Methyl glucoside hydrolysis catalyzed by β-glucosidase

Abstract

Sweet almond β-glucosidase is a retaining, family 1, glycohydrolase, catalyzing the highly efficient hydrolysis of a variety of glycosides. For example, the enzyme-catalyzed hydrolysis of methyl β- D-glucopyranoside is ≈4 × 10 15-times faster than the spontaneous hydrolysis at 25 °C. As with most enzymes, the dependence of k cat/ K m on pH is bell-shaped, indicating the importance of a protonated (acidic) residue and a deprotonated (nucleophilic) residue in its mechanism. Surprisingly, however, in D 2O the pD-independent k cat/ K m (=28 M − 1 s − 1 ) is essentially identical to the value obtained in H 2O, yielding a solvent kinetic isotope effect of DOD( k cat/ K m) lim = 1.05 (± 0.08). There is also no effect of substituting D 2O for H 2O on K m nor on the K i values for a variety of competitive inhibitors. The lack of a solvent kinetic isotope effect on k cat/ K m can be explained by a stepwise mechanism for the glucosylation of the enzyme. This mechanism involves a preequilibrium protonation of the glycosidic oxygen of the bound substrate followed by the rate-limiting cleavage of the glycosidic bond. Much of the enzymic rate enhancement is due to the stabilization, presumably by ionic interactions, of the protonated glucoside.