Solvent and α-secondary kinetic isotope effects on β-glucosidase

beta-Glucosidase from sweet almond is a retaining, family 1, glycohydrolase. It is known that glycosylation of the enzyme by aryl glucosides occurs with little, if any, acid catalysis. For this reaction both the solvent and alpha-secondary kinetic isotope effects are 1.0. However, for the deglucosylation reaction (e.g., k(cat) for 2,4-dinitrophenyl-beta-D-glucopyranoside) there is a small solvent deuterium isotope effect of 1.50 (+/- 0.06) and an alpha-secondary kinetic isotope effect of 1.12 (+/- 0.03). For aryl glucosides, k(cat)/K-M is very sensitive to the pK(a) of the phenol leaving group [beta(lg) approximate to -1; Dale et al., Biochemistry 25 (1986) 2522-2529]. With alkyl glucosides the beta(lg) is smaller (between -0.2 and -0.3) but still negative. This, coupled with the small solvent isotope effect on the pH-independent second-order rate constant for the glucosylation of the enzyme with 2,2,2-trifluoroethyl-beta-glucoside [(D2O)(k(cat)/K-M) = 1.23 (+/- 0.04)] suggests that there is more glycone-aglycone bond fission than aglycone oxygen protonation in the transition state for alkyl glycoside hydrolysis. The kinetics constants for the partitioning (between water and various alcohols) of the glucosyl-enzyme intermediate, coupled with the rate constants for the forward (hydrolysis) reaction provide an estimate of the stability of the glucosyl-enzyme intermediate. This is a relatively stable species with an energy about 2 to 4 kcal/mol higher than that of the ES complex. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. (C) 2015 Elsevier B.V. All rights reserved.