Conformational transitions between Na +-bound and K +-bound forms of (Na + + K +)-ATPase, studied with formycin nucleotides

Abstract

1. 1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na +-containing media can be displaced by the following ions (listed in order of effectiveness): Tl +, K +, Rb +, NH 4 +, Cs +. 2. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na + and predominantly K + media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. 3. The transition from a K +-bound form (E 2 · (K)) to an Na +-bound form (E 1 · Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E 2 · (K) accelerates its conversion to E 1 · Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K +-catalysed hydrolysis of E 2P, to a form in which it can be phosphorylated in the presence of Na +. 4. 4. The rate of the reverse transformation, E 1 · Na to E 2 · (K), varies roughly linearly with the K + concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K + are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K + with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E 2 · (K), containing occluded K +. 5. 5. Mg 2+ or oligomycin slows the rate of conversion of E 1 · Na to E 2 · (K) but does not significantly affect the rate of conversion of E 2 · (K) to E 1 · Na. 6. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na + and K + each involve the transfer of a phorpharyl group as well as a change in conformation between E 1 and E 2 forms of the enzyme or phosphoenzyme.