Involvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp strain GK16

Abstract

The glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase from Pseudomonas sp. strain GK16 is an (alpha beta)(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 alpha beta 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. (C) 2000 Elsevier Science B.V. All rights reserved.