Shift to the Na+ form of Na+/K+-transporting ATPase due to modification of the low-affinity ATP-binding site by Co(NH3)4ATP.

1. Inactivation of purified Na+/K+-transporting ATPase by the MgATP complex analogue Co(NH3)4ATP, which binds to the low-affinity ATP-binding site, results in the concomitant inhibition of the K+-activated p-nitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state. 2. Complete inactivation of Na+/K+-transporting ATPase by Co(NH3)4ATP does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the E1 conformation. 3. The enzyme binds eosin at the high-affinity ATP-binding site as measured by the change in eosin fluorescence. Eosin binding to the Co(NH3)4ATP-inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na1. Inactivation by Co(NH3)4ATP increased the half-maximal opposing effect of K+ on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP-inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high-affinity ATP-binding site. CrATP thus abolishes eosin binding. 4. It is concluded, that Co(NH3)4ATP interacts with Na+/K+-transporting ATPase in the E2 conformation and arrests it there. This affects eosin binding to the high-affinity ATP-binding site, since the K+ sensitivity is lost. A possible interpretation of these differing effects of Co(NH3)4ATP on partial reactions of Na+/K+-transporting ATPase is that the sodium pump works as an (alpha,beta)2 diprotomer. It is likely that the arrest of one alpha,beta promoter in the E2 conformational state by occupancy of the low-affinity ATP-binding site with Co(NH3)4ATP induces the Na+ form (E1 form) in the corresponding alpha,beta promoter, as is indicated by the unaffected ADP/ATP exchange and the response of the eosin fluorescence on Na+ and K+.