SNAPSHOTS ALONG AN ENZYMATIC-REACTION COORDINATE - ANALYSIS OF A RETAINING BETA-GLYCOSIDE HYDROLASE

The enzymatic hydrolysis of O-glycosidic linkages is one of the most d iverse and widespread reactions in nature and involves a classic ''tex tbook'' enzyme mechanism. A multidisciplinary analysis of a beta-glyco side hydrolase, the Ce15A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-inter mediate, and product complexes have been determined and their intercon versions analyzed kinetically, providing unprecedented insights into t he mechanism of this enzyme class. Substrate is bound in a distorted S -1(3) skew-boat conformation, thereby presenting the anomeric carbon a ppropriately for nucleophilic attack as well as satisfying the stereoe lectronic requirements for an incipient oxocarbenium ion. Leaving grou p departure results in the trapping of a covalent alpha-glycosylenzyme intermediate in which the sugar adopts an undistorted C-4(1) conforma tion. Finally, hydrolysis of this intermediate yields a product comple x in which the sugar is bound in a partially disordered mode, consiste nt with unfavorable interactions and low product affinity.