Tanaka Y, Tao W, Blanchard J S, Hehre E J
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1994 Dec 23;269(51):32306-12.
Secondary tritium and primary 14C kinetic isotope effects were measured for the hydrolysis of alpha-D-glucopyranosyl fluoride catalyzed by sugar beet seed alpha-D-glucosidase, forming alpha-D-glucose, and by Rhizopus niveus glucoamylase forming beta-D-glucose. The data provided a novel opportunity to model and directly compare the transition state structures for the hydrolysis of a substrate promoted with retention or inversion of configuration according to the enzyme catalyst. The isotope effects for the reaction catalyzed by each enzyme are most consistent with an SN1 rather than an SN2 mechanism. The modeled transition state structures for the hydrolysis promoted by the alpha-glucosidase and the glucoamylase both bear significant oxocarbonium ion character, with the D-glucosyl residue having a flattened 4C1 conformation and a C-1-O-5 bond order of 1.92, even though opposite D-glucose anomers were produced from the substrate. The transition states show some modest differences, but their general similarity strongly suggests that the stereochemical outcome of glycosylase reactions does not predict the transition state structure, nor does the transition state structure of such reactions predict the stereochemical outcome. The results support previously reported evidence for the separate topological control of product configuration by protein structures in these and other glycosylases.
测定了甜菜种子α-D-葡萄糖苷酶催化α-D-吡喃葡萄糖基氟水解生成α-D-葡萄糖以及雪白根霉葡糖淀粉酶催化其水解生成β-D-葡萄糖时的二级氚和一级14C动力学同位素效应。这些数据提供了一个新的机会，可根据酶催化剂对构型保留或构型翻转促进的底物水解的过渡态结构进行建模和直接比较。每种酶催化反应的同位素效应与SN1机制而非SN2机制最为一致。由α-葡萄糖苷酶和葡糖淀粉酶促进的水解的模拟过渡态结构均具有显著的氧鎓离子特征，D-葡萄糖基残基具有扁平的4C1构象且C-1-O-5键级为1.92，尽管底物产生了相反的D-葡萄糖端基异构体。过渡态显示出一些适度的差异，但其总体相似性强烈表明，糖基化酶反应的立体化学结果无法预测过渡态结构，此类反应的过渡态结构也无法预测立体化学结果。这些结果支持了先前报道的关于在这些及其他糖基化酶中蛋白质结构对产物构型进行独立拓扑控制的证据。
