A COVALENT ENZYME-SUBSTRATE ADDUCT IN A MUTANT HEN EGG-WHITE LYSOZYME(D52E)

A mutant hen egg white lysozyme, D52E, was designed to correspond to t he structure of the mutant T4 lysozyme T26E (Kuroki, R., Weaver, L. H. , and Matthews B. W. (1993) Science 262, 2030-2033) to investigate the role of the catalytic residue on the ct-side of the saccharide in the se enzymes, The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crys tallographic analysis showed that the covalent adduct was formed betwe en Glu-52 and the C-1 carbon of the N-acetylglucosamine residue in sub site D of the saccharide binding site, It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-s ubstrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme, It was confirmed that the substi tution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme, Although the po sition of the catalytic residue on the beta-side of the saccharide is quite conserved among hen egg white lysozyme, goose egg white lysozyme , and T4 phage lysozyme, the adaptability of the side chain on the alp ha-side of the saccharide is considered to be responsible for the func tional variation in their glycosidase and transglycosidase activities.