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.