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: kcat = 0.02 s−1 and KM = 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 kcatand KMsupport 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? Hint: consider Equation 2.2 (Coulomb’s Law).
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