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STEREOCHEMISTRY AND KINETICS OF THE HYDRATION OF 2-ACETAMIDO-D-GLUCAL BY BETA-N-ACETYLHEXOSAMINIDASES

TitleSTEREOCHEMISTRY AND KINETICS OF THE HYDRATION OF 2-ACETAMIDO-D-GLUCAL BY BETA-N-ACETYLHEXOSAMINIDASES
Publication TypeJournal Article
Year of Publication1994
AuthorsLai, ECK, Withers, SG
JournalBIOCHEMISTRY
Volume33
Pagination14743-14749
Date PublishedDEC 13
ISSN0006-2960
Abstract

Hydrolysis by three beta-N-acetylhexosaminidases (human placenta, jack bean, and bovine kidney) is shown to occur with the retention of anomeric configuration, most likely via a double-displacement mechanism involving the formation and hydrolysis of a glycosyl-enzyme intermediate. 2-Acetamido-D-glucal is shown to be a slow, tight-binding substrate for the jack bean enzyme, with V-max and K-m values of 0.48 +/- 0.01 unit/mg and 27 +/- 2.8 mu M, respectively. The same substrate is also bound Very tightly by the human and bovine enzymes, with K-i values of 8 and 25 mu M, respectively. All three enzymes hydrate 2-acetamido-D-glucal, yielding N-acetyl-D-glucosamine as the product in each case. This is the first time that proton transfer has been shown to occur from the top face during the hydration of a glycal by a retaining beta-glycosidase. Kinetic studies of this hydration reaction with the jack bean enzyme demonstrate that the tight binding observed is due to the formation of a high-affinity, reversible complex, and not due to the accumulation of a reaction intermediate. This indicates that correctly substituted glycals might act as transition state mimics and suggests approaches to the design of high-affinity inhibitors of beta-N-acetylhexosaminidases.

DOI10.1021/bi00253a012