Involvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16.
The glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase from Pseudomonas sp. strain GK16 is an (alphabeta)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. The newly formed N-terminal serine of the beta subunit plays an essential role as a nucleophile in enzyme activity. Chemical modification studies on the recombinant enzyme purified from Escherichia coli revealed the involvement of a single arginine and tryptophan residue, per alphabeta heterodimer of the enzyme, in the catalytic activity of the enzyme. Glutaric acid, 7-aminocephalosporanic acid (7-ACA) (competitive inhibitors) and GL-7-ACA (substrate) could not protect the enzyme against phenylglyoxal-mediated inactivation, whereas except for glutaric acid protection was observed in case of N-bromosuccinimide-mediated inactivation of the enzyme. Kinetic parameters of partially inactivated enzyme samples suggested that while arginine is involved in catalysis, tryptophan is involved in substrate binding.
Lee
Y S
YS
Graduate School of Biotechnology, Korea University, Seoul, South Korea.
Kim
H W
HW
Lee
K B
KB
Park
S S
SS
D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001120:Arginine; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D007700:Kinetics; D010399:Penicillin Amidase; D010658:Phenylglyoxal / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan
D002511:Cephalosporins; D011994:Recombinant Proteins; C081063:glutaryl-7-aminocephalosporanic acid; D014364:Tryptophan; D001120:Arginine; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase; D001974:Bromosuccinimide; D010658:Phenylglyoxal