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

Biochimica Et Biophysica Acta
Miaomiao Xie, Larry D Byers

Abstract

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) rea...Continue Reading

References

Oct 20, 1992·Biochemistry·J B Kempton, S G Withers
Dec 1, 1968·Proceedings of the National Academy of Sciences of the United States of America·F W DahlquistM A Raftery
Dec 1, 1994·Current Opinion in Structural Biology·J D McCarter, S G Withers
Aug 5, 2009·Biochimica Et Biophysica Acta·Emily B Golden, Larry D Byers

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