Site-directed Mutagenesis of the Active
Center of Penicillin Acylase from E. coli ATCC 11105
DAI Ming-Hua, WANG En-Duo, XIE Yong, JIANG Wei-Hong and ZHAO
Guo-Ping
( 1 Laboratory of Molecular Regulation for
Microbial Secondary Metabolism,Shanghai Institute of Plant Physiology the
Chinese Academy of Sciences, Shanghai 200032, China；2 State Key Laboratory of
Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of
Sciences, Shanghai 200031, China；3 Department of Biology,
Hong Kong Science and Technology University, Hong Kong )
Abstract 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 Km values were not changed,
the kcat 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.
Key Words penicillin acylase； site-directed mutagenesis； chemical modification； catalytic center
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