Site-directed Mutagenesis of the Active Center of Penicillin Acylase from E. coli ATCC 11105.
Site-directed mutagenesis and chemical modification were performed at Ser290 of the penicillin G acylase from E. coli ATCC11105. The Ser290 was substituted by Cys or Secys. Wild type and mutant proteins were purified, and the activities and kinetic constants of penicillin acylases for hydrolysis and synthesis were determined, respectively. Although their K(m) values were not changed, the k(cat) values of the thiol-PGA and seleno-PGA were decreased from 135s(-1) to 0.63s(-1) and 0.38s(-1) against NIPAB, and from 34.38s(-1) to 0.23s(-1) and 0.06s(-1) against penicillin G. Contrary to Choi's report(Choi K S (et al. J Bacteriology), 1992, 10 6270-6276), we found that hydrolysis activity was certainly kept in the mutant of penicillin acylase. In addition, the specific activities of synthesis were decreased by 5-fold and 20-fold, respectively.
Dai
Ming-Hua
MH
Laboratory of Molecular Regulation for Microbial Secondary Metabolism, Shanghai Institute of Plant Physiology, the Chinese Academy of Sciences, Shanghai 200032, China. wjiang@iris.sipp.ac.cn
Wang
En-Duo
ED
Xie
Yong
Y
Jiang
Wei-Hong
WH
Zhao
Guo-Ping
GP