intermediate enzyme substrate complex (A-X) then donates the group (X) to a second substrate (B) (B + X-E <-> B-X + E)

32. Covalent Catalysis Protein Kinases ATP + E + Protein <-> ADP + E + Protein-P A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO 4 - E-O-PO 4 - + Protein-OH <-> E + Protein-O- PO 4 -

33. F) Metal Ion Catalysis Thermolysin is an endoprotease with a catalytic Zn 2+ ion in the active site. The Zn 2+ ion stabilizes the buildup of negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl carbon.

34. How Do Active-Site Residues Interact to Support Catalysis? About half of the amino acids engage directly in catalytic effects in enzyme active sites Other residues may function in secondary roles in the active site: Raising or lowering catalytic residue p K a values Orientation of catalytic residues Charge stabilization Proton transfers via hydrogen tunneling

35. 3.3. Mechanism of Enzyme Action: Enzyme Kinetics Enzyme Kinetics Study of reaction rate and how they changes in response to change in experimental parameter is known as kinetics Amount of substrate present is one of the key factor affecting the rate of reaction catalyzed by an enzyme in vitro

36. Low of Mass Action

37. Zero Order Reaction The rate is independent of the concentration of any of the reactants

38. First Order Reaction

39. Second Order Reaction

40. Michaelis-Menten Kinetics The quantitative theory of enzyme kinetics was proposed by two scientists Leonor Michaelis and Maud Leonora Menten in 1913 E nzyme reactions in Michaelis – Menten kinetics theory occur in two stages: T he substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex The enzyme then catalyzes the chemical step in the reaction and releases the product

41. Enzyme Kinetics E + S ES E + P k 1 k 2 k -1 E = enzyme concentration S = Substrate concentration ES = Enzyme-substrate complex concentration P = product concentration k 1 = rate constant for formation of ES from E + S k -1 = rate constant for decomposition of ES to E + S k 2 = rate constant for decomposition of ES to E + P

42. Michaelis-Menton Plot Graphical relationship between reaction velocity and substrate concentration E + S ES E + P k 1 k -1 k 2 Zero order (High [S]) 1 st order (low [S])

43. Finding Initial Veocity , V o Rate at the start of an enzyme catalyzed reaction

44. Enzyme Kinetics Enzyme-substrate complex ( ES ) - a non-covalent complex formed when specific substrates fit into the enzyme active site When [S] >> [E], every enzyme binds a molecule of substrate (enzyme is saturated with substrate) Under these conditions the rate depends only upon [E], and the reaction is pseudo-first order E + S ES E + P k 1 k 2 k -1

45. The Michaelis-Menton Equation (1) Assume steady-state conditions: Rate of ES formation = Rate of ES decomposition (2) Define Michaelis constant: K M = (k -1 + k 2 ) / k 1 (3) The overall velocity of an enzyme-catalyzed reaction is given by rate of conversion of ES to E + P. v o = k 2 [ES] = k cat [ES]

46. Reaction Constituents

47. Michaelis-Menton Derivation 1. The overall rate of product formation: v = k 2 [ES] 2. Rate of formation of [ES]: v f = k 1 [E][S] 3. Rate of decomposition of [ES]: v d = k -1 [ES] + k 2 [ES] 4. Rate of ES formation = Rate of ES decomposition (steady state) 5. So: k 1 [E][S] = k -1 [ES] + k 2 [ES] E + S ES E + P k 1 k 2 k -1

48. Michaelis-Menton Derivation 6. In solving for [ES], use the enzyme balance to eliminate [E]. E T = [E] + [ES] 7. k 1 (E T - [ES])[S] = k -1 [ES] + k 2 [ES] k 1 E T [S] - k 1 [ES][S] = k -1 [ES] + k 2 [ES] 8. Rearrange and combine [ES] terms: k 1 E T [S] = (k -1 + k 2 + k 1 [S])[ES] k 1 E T [S] 9. Solve for [ES] = ----------------------- (k -1 + k 2 + k 1 [S])

49. Michaelis-Menton Derivation E T [S] 10. Divide through by k 1 : [ES] = ----------------------- (k -1 + k 2 )/k 1 + [S] 11. Defined Michaelis constant: K M = (k -1 + k 2 ) / k 1 12. Substitute K M into the equation in step 10. 13. Then substitute [ES] into v = k 2 [ES] from step1 and replace V max with k 2 E T to give: V max [S] v o = ----------- K M + [S]

50. Michaelis-Menton Plot Relates reaction velocity and substrate concentration V max [S] v o = ----------- K M + [S]

51. Lineweaver -Burke Plot Also called a double reciprocal plot 1 K M 1 1 --- = ----- • ---- + ----- v o V max [S] V max

52. k cat In an enzyme catalyzed reaction, the overall rate of product formation is v = k 2 [ES]. If all of the enzyme molecules are complexed with substrate (excess [S]) then the maximum velocity occurs and V max = k cat E T where k cat is the overall reaction rate constant. This can also be written as k cat = V max /E T. k cat is called the turnover number (TON). E + S ES E + P k 1 k 2 k -1

53. K M When K M = [S] when v o = 1/2 Vmax . K M @ k -1 / k 1 = K s (the enzyme-substrate dissociation constant) when k cat is small (<< either k 1 or k -1 ). Generally, the lower the numerical value of K M , the tighter the substrate binding. K M is used as a measure of the affinity of E for S.

54. = k cat (s -1 )

55. k cat /K M k cat /K M is taken to be a measure of the efficiency of an enzyme . Rewriting k cat /K M in terms of the kinetic constants gives: k cat k 1 k 2 ---- = ----------- K M k -1 + k 2 So, where k 2 is small, the denominator becomes k -1 and k cat /K M is small.

56. k cat /K M k cat k 1 k 2 ---- = ----------- K M k -1 + k 2 And where k 2 is large, the denominator becomes k 2 and k cat /K M is limited by the value of k 1 or formation of the ES complex. This formation is in turn limited by the rate of diffusion of S into the active site of E. So, the maximum value for this second-order rate constant (k cat /K M ) is the rate of diffusion (~ 10 9 sec -1 M -1 ).

57. Enzyme Inhibition Noncovalent binding : Competitive (I binds only to E) Uncompetitive (I binds only to ES) Noncompetitive (I binds to E or ES) Covalent binding – irreversible Group Specific Substrate Analogs Suicide

58. Competitive Inhibition

59. Competitive Inhibition (I binds only to E)

60. Uncompetitive Inhibition

61. Uncompetitive Inhibition (I binds only to ES)

62. Noncompetitive Inhibition

63. Noncompetitive Inhibition (I binds to E or ES)

64. Irreversible - Group Specific

65. Irreversible - Group Specific

66. Irreversible - Substrate Analog

67. Irreversible - Substrate Analog

68. Irreversible - Suicide

69. Transition State Analog