[Studies on the function of Ser579 and Arg580 in beta-subunit of penicillin G acylase with the method of site-specific mutagenesis].
According to the comparison of amino acid sequence between PGA (Penicillin G Acylase) and PBPs (Penicillin Binding Protein), We suggest that No. 565-595 peptide fragment in beta-subunit of PGA may be a substrate-binding site of enzyme. Plasmid pTZGA was constructed by cloning the 2.6 kb PGA gene of pWGA into phagemid pTZ18U The technique of site-specific mutagenesis was used to study the role of residue No. 579 (Ser) and No. 580 (Arg) of PGA. Four kinds of mutants were obtained (Ser579-->Gly579, Arg580-->Gly580, Arg580-->Glu580, Arg580-->Lys580), both Glu580 and Gly580 mutants showed no activity of enzyme and Lys580 mutant remained 30% and Gly579 mutant kept 70% activity of wilde type. The same protein expression of four mutants according to the results of ELISA indicate that mutation does not affect the expression of PGA, but Arg580 residue may be essential for substrate-binding or catalysis of PGA.
Fei
J
J
Shanghai Institute of Cell Biology, Academia Sinica.
Lin
Q C
QC
Cai
G Q
GQ
Zhang
M H
MH
Huang
Q
Q
Wang
Y K
YK
Guo
L H
LH
Zhang
Q J
QJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006602:Hexosyltransferases; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000235:genetics*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010399:Penicillin Amidase