A study of the vanadate-trapped state of the (Na,K)-ATPase. Evidence against interacting nucleotide site models.

The state of the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) which binds vanadate was investigated by taking advantage of the slow rate of vanadate release (koff = 0.32/h at 25 degrees C and 0.045/h at 4 degrees C). Vanadate release from the enzyme could be accelerated approximately 2-fold by addition of K+ and 50-fold by addition of Na+. The K+ effect saturated hyperbolically with a K1/2 of 0.5 mM, while the Na+ effect had a sigmoidal activation curve and K1/2 of 250 mM. These results indicate that either Na+ or K+ can equilibrate with the vanadate-"trapped" enzyme prior to vanadate release. In the presence of vanadate, a saturable Mn2+ binding site could be detected with a dissociation constant of 120 nM. When 54Mn2+ was added during incubation of the enzyme with vanadate, 1 mol of 54Mn2+ could be trapped/mol of vanadate trapped and the two metals dissociated in parallel. This result indicates a single divalent cation site is involved in stabilizing vanadate (and probably phosphate) binding. Addition of 1 to 4 mM of ATP to the vanadate-trapped enzyme had no affect on the rate of vanadate release. Also, the high affinity ATP site could not be detected in equilibrium-binding studies with the vanadate-trapped enzyme. Since kinetic experiments indicate that vanadate binding is competitive with the low affinity ATP site (Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978) J. Biol. Chem. 253, 7361--7368) and the high affinity ATP site is absent on vanadate-trapped enzyme, it appears unlikely that ATP can occupy either a high or low affinity site on the vanadate-trapped enzyme. We show that nonlinear Lineweaver-Burk plots for ATP hydrolysis can be explained by a single hydrolysis site which exhibits a low affinity for ATP prior to the rate-limiting E2 to E1 conformational change, but a high affinity for ATP following the conformational change.