Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover.

Based on work of Post et al. (Post, R. L. Toda, G., and Rogers, F.N. (1975) J. Biol, Chem. 250, 691-701), we studied the E2 form reactivity of Na(+),K(+)-ATPase (EC 3.6.1.37) during Na(+)-ATPase turnover by following ATP hydrolysis with and without P(i) and enzyme phosphorylation from P(i) at 20 degrees C. For theoretical calculations we employed the Albers-Post scheme assuming that, even with no K+, E2 exhibits the ATP regulatory site. Using available rate constants the model predicts: (i) without P(i), Na(+)-ATPase displays a single high affinity ATP site but becomes double Michaelian (with high and low ATP affinity) when P(i) is present. (ii) Phosphorylation from P(i) can be detected during Na(+)-ATPase (t 1/2 about 400 ms); the KmP(i) is substantially higher that the KdP(i). (iii) P(i) incorporation is reduced by ATP acting with low affinity; this does not require an increase in the E2-E1 transition rate. (iv) The KmATP of the regulatory site is augmented when [P(i)] increases. The experimental observations, using partially purified pig kidney enzyme, agreed with the predictions. In addition they showed that: (i) extracellular Na+ can prevent P(i) incorporation; this effect is additive with that of ATP but with independent Ki values. (ii) Mg2+ stimulates P(i) incorporation with low affinity (Km of 1.5 mM). (iii) beta, gamma-Methyleneadenosine 5'-triphosphate and palmitoyl-CoA antagonize P(i) inhibition of Na(+)-ATPase. These results agree with a model where the Na(+),K(+)-ATPase and Na(+)-ATPase cycles share most of their intermediate steps and enzyme conformations.