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

• The α-secondary kinetic isotope in the deglucosylation of β-glucosidase is 1.12.
• There is a small, negative, βlg on the glucosylation of the enzyme with alkyl glucosides.
• There is a small solvent kinetic isotope effect of 1.23 on kcatKM with trifluoroethyl-β- glucoside.
• The energy of the glucosyl-enzyme intermediate is only 2–4 kcal/mol higher than that of the ES complex.

β-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 α-secondary kinetic isotope effects are 1.0. However, for the deglucosylation reaction (e.g., kcat for 2,4-dinitrophenyl-β-D-glucopyranoside) there is a small solvent deuterium isotope effect of 1.50 (± 0.06) and an α-secondary kinetic isotope effect of 1.12 (± 0.03). For aryl glucosides, kcat/KM is very sensitive to the pKa of the phenol leaving group [βlg ≈ − 1; Dale et al., Biochemistry25 (1986) 2522–2529]. With alkyl glucosides the βlg 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-β-glucoside [D2O(kcat/KM) = 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.

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