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Merck

Methyl glucoside hydrolysis catalyzed by beta-glucosidase.

Biochimica et biophysica acta (2009-08-05)
Emily B Golden, Larry D Byers
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Millipore
Methyl α-D-glucopyranoside, ≥99.0%, suitable for microbiology
Sigma-Aldrich
Methyl α-D-glucopyranoside, ≥99% (GC)
Sigma-Aldrich
Methyl β-D-glucopyranoside, ≥99% (HPLC and GC)