Solvent Isotope Effects in H2O−D2O Mixtures (Proton Inventories) on Serine-Protease-Catalyzed Hydrolysis Reactions. Influence of Oxyanion Hole Interactions and Medium Effects

Rates of hydrolysis of succinyl-Ala-Ala-Pro-Phe- p -nitroanilide catalyzed by subtilisin and its N155G mutant were measured in H 2 O, D 2 O, and “HDO” (1:1 H 2 O:D 2 O). The solvent isotope effects (proton inventories) determined by these data showed no differences between the wild-type and mutant enzymes, despite the fact that the mutation removes a hydrogen-bonding interaction in the oxyanion hole of the enzyme worth two orders of magnitude in reaction rate. This suggests that curvature previously observed (ref 10) in the proton inventory for a reaction catalyzed by methyl chymotrypsin is also not due to oxyanion hole interactions, and this curvature can in fact be accounted for by a medium effect. Proton inventory analysis of the isotope effects for subtilisin and its mutant also indicate the presence of strong medium effects in those systems.