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INACTIVATION OF A BETA-GLUCOSIDASE THROUGH THE ACCUMULATION OF A STABLE 2-DEOXY-2-FLUORO-ALPHA-D-GLUCOPYRANOSYL ENZYME INTERMEDIATE - A DETAILED INVESTIGATION

TitleINACTIVATION OF A BETA-GLUCOSIDASE THROUGH THE ACCUMULATION OF A STABLE 2-DEOXY-2-FLUORO-ALPHA-D-GLUCOPYRANOSYL ENZYME INTERMEDIATE - A DETAILED INVESTIGATION
Publication TypeJournal Article
Year of Publication1992
AuthorsSTREET, IP, KEMPTON, JB, Withers, SG
JournalBIOCHEMISTRY
Volume31
Pagination9970-9978
Date PublishedOCT 20
ISSN0006-2960
Abstract

The inactivation of glycosidases by 2-deoxy-2-fluoroglycosides has been shown previously to occur via the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucopyranosyl enzyme intermediate {[}Withers, S. G., & Street, 1. P. (1988) J. Am. Chem. Soc. 110, 8551]. Further characterization of this process with Agrobacterium beta-glucosidase is described, and the range of glycosides engaging in this behavior is examined. Inactivation is shown to be accompanied by the release of a stoichiometric ``burst{''} of aglycon, thereby providing a new class of active site titration agents for glycosidases. The rate of inactivation is shown to be very strongly dependent on the leaving group ability of the aglycon, the slowest inactivator studied (p-nitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside) yielding only partial inactivation due to turnover of the intermediate becoming competitive with its formation. Such turnover of the intermediate is shown to be greatly accelerated by transglycosylation to a suitable glycoside bound in the aglycon site, resulting in the release of a disaccharide product which was isolated and characterized. The pH dependences of both the formation and the hydrolysis of the 2-deoxy-2-fluoroglycosyl-enzyme closely resemble those of each step for normal catalysis, indicating that the same catalytic groups are involved in both processes. A model system for the partial ``steady-state{''} inactivation observed previously {[}Withers, S. G., Rupitz, K., & Street, I. P. (1988) J. Biol. Chem. 263, 79291 with certain other glycosidases was established by incubating the enzyme with an inactivator known to undergo relatively rapid transglycosylation in the presence of various concentrations of a suitable reactivator. The extent of steady-state inactivation observed in each case agreed closely with that predicted according to the kinetic scheme proposed.

DOI10.1021/bi00156a016