Na+/K(+)-ATPase: modes of inhibition by Mg2+

Adding 15 mM free Mg2+ decreased Vmax of the Na+/K(+)-ATPase reaction. Mg2+ also decreased the K0.5 for K+ activation, as a mixed inhibitor, but the increased inhibition at higher K+ concentrations diminished as the Na+ concentration was raised. Inhibition was greater with Rb+ but less with Li+ when these cations substituted for K+ at pH 7.5, while at pH 8.5 inhibition was generally less and essentially the same with all three cations: implying an association between inhibition and ion occlusion. On the other hand, Mg2+ increased the K0.5 for Na(+)-activation of the Na+/K(+)-ATPase and Na(+)-ATPase reactions, as a mixed inhibitor. Changing incubation pH or temperature, or adding dimethylsulfoxide affected inhibition by Mg2+ and K0.5 for Na+ diversely. Presteady-state kinetic studies on enzyme phosphorylation, however, showed competition between Mg2+ and Na+. In the K(+)-phosphatase reaction catalyzed by this enzyme Mg2+ was a (near) competitor toward K+. Adding Na+ with K+ inhibited phosphatase activity, but under these conditions 15 mM Mg2+ stimulated rather than inhibited; still higher Mg2+ concentrations then inhibited with K+ plus Na+. Similar stimulation and inhibition occurred when Mn2+ was substituted for Mg2+, although the concentrations required were an order of magnitude less. In all these experiments no ionic substitutions were made to maintain ionic strength, since alternative cations, such as choline, produced various specific effects themselves. Kinetic analyses, in terms of product inhibition by Mg2+, require Mg2+ release at multiple steps. The data are accommodated by a scheme for the Na+/K(+)-ATPase with three alternative points for release: before MgATP binding, before K+ release and before Na+ binding. The latter alternatives necessitate two Mg2+ ions bound simultaneously to the enzyme, presumably to divalent cation-sites associated with the phosphate and the nucleotide domains of the active site.