Chapter 8, Problem 12P

At 37 ^oC, the serine protease subtilisin has k_cat = 50 S-1 and K_M = 1.4 x 10-4 M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues repotted (1986, Phil Trans. R Soc. Lond. A 317: 415-423) the following kinetic parameters for the N155T mutant of subtilisin:
k_cat = 0.02 S-1 and
K_M = 2 x 10-4 M.

a. Subtilisin is used in some laundry detergents to help remove protein-type stains. What unusual kind of stability does this suggest for subtilisin?
b. Subtilisin does have a problem in that it becomes inactivated by oxidation of a methionine close to the active site. Suggest a way to make a better subtilisin.
c. Is the effect of the N155T mutation what you would expect for a residue that makes up part of the oxyanion hole? How do the reported values of k_cat and K_M support your answer?
d. Assuming that the T 155 side chain cannot H-bond to the oxyanion intermediate, by how much (in kJ / mol) does N155 appear to stabilize the transition state at 37 ^oC?
e. The value you calculated in part (d) represents the strength of the H-bond between N155 and the oxyanion in the transition state. This value is higher than typical H-bonds in water. How might this observation be rationalized? Hint: Consider Equation 2.2 (Coulomb's Law).