KINETIC STUDIES OF ADENYLOSUCCINATE SYNTHETASE: METAL ACTIVATION, ISOTOPE EXCHANGE AND PULSE-CHASE
COOPER, BRUCE FARIEL
Doctor of Philosophy
Adenylosuccinate synthetase catalyzes the reaction: IMP + aspartate + GTP () AMP-S + GDP + Pi. The enzyme required a divalent cation for activity. When the concentration of GTP was equal to the concentration of the metal ion the order of activity followed Mn:GTP > Mg:GTP > Ca:GTP. However, when the metal concentration exceeded the concentration of GTP the order of activity was Mg:GTP > Mn:GTP > Ca:GTP. The increased activity associated with excess concentrations of Mg('2+) and Ca('2+) was due to metal binding at a second site. The reaction with Mn:GTP showed no activation by excess metal concentrations. Kinetic studies with Mg('2+) as a varied substrate yielded a K(,m) value of 140 (mu)M for Mg('2+). The K(,m) determined for aspartate, from experiments when Mg('2+) was varied, was lower than previously reported values for the basic isozyme. The K(,m) for GTP from these studies was identical to previously reported values. A K(,m) for IMP could not be determined by varying Mg('2+) concentration due to the nonlinearity of the double reciprocal plots. In the presence of 1.0 mM excess Mn('2+), the K(,m)'s for aspartate, IMP and GTP were lower than those determined in the presence of excess concentrations of Mg('2+). The reaction mechanism in the presence of Mn('2+) is consistent with steady state binding of aspartate followed by random binding of IMP and GTP. Results of kinetic and inhibitor studies at varying concentrations of Mg('2+) suggested that the second Mg('2+) ion was involved in the formation of the E-IMP-Mg:GTP complex. The metal ion in the second site was closely associated with the Me:GTP binding site since activation of the reaction when Mg:GTP was a substrate occurred only when Mg('2+) occupied the second site. Mg('2+) in the second site can not activate the reaction involving Mn:GTP. Ca('2+) can compete with Mg('2+) for the second site but Ca('2+) did not activate the reaction when Mg:GTP was a substrate. A comparison of the acidic and basic isozymes of adenylosuccinate synthetase from rat tissue showed that the two isozymes had similar metal specificities although the acidic isozyme required a lower excess of Mg('2+) for maximum activity. Isotope exchange at equilibrium and pulse-chase studies using the basic isozyme were consistent with a fully random sequential mechanism involving rapid equilibrium binding of aspartate and steady state binding of GTP and IMP. The reverse reaction may involve rapid equilibrium binding of the products (GDP, Pi and adenylosuccinate). The experiments were performed in the presence of excess Mg('2+).