Studies on the active site of Escherichia coli adenylosuccinate synthetase by chemical modification and site-directed mutagenesis

Reaction of E. coli adenylosuccinate synthetase with thiol reagents leaded to modification of one cysteine residue per subunit without significant loss of enzyme activity. Modification of a second cysteine residue occurred under 3.4 M urea and resulted in complete loss of enzyme activity. The remaining two cystein residues were modified only after treatment with 8 M urea. The reactive cystein had been identified as Cys[superscript]291, and the thiol exposed with 3.5 M urea is Cys[superscript]344. When Cys[superscript]344 was replaced by either serine or alanine, the mutant enzymes were as active as the wild type enzyme. These findings point to the nonessential roles of sulfhydryl groups in the enzyme;Incubation of the enzyme with low concentrations of pyridoxal 5[superscript]\u27-phosphate (PLP) resulted in a rapid loss of activity. Stoichiometry and peptide isolation studies showed that two lysine residues were modified. Complete protection of the enzyme against inactivation can be observed only in the presence of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping revealed that the lysine shielded by GTP is Lys[superscript]140. Replacing the Lys[superscript]140 with Ile[superscript]140 resulted in total loss of the activity. These results suggest that Lys[superscript]140 may play an important role in enzymatic activity;Chemical modification of the enzyme with phenylglyoxal resulted in an inhibition of enzyme acitivity. The substrates, GTP or IMP, can partially protect the enzyme against inactivation. The other substrate, aspartate, had no such effect. In the presence of both IMP and GTP, nearly complete protection of the enzyme against inactivation can be observed. Stoichiometry studies showed that only 1 reactive arginine residue was modified and this arginine residue could be shielded by GTP and IMP. Sequence analysis indicated that Arg[superscript]147 is the site of phenylglyoxal modification. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild type system, whereas the K[subscript] m for aspartate exhibits no significant change compared with the native enzyme. In addition, k[subscript] cat of the R147L mutant decreased by a factor of 1.3 x 10[superscript]4. These results indicate that Arg[superscript]147 is critical for enzyme catalysis