1. Catalytic Mechanisms

2. To understand how enzymes work at the molecular level.
Objective
To understand how enzymes work at the molecular level.
Ultimately requires total structure determination, but can learn much through biochemical analysis.

3. To Be Explained Specificity Catalysis For specific substrates
Amino acids residues involved
Catalysis
Mechanisms
Amino acids involved/Specific role(s)

4. Enzyme Binding Sites Active Site: Regulatory Site:
Substrate Binding Site + Catalytic Site
Regulatory Site:
a second binding site,
Binding by regulatory molecule affects the active site
alter the efficiency of catalysis
improve or inhibit

5. General Characteristics
Three dimensional space
Occupies small part of enzyme volume
Clefts or crevices
Ligands (substrate or effector) bound by multiple weak interactions
Specificity depends on precise arrangement of atoms in active site

6. Models
Induced Fit
Lock and Key

7. Identification and Characterization of Active Site
Structure: size, shape, charges, etc.
Composition: identify amino acids involved in binding and catalysis.

8. Binding or Positioning Site (Trypsin)

9. Binding or Positioning Site (Chymotrypsin)

10. Catalytic Site (e.g. Chymotrypsin)

11. Probing the Structure of the Active Site
Model Substrates

12. Model Substrates (Chymotrypsin)

13. Peptide Chain?
All Good Substrates!

14. a-amino group?
Good Substrate!

15. Side Chain Substitutions
Good Substrates
t-butyl-
Cyclohexyl

16. Conclusion Bulky Hydrophobic Binding Site

17. Probing the Structure of the Active Site
Competitive Inhibitors

18. Arginase

19. Good Competitive Inhibitors

20. Poor Competitive Inhibitors
All Three Charged Groups are Important

21. Conclusion Active Site Structure of Arginase

22. Identifying Active Site Amino Acid Residues
Covalent modification of residues
Inactivation of enzyme
Site directed mutagenesis

23. Mechanisms of Catalysis
Acid-base catalysis
Covalent catalysis
Metal ion catalysis
Proximity and orientation effects
Preferential binding (stabilization) of the transition state

24. Addition or removal of a proton by side chains
Acid-Base Catalysis
Addition or removal of a proton by side chains

25. General Acids and Bases

26. Keto-Enol Tautomerization
Acid-Base Catalysis
Keto-Enol Tautomerization

27. Uncatalyzed Reaction

28. General Acid Catalysis

29. General Base Catalysis

30. Ribonuclease A
Figure 11-10

31. Mechanism of RNase A
Get specificity, because DNA doesn’t have an OH group on carbon 2.
Figure part 1

32. Mechanism of RNase A
Figure part 2

33. Covalent Catalysis (Nucleophilic catalysis) (Principle)
Involves a transient covalent bond between the enzyme and the substrate
Usually by the nucleophilic attack of the substrate by the enzyme

34. Covalent Catalysis (Principle)
Slow
H2O + A–B ——> AOH + BH
A-B + E-H ——> E-A + BH
E-A + H2O ——> A-OH + E-H
Fast
NOTE: New Reaction Pathway

35. Covelent Catalysis

36. The Schiff Base

37. Metal Ion Catalysis Charge stabilization Water ionization
Charge shielding

38. Metal Ion Catalysis Metalloenzymes: tightly bound metal ions
Catalytically essential
Fe2+, Fe3+, Cu2+, Mn2+, and Co2+
Metal-activated enzymes: loosely bound metal ions (from solution or with substrate)
Structural metal ions:
Na+, K+, and Ca2+
Both: Mg2+ and Zn2+

39. Carbonic Anhydrase

40. Carbonic Anhydrase

41. Proximity and Orientation Effects Rate of a reaction depends on:
Number of collisions
Energy of molecules
Orientation of molecules
Reaction pathway (transition state)

42. Proximity
V = k[A][B]
[A] and [B] = ~13M on enzyme surface

43. Biomolecular Reaction of Imidazole with p-Nitrophenylacetate (Intermolecular)
Page 336

44. Intramolecular Rate = 24x Intermolecular Rate
Intramolecular Reaction of Imidazole with p-Nitrophenylacetate (Intramolecular)
Within an active site imidazole can be in that proximity to p-NP
Intramolecular Rate = 24x Intermolecular Rate
Page 336

45. Orientation

46. Geometry of an SN2 Reaction
Figure 11-14

47. Preferrential Binding of Reaction Intermediate
Stabilize Transition State
Electrostatic stabilization of developing charge
Relief of induced bond angle strain
Enhancement of weak interactions between enzyme and intermediate.
Draw out diagram.

48. Steric Strain in Organic Reactions
Reaction Rate: R=CH3 is 315x vs R=H
Page 338

49. Effect of Preferential Transition State Binding
Figure 11-15

50. Transition State Analogs
Powerful Enzyme Inhibitors

51. Proline Racemase (planar transition state)
Page 339

52. Transition State Analogs of Proline
Binding = 160x versus Proline
Page 339

53. Chymotrypsin Trypsin Elastase etc.
Serine Proteases
Chymotrypsin
Trypsin
Elastase
etc.

54. Convergent Evolution

55. Substrate Specificity

56. Mechanism of Chymotrypsin
p-nitrophenolate

57. X-Ray Structure of Bovine Trypsin (Ribbon Diagram)

58. Active Site Residues of Chymotrypsin (Catalytic Triad)
Figure 11-26

59. Catalytic Mechanism of the Serine Proteases
Catalytic Triad

60. Catalytic Mechanism of the Serine Proteases
Figure part 2

61. Catalytic Mechanism of the Serine Proteases

62. Catalytic Mechanism of the Serine Proteases

63. Catalytic Mechanism of the Serine Proteases

64. Catalytic Mechanism of the Serine Proteases

65. Catalytic Mechanism of the Serine Proteases

66. Transition State Stabilization in the Serine Proteases
Figure 11-30a

67. Transition State Stabilization in the Serine Proteases
Figure 11-30b

68. Mechanism of Chymotrypsin
p-nitrophenolate
New Reaction Pathway
(versus uncatalyzed reaction)