BCH210H1 Midterm: Study notes for midterm

The serine protease, subtilisin, is used in some laundry detergents to help remove protein-type stains.

(a) What unusual kind of stability does this suggest for subtilisin?

(b) What is the structure of the catalytic site in subtilisin?

(c) Draw the pH profile for subtilisin activity, if the assumed pH of hot tap water in a washer is ca. 6.5.

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.

You wish to design a new serine protease that will cleave polypeptide chains to create a series of fragments that contain either cysteine or serine residues at their C-termini. Which of the following characteristics of the new protease is most important to achieve this result?