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 P i during Na + ‐ATPase turnover and that obtained from P i in the absence of ATP, Na + and K + was studied. The phosphoenzyme formed from P i 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 m m 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 P i during Na + ‐ATPase activity were indistinguishable from each other. In K + ‐free medium containing 130 m m Na + , the decays followed a single exponential with a rate constant of 0.45 s −1 . The addition of 20 m m 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 E 2 P forms obtained with ATP and P i 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 P i showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na + ‐ATPase turnover share the same intermediates.