1. For the amide substrate, the formation of the acyl-enzyme intermediate is slower than the hydrolysis of the acyl-enzyme intermediate, and so no burst is observed. A burst is observed for ester substrates; the formation of the acyl-enzyme intermediate is faster, leading to the observed burst.

2. The histidine residue in the substrate can substitute to some extent for the missing histidine residue of the catalytic triad of the mutant enzyme.

3. No. The catalytic triad works as a unit. After this unit has been made ineffective by the mutation of histidine to alanine, the further mutation of serine to alanine should have only a small effect.

4. The substitution corresponds to one of the key differences between trypsin and chymotrypsin, and so trypsin-like specificity (cleavage after lysine and arginine) might be predicted.

A11

In fact, additional changes are required to effect this specificity change.

5. Imidazole is apparently small enough to reach the active site of carbonic anhydrase and compensate for the missing histidine. Buffers with large molecular components cannot do so, and the effects of the mutation are more evident.

6. No. The odds of such a sequence being present are approximately 1 in 4¹⁰ = 1,048,576. Because a typical viral genome has only 50,000 bp, the target sequence would be unlikely to be present.

7. No, because the enzyme would destroy the host DNA before protective methylation could take place.

8. No. The bacteria receiving the enzyme would have their own DNA destroyed because they would likely lack the appropriate protective methylase.

9. EDTA will bind to Zn²⁺ and remove the ion, which is required for enzyme activity, from the enzyme.

10. (a) The aldehyde reacts with the active-site serine. (b) A hemiacetal is formed.

11. Trypsin.

12. The reaction is expected to be slower by a factor of 10 because the rate depends on the pK_a of the zinc-bound water. k_cat = 60,000s⁻¹.

13. EDTA binds the magnesium necessary for the reaction.

14. ATP hydrolysis is reversible within the active site. ATP hydrolysis takes place within the active site with the incorporation of ¹⁸O, ATP is re-formed, and the ATP is released back into solution.

15. If the aspartate is mutated, the protease is inactive and the virus will not be viable.

16. Water substitutes for the hydroxyl group of serine 236 in mediating proton transfer from the attacking water and the γ-phosphoryl group.

17. For subtilisin, the catalytic power is approximately 30 s⁻¹/10⁻⁸ s⁻¹ = 3 × 10⁹. For carbonic anhydrase (at pH 7), the catalytic power is approximately 500,000 s⁻¹/0.15 s⁻¹ = 3.3 × 10⁶. Subtilisin is the more powerful enzyme by this criterion.

18. The triple mutant still catalyzes the reaction by a factor of approximately 1000-fold compared to the uncatalyzed reaction. This could be due to the enzyme binding the substrate and holding it in a conformation that is susceptible to attack by water.

19. (a) Cysteine protease: The same as Figure 9.8, except that cysteine replaces serine in the active site and no aspartate is present.

(b) Aspartyl protease:

(c) Metalloprotease: