Solvent isotope effects in H2O-D2O mixtures (proton inventories) on serine-prolease-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 H2O, D2O and ''HDO'' (1:1 H2O:D2O). The solvent isotope effects (proton inventories) determined by these data showed no differences between the wild type and mutant enzymes, despite the fact hat 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 fur subtilisin and its mutant also indicate the presence of strong medium effects in those systems.