1. Enzyme Rate Enhancement
How do enzymes work to catalyze reactions?

2. Enzyme Classes by Reaction Type

3. Oxidation-Reduction Reaction: Oxidoreductase

4. Functional Group Transfer: Transferase

5. Cutting with Water: Hydrolysis

6. Group Elimination with Double Bond Formation: Lyase

7. Compound Conversion to an Isomeric Form: Isomerase
Triose phosphate isomerase

8. Bond Formation with ATP Hydrolysis: Ligase
DNA Ligase -- nucleotide base coupling with loss of PPi

9. Chymotrypsin Catalyzes Peptide Hydrolysis
Enzyme specificity in terms of:
Site of hydrolysis and
Substrate reactivity
How can a reaction be pushed in the forward direction?

10. Reactions that are Spontaneous in the Forward and Reverse Direction
How do enzymes affect the energy diagram of a reaction?

11. Enzymes Decrease the Activation Energy
How can enzymes lower the transition state?
Is external energy required?

12. Mechanisms of Enzyme Catalysis
Acid-base, covalent, metal ion, orientation and proximity

13. Ketone-Enol Conversion
What mechanism of enzyme catalysis is operative here?

14. Amino Acid Residues that Function in Acid-Base Catalysis

15. Conventions for Depicting Reaction Mechanisms
Curved arrow indicates electron rearrangement during a reaction

16. Covalent Catalysis
A formal positive charge favors electron flow to the nitrogen group

17. Covalent Catalysis

18. Amino Acids that can Participate in Covalent Catalysis

19. Metal Ion Catalysis

20. Components that Facilitate Enzyme Catalysis
What is the cofactor in ATP hydrolysis?
What is the co-substrate in an oxidation-reduction reaction?

21. Enzymes Decrease the Activation Energy

22. Mechanisms of Enzyme Catalysis
Acid-Base Catalysis
Covalent Catalysis
Metal Ion Catalysis
Orientation/Proximity Effects
Preferential Transition- State Binding

23. Proximity and Orientation Effects Facilitate Catalysis

24. Chymotrypsin Specificity
Cleaves peptides on the C-terminus side of hydrophobic residues (e.g. Phe, Tyr and Try)

25. Active Site Mapping via Irreversible Inhibitors
Diisopropylphosphofluoridate (DIPF) inhibits chymotrypsin by modifying 1 of 28 serine residues

26. Active Site Mapping via Irreversible Inhibitors

27. Covalent Catalysis for Chymotrypsin: a Two Step Process
Enzyme acylation with leaving group departure
Enzyme deacylation
What is the leaving group with Chymotrypsin?

28. Chymotrypsin Catalysis Proceeds via a Two-Step Mechanism
Chromogenic substrate for kinetic studies
Why is this compound not an ideal substrate mimic?

29. Chymotrypsin Catalytic Triad
Catalytic triad serves as the site of catalysis
Aspartate and histidine contribute serine’s basicity
Serine serves as a nucleophile in covalent catalysis
What type of catalysis occurs?

30. Mechanism of Peptide Hydrolysis in Chymotrypsin
Substrate binding via nucleophilic attack

31. Mechanism of Peptide Hydrolysis in Chymotrypsin
Polypeptide original C-side serves as leaving group

32. Mechanism of Peptide Hydrolysis in Chymotrypsin
Water attacks original N-side of polypeptide

33. Mechanism of Peptide Hydrolysis in Chymotrypsin
Polypeptide original N-side serves as leaving group and enzyme is regenerated

34. Chymotrypsin Hydrolysis

35. Tetrahedral-Intermediate Stabilization in Chymotrypsin
H-bonds ideally positioned in the oxyanion hole stabilize the sp3 transition state

36. Chymotrypsin Specificity Pocket
Large structural pocket lined with hydrophobic amino acids favors bulky hydrophobic residues

37. Serine Proteases Differ in Little Except Their Specificity Pockets
Chymotrypsin Trypsin Elastase

38. Substrate Specificity Observed with each Proteolytic Enzyme
Papain cleave peptides non-selectively
Trypsin cleaves carboxyl side of bulky + charged R- groups
Chymotrypsin cleaves carboxyl side of bulky aromatic R-groups
Thrombin

39. Divergent Evolution
Percent Sequence Identity among Three Serine Proteases
Bovine trypsin %
Bovine chymotrypsinogen 53%
Porcine elatase 48%
Common ancestor with retention of overall structure and catalytic mechanism

40. Convergent Evolution Bovine versus bacterial serine protease
No sequence or structural similarity but the same catalytic triad and oxyanion hole in the active site

41. Enzyme-Substrate Binding Critical for Catalysis
Lock and Key Model
Enzyme Active Site
3-D cleft or crevice
Small part of enzyme
Unique micro-environment
Substrate binding by weak forces
Induced Fit Model

42. Substrate-Induced Enzyme Conformational Change

43. Inhibition by Transition State Analogs
Pyrrolidine the natural substrate binds 160 less tightly than pyrrole a transition state analog.
What is the favored enzyme binding geometry?

44. Rate of Enzyme Catalysis
Explain why enzyme activity increases with temperature and then precipitously drops off

45. RNAas A Digestive Enzyme Cleaving Mechanism
Why does ribonuclease catalyzes the hydrolysis of RNA but not DNA

46. Conversion of Adenosine to Inosine
What does the much greater binding affinity of 1,6-dihydropurine ribonucleoside than the substrate indicate about the enzyme mechanism?

47. Chapter 6 Problems: 1, 3, 7, 9, 11, 15, 20, 23, 25 and 37