Transient kinetics of (Na⁺ + K⁺)-ATPase studied with a fluorescent substrate

Abstract

WE describe experiments, with a purified (Na⁺+K⁺)-ATPase, designed to investigate details of the enzymatic process that couples ATP hydrolysis to the active transport of Na⁺ and K⁺ ions. We have found that formycin triphosphate^1–3 (FTP), an analogue of ATP previously used in studies of myosin ATPase⁴, is a substrate for the (Na⁺+K⁺)-ATPase, and that the binding of FTP or of formycin diphosphate (FDP) to the enzyme is accompanied by a two- to threefold enhancement of nucleotide fluorescence. The change in strength of the fluorescent signal has allowed us to measure equilibrium binding of the nucleotides to the enzyme, and using stopped-flow fluorimetry we have been able to measure the rates of binding and release. FTP and FDP bind with a high affinity and may be displaced by excess ATP or by K⁺ ions. During turnover, at least with FTP concentrations up to 24 µM, the FDP is released (or its fluorescence is quenched) before the rate-limiting step of the overall reaction, both in the presence and absence of K⁺ ions. When turnover is prevented by the absence of magnesium, the enzyme is still able to change its conformation according to whether Na⁺ or K⁺ is the predominant alkali-metal ion⁵. Because the Na and K conformations have different nucleotide affinites, we have been able to measure the rates of the conformational changes by monitoring the decrease or increase in fluorescence associated with the net release or binding of nucleotide. Finally, the effects of alterations in FTP concentration on the time course of the fluorescence changes have provided a clue to the non-phosphorylating role of ATP.