This study reports that two Mg2+ ions are required for Escherichia coli adenylosuccinate synthetase activity. The first metal ion is presumably coordinated with beta- and gamma-phosphoryl groups of GTP to provide an electron sink, and the second one seems to interact with aspartate in the enzyme active site. Regarding the latter metal ion, kinetic studies show that aspartate and the second Mg2+ ion bind to the enzyme active site randomly with a kcat value of 1.47 s-1 and with Km values for aspartate and Mg2+ of 225 and 114 microM, respectively. The dissociation constants for aspartate and Mg2+ of the enzyme.GTP.IMP.(aspartate or Mg2+) complex are 79.2 and 40.0 microM, respectively. However, variable amounts of aspartate or Mg2+ did not show any significant changes in the Km values for GTP and IMP. Kinetic studies using Mn2+ and Ca2+ ions indicate that the kcat values (0.930 and 0.235 s-1, respectively) were slightly decreased compared with the value obtained using Mg2+; however, the Km values for aspartate and GTP in the presence of Mn2+ and Ca2+ were significantly decreased compared with those obtained using Mg2+ ion (4.5 and 4.6 times for Mn2+ ion and 5.6 and 5.8 times for Ca2+ ion, respectively). On the other hand, the Km values for IMP were not significantly changed (1.9 and 1.8 times for Mn2+ and Ca2+ ions, respectively). Taken together, these kinetic results imply that aspartate may interact with Mg2+ to form a Mg.aspartate complex in the enzyme active site. An inhibition study of the enzyme with ZnCl2 (its Ki value is 29 nM) also suggested that Zn2+ competes with aspartate as well as Mg2+, implying that Zn2+ might form a complex with aspartate in the active site. On the basis of these results, it is suggested that Mg.aspartate complex formation in the active site of adenylosuccinate synthetase may be important in activation of the protonated amino group of aspartate, enhancement of the enzyme's binding affinity, and its specificity for aspartate.