|Title||STEREOCHEMISTRY AND KINETICS OF THE HYDRATION OF 2-ACETAMIDO-D-GLUCAL BY BETA-N-ACETYLHEXOSAMINIDASES|
|Publication Type||Journal Article|
|Year of Publication||1994|
|Authors||Lai, ECK, Withers, SG|
|Date Published||DEC 13|
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.