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

Hydrolysis by three beta-N-acetylhexosaminidases (human placenta, jack bean, and bovine kidney) is shown to occur with the retention of anom eric configuration, most likely via a double-displacement mechanism in volving 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 u nit/mg and 27 +/- 2.8 mu M, respectively. The same substrate is also b ound 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-g lucal, yielding N-acetyl-D-glucosamine as the product in each case. Th is 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-gly cosidase. Kinetic studies of this hydration reaction with the jack bea n enzyme demonstrate that the tight binding observed is due to the for mation of a high-affinity, reversible complex, and not due to the accu mulation of a reaction intermediate. This indicates that correctly sub stituted glycals might act as transition state mimics and suggests app roaches to the design of high-affinity inhibitors of beta-N-acetylhexo saminidases.