Question

Subtilisin (Mr 27600) is a bacterial protease that can catalyzehydrolysis of certain amino acid esters and amides. Subtilisin doeshave a problem, in that it becomes inactivated by oxidation of amethionine Met-222. Subtilisin mutant with Cys replacing Met-222shows 138% of activity of the wild type (due to increase in kcatfor mutant). Subtilisin mutants with either Ala, Ser, Gly, or Thrin place of Met-222 exhibit enzymatic activity of 53, 35, 30, and28% of subtilisin, respectively, while any other amino acidreplacement leads to a significant reduction in enzymatic activityof mutant (0.3 - 15% of subtilisin activity). Explain in detail thereason(s) for such a loss of activity in:

a). Oxidized subtilisin
b). Ala, Ser, Gly, and Thr mutants
c). All other mutants.

Answer the questions:
d). Why Cys-222 mutant may have higher kcat than a wild-type enzyme?
e). Why this (Cys-222) mutation has not been selected as the bestin evolution ?

**Any help whatsoever would be appreciated.