A Covalent Enzyme-Substrate Adduct in a Mutant Hen Egg White Lysozyme (D52E)

Ryota Kuroki,Yuji Ito,Yoichi Kato,Taiji Imoto

doi:10.1074/jbc.272.32.19976

Abstract

A mutant hen egg white lysozyme, D52E, was designed to correspond to the 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 alpha-side of the saccharide in these enzymes. The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crystallographic analysis showed that the covalent adduct was formed between Glu-52 and the C-1 carbon of the N-acetylglucosamine residue in subsite D of the saccharide binding site. It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-substrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme. It was confirmed that the substitution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme. Although the position 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 alpha-side of the saccharide is considered to be responsible for the functional variation in their glycosidase and transglycosidase activities.

Highlights

It is known that the catalytic sites of most glycosidases have common features
It is known that the kind of catalytic residue located on the ␣-side of the saccharide seems more adaptable because the acidic residue corresponding to Asp-52 of HEWL was not found on the ␣-side of the saccharide in GEWL (13), mutant HEWL (14), or mutant T4L (15–17)
Asp-20 in T4L has been considered to correspond to Asp-52 in HEWL (8), these residues were excluded from the structural comparison because the catalytic role of these aspartic acids were recently proposed to be different (11, 12)

Summary

EXPERIMENTAL PROCEDURES

Materials—The wild type and D52E mutant lysozymes were prepared by expression and secretion from yeast as described previously (18). Molecular mass determination of the proteins eluted from the reverse phase column was carried out on a Finnigan TSQ7000 mass spectrometer (Finnigan MAT, San Jose, CA) with an ion-spray interface and a quadruple mass analyzer with an upper mass limit of m/z ϭ 4000 Da. Large Scale Preparation of the D52E Mutant with a Covalent Adduct for Crystallization—One mg of D52E mutant was allowed to react with 1 mg of (NAG) at pH 5.0 and 40 °C for 2 days. Crystallization and Tertiary Structure Analysis of the D52E Mutant Lysozyme with and without Saccharide Adduct—Crystallization of the apo-D52E mutant and the D52E mutant with a covalent adduct were performed using approximately the same method as that of the wild type reported previously (6, 19). In the case of the crystallization of D52E mutant with a covalent adduct, no extra (NAG)

Subsitea CCDDD

RESULTS

Reflections included

DISCUSSION