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Protein Engineering, Vol. 12, No. 4, 327-331, April 1999
© 1999 Oxford University Press

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

Yuji Ito1,2, Ryota Kuroki3, Yoko Ogata1, Yoshio Hashimoto1, Kazuhisa Sugimura2 and Taiji Imoto1,4

1 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812–8582, 2 Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto 890–0065 and 3 Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1–13–5, Fukuura, Kanazawa-ku, Yokohama 236, Japan

We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme–substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4.5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.

Keywords: catalyst redesign/glycosidase/glycosyl adduct/intermediate

4 To whom correspondence should be addressed. Email: imoto{at}imml.phar.kyushu-u.ac.jp


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