A COVALENT ENZYME-SUBSTRATE INTERMEDIATE WITH SACCHARIDE DISTORTION IN A MUTANT T4 LYSOZYME

The glycosyl-enzyme intermediate in lysozyme action has long been cons idered to be an oxocarbonium ion, although precedent from other glycos idases and theoretical considerations suggest it should be a covalent enzyme-substrate adduct. The mutation of threonine 26 to glutamic acid in the active site cleft of phage T4 lysozyme (T4L) produced an enzym e that cleaved the cell wall of Escherichia coli but left the product covalently bound to the enzyme. The crystalline complex was nonisomorp hous with wild-type T4L, and analysis of its structure showed a covale nt linkage between the product and the newly introduced glutamic acid 26. The covalently linked sugar ring was substantially distorted, sugg esting that distortion of the substrate toward the transition state is important for catalysis, as originally proposed by Phillips. It is al so postulated that the adduct formed by the mutant is an intermediate, consistent with a double displacement mechanism of action in which th e glycosidic linkage is cleaved with retention of configuration as ori ginally proposed by Koshland. The peptide part of the cell wall fragme nt displays extensive hydrogen-bonding interactions with the carboxyl- terminal domain of the enzyme, consistent with previous studies of mut ations in T4L.