Human adenylosuccinate synthetase. Partial purification, kinetic and regulatory properties of the enzyme from placenta.

Human adenylosuccinate synthetase (IMP:aspartate ligase (GDP) EC 6.3.4.4) has been purified 1 IO-fold from placenta. The partially purified preparation was stable and free of nucleotidase activity, permitting study of substrate kinetics and regulatory properties. The Michaelis constants for the substrates, IMP, GTP, and aspartate were 37 PM, 31 PM, and 0.95 m&x, respectively. Based on studies of initial velocity and product or end product inhibition, the enzyme kinetic mechanism was compatible with a sequential rapid equilibrium fully random mechanism. Human adenylosuccinate synthetase was inhibited by a variety of purine nucleotides; the corresponding nucleosides and bases were ineffective. Inhibitory effectiveness decreased with the series mono > di > triphosphates. The mechanism of inhibition by specific purine nucleotides revealed that adenylosuccinate, AMP, and XMP were competitive inhibitors with respect to IMP with K; values of 57 PM, 170 PM, and 140 PM, respectively. GMP and GDP were competitive with respect to GTP with Ki values of 10 PM and 45 @u, respectively. These findings suggest that human adenylosuccinate synthetase activity, despite its importance in a branched pathway, is not regulated by a highly specific molecular control mechanism.