Demonstration of carbonium ion intermediate during lysozyme catalysis.

X-ray analysis studies1' 2 on the relative modes of association of various inhibitors and substrates with lysozyme have led to a proposal for the catalytic mechanism of the enzyme.3'4 This proposal suggests that the enzyme catalyzes the formation of a carbonium ion at C1 of the glycosidic bond undergoing hydrolysis and, further, provides it with steric and electrostatic stabilization. To date, little evidence of a chemical nature has been available to test the mechanistic proposal. It has recently been shown5 that hen egg-white lysozyme-catalyzed hydrolysis of a glycosidic bond proceeds with quantitative retention of configuration (>99.7%). This result eliminates a single displacement mechanism for the enzyme6 and leaves the following: (1) a carbonium-ion mechanism whose enzyme-bound orientation would allow reaction with solvent from only one side (i.e., frontal attack); (2) any mechanism involving an even number of displacements at C1 of the glycoside being cleaved, e.g., the double-displacement mechanism7 which could involve a covalent enzyme-substrate intermediate; and (3) a variation of (2) in which the acetamido carbonyl group of the glycoside would displace the aglycone with formation of an oxazoline intermediate. Evidence for this mechanism has been obtained in model studies of N-acetyl-Dglucosamine derivatives8 and in aryl-2-acetamido-2-deoxy-,B-D-glucopyranoside spontaneous hydrolysis.9 10 Such a scheme has been suggested as a mechanism to explain lysozyme catalysis.9-13 We have recently shown that lysozyme catalyzes the hydrolysis of aryl-4-O(2-acetamido-2-deoxy-j3-D-glucopyranosyl)-f,-D-glucopyranosides (I) with release of the aglycone.14 Furthermore, participation by the C2-OH as a nucleophilic neighboring group was ruled out since the analogous 2-deoxy-D-glucose