Methyl glucoside hydrolysis catalyzed by beta-glucosidase.

Sweet almond beta-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 beta-D-glucopyranoside is approximately 4 x 10(15)-times faster than the spontaneous hydrolysis at 25 degrees 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(2)O the pD-independent k(cat)/K(m) (= 28M(-1) s(-1)) is essentially identical to the value obtained in H(2)O, yielding a solvent kinetic isotope effect of (DOD)(k(cat)/K(m))(lim) = 1.05 (+ or - 0.08). There is also no effect of substituting D(2)O for H(2)O 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.