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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-gluc...
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Published in: | Biochemistry (Easton) 1994-12, Vol.33 (49), p.14743-14749 |
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Main Authors: | , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that cite this one |
Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi00253a012 |