title: Mutagenesis of N-terminal Amino Acid Residues in beta-subunit of Glutaryl-7-amino-cephalosporanic Acid Acylase C130.
abstract: In many GL-7ACA acylases, the first Ser residue at the N-terminal of beta-subunit is the catalytic center. In order to investigate relationship between the N-terminal structure and catalytic activities, peptide replacement and site-directed mutagenesis were performed at the N-terminal of beta-subunit of GL-7ACA acylase C130. When the N-terminal 8 amino acid residues of C130 were replaced by the corresponding sequence of penicillin acylases PAC and PGA, respectively, the first mutant B8PAC lost the activity of the acylase, and the second mutant B8PGA had lower activity with the K(m) value increasing from 0.44x10(-3)mol.L(-1) to 0.55x10(-3) mol.L(-1), and the k(cat) decreasing from 4.92 s(-1) to 1.64 s(-1). Although the substitution of Trp (beta4) by Tyr did not change the K(m) value, the k (cat) decreased to 2.29 s(-1). When the Trp was substitued by Leu, both the K ( m ) and k ( cat ) values decreased. Compared with the wild type, mutations of Ser (beta3) to Met, Ala and Cys caused decrease of K(m) values by 52.27%, 43.18% and 38.64%, respectively. Mutation of Asn (beta2) to Gln caused the K ( m ) value being increased by 5-fold, and k ( cat ) decreased by 10-fold. These results suggested that the N-terminal amino acid residues of beta-subunit in GL-7ACA acylase C130 are important for enzyme function.
authors.affiliation: Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai, 200032, China. wjiang@iris.sipp.ac.cn
authors.forename: N
authors.initials: N
authors.lastname: Zhang
authors.forename: X M
authors.initials: XM
authors.lastname: Ding
authors.forename: X
authors.initials: X
authors.lastname: Huang
authors.forename: E D
authors.initials: ED
authors.lastname: Wang
authors.forename: Y L
authors.initials: YL
authors.lastname: Yang
authors.forename: G P
authors.initials: GP
authors.lastname: Zhao
authors.forename: W H
authors.initials: WH
authors.lastname: Jiang