Mutagenesis of
N-terminal Amino Acid Residues in β-subunit of Glutaryl-7-amino-cephalosporanic
Acid Acylase C130
ZHANG Ni, DING Xiao-Ming, HUANG Xi, WANG En-Duo1,
YANG Yun-Liu, ZHAO Guo-Ping, JIANG Wei-Hong*
( Institute of Plant Physiology and Ecology, Shanghai Institutes for
Biological Sciences, the Chinese Academy of Sciences, Shanghai, 200032,
China； 1Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of
Sciences, Shanghai 200031,
China )
Abstract In many
GL-7ACA acylases, the first Ser residue at the N-terminal of β-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 β-subunit of GL-7ACAacylase
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 Km value increasing from
0.44×10-3mol・L-1 to 0.55×10-3 mol・L-1,
and the kcat decreasing from 4.92 s-1 to 1.64 s-1.
Although the substitution of Trp (β4) by Tyr did not change the Km
value, the kcat decreased to 2.29 s-1. When the
Trp was substitued by Leu, both the Km and kcat
values decreased. Compared with the wild type, mutations of Ser (β3) to Met,Ala
and Cys caused decrease of Km values by 52.27%, 43.18% and
38.64%, respectively. Mutation of Asn (β2) to Gln caused the Km
value being increased by 5-fold, and kcat decreased by
10-fold. These results suggested that the N-terminal amino acid residues of
β-subunit in GL-7ACA acylase C130 are important for enzyme function.
Key words GL-7ACA acylase； mutagenesis； catalytic center
*Corresponding author： Tel, 86-21-64170709； Fax, 86-21-64042385； e-mail, [email protected]