Steady-state kinetic analysis of the Na+/K+-ATPase. The inhibition by potassium and magnesium.

At micromolar ATP, low (< 5 mM) concentrations of K+ activate the Na+/K+-ATPase to an extent that is substantially reduced compared to that observed at more physiological concentrations of the nucleotide. At higher concentrations of K+, activation is replaced by partial inhibition. Inhibition is not due to the displacement of Na+ by K+, its main causes being a decrease of the Vm of the high-affinity component and the increase in the apparent Km of the low-affinity component of the substrate curve of the Na+/K+-ATPase. The apparent affinity for inhibition by K+ is highly dependent on Mg2+. In the presence of an excess K+, Mg2+ decreases towards zero the Vm of the high-affinity component and acts as a dead-end inhibitor of the low-affinity component of the substrate curve of the Na+/K+-ATPase. These results can be explained assuming that binding of an additional K+ to the E2 conformer of the Na+/K+-ATPase allows low-affinity binding of Mg2+ with the formation of a dead-end complex. In the case of Na+-ATPase activity and for concentrations of ATP within the range of the substrate curve of this activity (0-2.5 muM), Mg2+ in concentrations up to 60 mM has no effect on ATPase activity at high (100 mM) [Na+]. At lower [Na+], Mg2+ becomes a low-affinity inhibitor of Na+-ATPase. Inhibition follows a pattern that is different from inhibition of Na+/K+-ATPase activity and is consistent with a mechanism in which Mg2+ acts both as a dead-end and as product inhibitor.