title: Solvent and α-secondary kinetic isotope effects on β-glucosidase.
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) 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.
authors.affiliation: Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Xie
authors.affiliation: Department of Chemistry, Tulane University, New Orleans, LA 70118, USA. Electronic address: byers@tulane.edu.
authors.forename: Larry D
authors.initials: LD
authors.lastname: Byers
mesh_terms: D000473:Alkanes / Q000737:chemistry; D055162:Biocatalysis; D003903:Deuterium / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D000068181:Prunus dulcis / Q000201:enzymology; D012997:Solvents / Q000737:chemistry*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000473:Alkanes; D006027:Glycosides; D007554:Isotopes; D010940:Plant Proteins; D012997:Solvents; D003903:Deuterium; D001617:beta-Glucosidase
keywords: 2,4-Dinitrophenyl β-D-glucopyranoside; Glucosyl-enzyme energy; Secondary kinetic isotope effect; β-glucosidase