Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

We previously demonstrated by X-ray crystallography and electrospraymass spectrometry that D52E mutant hen lysozyme formed a covalentenzyme–substrate adduct on reaction with N-acetylglucosamineoligomer. This observation indicates that D52E lysozyme mayacquire a catalytic pathway via a covalent adduct. To explainthis pathway, the formation and hydrolysis reactions of thecovalent adduct were investigated. Kinetic analysis indicatedthat the hydrolysis step was the rate-limiting step, 60-foldslower than the formation reaction. In the formation reaction,the pH dependence was bell-shaped, which was plausibly explainedby the functions of the two catalytic pK_as of Glu35 and Glu52.On the other hand, the pH dependence in the hydrolysis was sigmoidalwith a transition at pH 4.5, which was identical with the experimentallydetermined pK_a of Glu35 in the covalent adduct, indicating thatGlu35 functions as a general base to hydrolyze the adduct. Toimprove the turnover rate of D52E lysozyme, the mutation ofN46D was designed and introduced to D52E lysozyme. This mutationreduced the activation energy in the hydrolysis reaction ofthe covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C butdid not affect the formation reaction. Our data may providea useful approach to understanding the precise mechanism ofthe function of natural glycosidases, which catalyze via a covalentadduct.