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 electrospray mass s pectrometry that D52E mutant hen lysozyme formed a covalent enzyme-substrat e adduct on reaction with N-acetylglucosamine oligomer, This observation in dicates that D52E lysozyme may acquire a catalytic pathway via a covalent a dduct, To explain this pathway, the formation and hydrolysis reactions of t he covalent adduct were investigated. Kinetic analysis indicated that the h ydrolysis step was the rate-limiting step, 60-fold slower than the formatio n reaction. In the formation reaction, the pH dependence was bell-shaped, w hich was plausibly explained by the functions of the two catalytic pK(a)s o f Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis w as sigmoidal with a transition at pH 4.5, which was identical with the expe rimentally determined pK(a)s of Glu35 in the covalent adduct, indicating th at Glu35 functions as a general base to hydrolyze the adduct, To improve th e turnover rate of D52E lysozyme, the mutation of N46D was designed and int roduced to D52E lysozyme. This mutation reduced the activation energy in th e hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40 degrees C but did not affect the formation reaction, Our data may provid e a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.