Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis.
Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.
Dong
Q
Q
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Liu
F
F
Myers
A M
AM
Fromm
H J
HJ
D000265:Adenylosuccinate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000737:chemistry; D007291:Inosine Monophosphate / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010658:Phenylglyoxal / Q000494:pharmacology; D014357:Trypsin
D007291:Inosine Monophosphate; D006160:Guanosine Triphosphate; D001120:Arginine; D014357:Trypsin; D000265:Adenylosuccinate Synthase; D010658:Phenylglyoxal