Initial Rate Studies of Adenylosuccinate Synthetase with Product and Competitive Inhibitors

The kinetic mechanism of action of adenylosuccinate synthetase from Escherichia coli, strain B, was investigated from initial rate studies. The kinetic data are consistent with a sequential mechanism for the enzyme. A rapid equilibrium, fully random mechanism was found to be in harmony with the kinetic findings from studies with competitive and product inhibitors. This result serves to explain why partial exchange reactions are not observed with adenylosuccinate synthetase. It is not, however, possible to ascertain the nature of the catalytic transition state. It was observed that competitive inhibitors for a single substrate acted as noncompetitive inhibitors relative to the other substrates. Similarly, guanosine diphosphate functions as a competitive product inhibitor of GTP, but shows noncompetitive inhibition patterns for inosine monophosphate and aspartate. Adenylosuccinate was found to be competitive with respect to IMP and noncompetitive relative to the other substrates. A procedure is presented for replotting initial rate data for three-substrate systems which permits a choice of mechanism to be made from among sequential mechanisms.