The kinetics of adenylosuccinate lyase.

Initial rate kinetics, product inhibition patterns, and equilibrium kinetics all indicate that cleavage of adenylosuccinate (AMP-S) by yeast adenylosuccinate lyase proceeds primarily by a reaction in which fumarate leaves the enzyme before AMP and cannot bind to free enzyme at kinetically significant concentrations. The alternate path, in which AMP leaves first, could be detected at equilibrium by the existence of AMPgAMP-S exchange at saturating levels of fumarate and AMP-S. This exchange was less than onetenth the rate of fumarate+AMP-S exchange. Moreover, evidence was obtained that the high concentrations of fumarate and AMP-S enhanced the AMP exchange rate in a manner unrelated to their roles as substrates. Thus, under steady state conditions, the contribution of the alternate path must be very small. The steady state analysis also provided evidence that breakages of the C-N and C-H bonds of the substrate are not the slowest steps of the over-all reaction. The inability of free enzyme to bind fumarate at kinetically significant concentrations is supported by failure of other dicarboxylic acids to inhibit the enzyme. However, the succinyl group of AMP-S plays a role in attachment to the enzyme, since AMP-S binds 8 times more strongly than AMP, as indicated by their Ki values. It is suggested that the free enzyme exists principally in a conformation that does not bind dicarboxylic acids, but that attachment of AMP or of the AMP portion of AMP-S promotes conversion to a form that can bind fumarate or the succinyl portion of AMP-S. The resultant inability of the enzyme to be inhibited by dicarboxylic acids may be of biological value.