1. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
Chapter 9:
Enzymes:
Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company

2. Types of Catalysis Covalent Acid-Base
General acid-base (Bronstead acid or base, HA or A-)
Specific acid-base (solvent, e.g. water, H+ or OH-)
3. Metal ion
4. Binding Effects
1. Approximation (proximity)
2. Transition state stabilization

3. Proteases or Peptidases
These enzymes cleave peptide bonds.
Remember that a peptide bond is an amide bond
and an amide is the least reactive carboxylic
acid derivative. It is very resistant to hydrolysis.
Enzymes catalyze this reaction in milliseconds.
protease

4. Chymotrypsin Chymotrypsin is an intestinal protease that
Recognizes and binds non-polar sidechains,
primarily aromatic sidechains: Phe, Tyr, Trp
Cleaves
slower
Cleaves

5. A Chromogenic Substrate
Cleaves
Esterase activity
Yellow Color

6. Covalent Inhibition at Ser-195 of Chymotrypsin

7. Mechanism Covalent catalysis – two steps Fast Slow A covalent
intermediate

8. Active Site Titration
Deacylation is slow
Acylation is rapid

10. Catalytic Triad in Chymotrypsin
The cataytic triad makes Ser-195 the only acidic
Ser in chymotrypsin.
The developing alkoxide is an excellent nucleophile.

11. Mechanism
Attack at the
Peptide bond

12. Stabilization of the tetrahedral intermediate
The O- forms ion-dipoles with two peptide N-H
hydrogens in the oxyanion hole.

13. Reform carbonyl and release N-terminus

14. Dissociation of the N-Terminus

15. New substrate (water) enters

16. Stabilization of the tetrahedral intermediate
Again, the O- forms ion-dipoles with two
peptide N-H hydrogens in the oxyanion hole.

17. Reform carbonyl and release C-terminus

18. Dissociation of the C-Terminus

19. Catalytic triad:
Asp 102…His 57…Ser 195

21. Hydrophobic binding pocket in chymotrypsin

22. Binding in other seryl enzymes

23. Papain – A Cysteine Protease

24. A Cysteine Protease with Substrate

25. Renin - An Aspartyl Protease

26. An Aspartyl Protease with Substrates

27. Pepsin - An Aspartyl Protease
A Typical Mechanism
Substrates in
Note the perturbed pKa values. Sidechain pKa of Asp is 3.86

28. Pepsin - Mechanism Tetrahedral intermediate General acid &
base catalysis
Tetrahedral
intermediate

29. Pepsin - Mechanism H on Asp32 moves to Asp215 followed by Peptide bond
general acid & base
catalysis
Peptide bond
cleavage

30. Pepsin - Mechanism H on Asp32 moves to Enzyme is ready
Asp215 and products leave the active site
Enzyme is ready
for substrate

31. A Dimeric Aspartyl Protease
Flaps/trap doors to retain substrate
HIV Protease

32. Thermolysin – A Metalloprotease

33. A Metalloprotease with Substrate

34. Carbonic Anhydrase, a Zn++ enzyme

35. Carbonic Anhydrase reaction

36. Carbonic Anhydrase Mechanism
Proton release
His64 assists
in H+ removal
HCO3-
released.
H2O
enters
CO2
enters
HO- attacks CO2

37. His64 Participation

38. Carbonic Anhydrase pH – Rate Profile
pKa of Zinc bound HOH
changes from 15.7 to ~7

39. Restriction Enzymes
These enzymes are endonucleases that cleave foreign DNA. They “restrict” invasion by foreign DNA by destroying it.
They cleave at specific base sequences (recognition sites). Host DNA with the same sequence is protected by methylation.
These are Class 3 enzymes, hydrolases, EC 3.x.x.x).

40. Restriction Enzymes
There are three types of restriction enzymes: I, II & III.
Types I and III require ATP for hydrolysis.
Type II does not and Type II enzymes are the ones used for cloning and sequencing DNA.
Hydrolysis by Type II enzymes generates a 3’ OH and 5’ phosphate.
Cleavage
Attack

41. Inversion of Configuration
Here one oxygen has been replaced with sulfur in order to determine the stereochemistry of reaction, i.e. inversion vs retention of configuration.

42. Mg++ needed by the Type II enzyme
Cleavage site
A segment
of DNA

43. Restriction sites are Palindromes

44. H-Bonding of enzyme-DNA site

45. Methylation prevents H-bonding with DNA substrate

46. Nucleoside Monophosphate (NMP) kinases
Adenylate kinase:
Mg++
ATP + AMP < == > 2 ADP
Guanylate kinase:
ATP + GMP < == > ADP + GDP

47. NMP kinase reaction

48. ATP:Mg++ Complexes
Most enzymes that require ATP, actually require ATP:Mg++ as substrate and will not use ATP alone.
Kinases are of this type.
Isomeric forms

49. Hexacoordinate Mg++

52. Additional conformational
change occurs after NMP
binds.

53. P Loop X X X X
Conserved G- X-X-X-X-G-K

54. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
End of Chapter 9
Copyright © 2007 by W. H. Freeman and Company