S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet from ATP and L-methionine with subsequent hydrolysis of the bound tripolyphosphate intermediate. Maximal activity requires the presence of two divalent and one monovalent cation per active site. Recently, the x-ray structure of the Escherichia coli AdoMet synthetase was solved, and the positions of the two Mg2+ binding sites were identified. Based on additional spherical electron density, the K+ binding site was postulated to be a nearby site where the uranyl heavy atom derivative also bound in the crystal. The side chain of glutamate 42 is within ligation distance of the metals. Mutagenesis of glutamate 42 to glutamine (E42QMetK) abolished monovalent cation activation and produced an enzyme that has kinetic properties virtually identical to those of K(+)-free wild type AdoMet synthetase in both the overall AdoMet synthetase reaction and in the hydrolysis of tripolyphosphate. Thus, there is a approximately 100-fold decrease in the Vmax for AdoMet synthesis and large increases in the Km values for both substrates. In contrast there is only a 2-fold decrease in Vmax for tripolyphosphate hydrolysis. The uranyl ion, UO2(2+), is a competitive inhibitor with respect to K+ (Ki = 350 nM) and is the first ion to bind at this site and inhibit the enzyme. The UO2(2+) inhibition is reversible and tight-binding, and results from UO2(2+) and not UO2(2+)-ATP. Analogous to K+ activation, UO2(2+) predominantly inhibits AdoMet formation rather than tripolyphosphate hydrolysis. The kinetic results indicate that UO2(2+) inhibition is likely to result from interference with productive ATP binding. UO2(2+) remains a tight-binding inhibitor of the E42Q mutant, which suggests that K+ and UO2(2+) have different ligation preferences when bound in the monovalent cation binding pocket. The results support the model that glutamate 42 provides ligands to the K+ and has a major role in monovalent cation binding.