|Title||SUBSTRATE-INDUCED INACTIVATION OF A CRIPPLED BETA-GLUCOSIDASE MUTANT - IDENTIFICATION OF THE LABELED AMINO-ACID AND MUTAGENIC ANALYSIS OF ITS ROLE|
|Publication Type||Journal Article|
|Year of Publication||1995|
|Authors||GEBLER, JC, TRIMBUR, DE, WARREN, AJ, AEBERSOLD, R, NAMCHUK, M, Withers, SG|
|Date Published||NOV 7|
The beta-glucosidase from Agrobacterium sp. catalyzes the hydrolysis of beta-glucosides via a covalent alpha-D-glucopyranosyl-enzyme intermediate involving Glu358. Hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside by the low activity Glu358Asp mutant of Agrobacterium beta-glucosidase is accompanied by time-dependent inactivation of the enzyme. Through kinetic studies, labeling, and sequence analysis, inactivation is shown to be a consequence of the occasional (1 time in 1100) attack of Tyr298 on the anomeric center of the substrate, in place of the catalytic nucleophile, with formation of a stable alpha-D-glucopyranosyl tyrosine residue. Tyr298 is conserved throughout family 1 of glycoside hydrolases, an indication of a possible role in catalysis. Results of a kinetic analysis of the Tyr298Phe mutant are consistent with a function of Tyr298 in both orienting the nearby nucleophile Glu358 and stabilizing its deprotonated state in the free enzyme.