At 37 C, the serine protease subtilisin has kcat = 50 s-1 and KM = 1.4 × 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 reported (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 × 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 kcat and KM support your answer?
d. Assuming that the T155 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 C?
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?