Stereochemistry and kinetics of the hydration of 2-acetamido-D-glucal by beta-N-acetylhexosaminidases.
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 Vmax and Km values of 0.48 +/- 0.01 unit/mg and 27 +/- 2.8 microM, respectively. The same substrate is also bound very tightly by the human and bovine enzymes, with Ki values of 8 and 25 microM, 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.
Lai
E C
EC
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Withers
S G
SG
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D007887:Fabaceae; D006595:Hexosamines / Q000378:metabolism* / Q000494:pharmacology; D006596:Hexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010946:Plants, Medicinal; D011485:Protein Binding; D013237:Stereoisomerism
D006595:Hexosamines; C019231:2-acetamidoglucal; D006596:Hexosaminidases