Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments.

The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.