Xie Miaomiao, Byers Larry D
Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
Biochim Biophys Acta. 2015 Nov;1854(11):1776-81. doi: 10.1016/j.bbapap.2015.02.015. Epub 2015 Mar 11.
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.
甜杏仁中的β-葡萄糖苷酶是一种保留型的1家族糖水解酶。已知芳基葡萄糖苷对该酶的糖基化反应几乎没有酸催化作用（即便有，作用也很小）。对于此反应，溶剂效应和α-二级动力学同位素效应均为1.0。然而，对于去糖基化反应（例如，2,4-二硝基苯基-β-D-吡喃葡萄糖苷的kcat），存在较小的溶剂氘同位素效应，为1.50（±0.06），α-二级动力学同位素效应为1.12（±0.03）。对于芳基葡萄糖苷，kcat/KM对酚离去基团的pKa非常敏感[βlg≈ -1；戴尔等人，《生物化学》25 (1986) 2522 - 2529]。对于烷基葡萄糖苷，βlg较小（在 -0.2至 -0.3之间）但仍为负值。这一点，再加上溶剂同位素对酶与2,2,2-三氟乙基-β-葡萄糖苷糖基化反应的pH无关二级速率常数的较小影响[D2O(kcat/KM)=1.23 (±0.04)]，表明在烷基糖苷水解的过渡态中，糖苷配基-糖基键的断裂比糖苷配基氧的质子化更多。葡萄糖基酶中间体在水和各种醇之间的分配动力学常数，与正向（水解）反应的速率常数相结合，可估算葡萄糖基酶中间体的稳定性。这是一个相对稳定的物种，其能量比ES复合物的能量高约2至4千卡/摩尔。本文是名为：理论与实验中的酶过渡态的特刊的一部分。
