Past exam of the chemistry B course of the University of Cambridge / 2024 / Paper 2 / B10 / d / i / Solution

His238 contributes strongly to turnover, with a plausible acid/base or electrostatic role near the attacking water. Replacing its imidazole by the methyl side chain of alanine removes both proton-transfer capacity and a positioned polar group, so the large fall in turnover number fits impaired chemical catalysis. A candidate role is acceptance of a proton from zinc-bound water, or stabilization and subsequent proton shuttling around the attacking oxygen.

Little change in the Michaelis constant suggests that the major kinetic defect is not a gross loss of substrate recognition, but $K_M$ is not generally a binding dissociation constant: $K_M=(k_{-1}+k_{\mathrm{cat}})/k_1$ for the elementary scheme. Nor does reduced $k_{\mathrm{cat}}$ uniquely prove that His238 is the initial general base. Purified-protein controls should check fold, zinc occupancy and active concentration. Importantly, primary mouse-enzyme experiments found that His238 mutants could still hydrate a substrate analogue, directly illustrating this mechanistic limitation.^[ref]