Breakdown of Na+/K+‐exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+‐ATPase activity.

The reactivity towards Na+ and K+ of Na+/K+‐ATPase phosphoenzymes formed from ATP and Pi during Na+‐ATPase turnover and that obtained from Pi in the absence of ATP, Na+ and K+ was studied. The phosphoenzyme formed from Pi in the absence of cycling and with no Na+ or K+ in the medium showed a biphasic time‐dependent breakdown. The fast component, 96% of the total EP, had a decay rate of about 4 s−1 in K+‐free 130 mm Na+, and was 40% inhibited by 20 mm K+. The slow component, about 0.14 s−1, was K+ insensitive. Values for the time‐dependent breakdown of the phosphoenzymes obtained from ATP and from Pi during Na+‐ATPase activity were indistinguishable from each other. In K+‐free medium containing 130 mm Na+, the decays followed a single exponential with a rate constant of 0.45 s−1. The addition of 20 mm K+ markedly increased the decays and made them biphasic. The fast components had a rate of ≈ 220 s‐1 and accounted for 92–93% of the total phosphoenzyme. The slow components decayed at a rate of about 47–53 s−1. A second group of experiments examined the reactivity towards Na+ of the E2P forms obtained with ATP and Pi when the enzyme was cycling. In both cases, the rate of dephosphorylation was a biphasic function of [Na+]: inhibition at low [Na+], with a minimum at about 5 mm Na+, followed by recovery at higher [Na+]. Although qualitatively similar, the phosphoenzyme formed from Pi showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na+‐ATPase turnover share the same intermediates.