A characterization of vanadate interactions with the (Na,K)-ATPase. Mechanistic and regulatory implications.

The interaction of vanadium in the +5 oxidation state (vanadate) with purified dog kidney Na+ and K+-stimulated adenosine trlphosphatase [(Na,K)-ATPase] has been studied using equilibrium binding, steady state kinetics, and measurements of relaxation kinetics. Vanadate binds to one high affinity site (Kl = 4 no) and one low afinity site (Kz = 0.5 pM) per enzyme molecule (Le. per ouabain binding site) under optimal conditions. Saturation of the high affinity site is sufficient to cause greater than 95% inhibition of (Na,K)-ATPase activity. Competitive inhibition experiments indicate that the high affmity vanadate site is identical with a low affinity ATP site (K,,, = 3 mM) on the enzyme. The low affinity vanadate site is responsible for inhibition of Na+-stimulated adenosine triphosphatase activity and is probably the high affinity ATP site associated with sodium-dependent protein phosphorylation. In the presence of vanadate, the enzyme has a higher affinity for Mg+ and K+ as judged by steady state kinetics measurements. The K+ sites were investigated using equilibrium binding of *6Rb+ (a potassium congener) in the presence and absence of vanadate. Vanadate increased the affinity of two Rb+ sites (per ouabain binding site) approximately &fold (from & = 0.77 m&I to 0.15 mM). It is proposed that the unusually high affinity for vanadate is due to its ability to form a trigonal bipyramidal structure analogous to the transition state for phosphate hydrolysis. On the basis of these data, an alternating site model for ATP hydrolysis is advanced.