An Equivalent Site Mechanism for Na+ and K+ Binding to Sodium Pump and Control of the Conformational Change Reported by Fluorescein 5'-Isothiocyanate Modification

We have previously shown that two K+ must bind to cause the conformational change in Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase reported by fluorescein 5'-isothiocyanate modification and that the ratio of the macroscopic K+ dissociation constants is 4 [Smirnova, I. N., & Faller, L. D. (1993) J. Biol. Chem. 268, 16120-16123]. Since 4 is the ratio expected for random binding to two identical and independent sites, in this paper we propose a two-equivalent-site mechanism for Na+ as well as K+ binding to sodium pump and control of the conformational change between E1 and E2. The equivalent site mechanism is tested by fitting algebraic equations for the empirical first-order rate constant and for the magnitude of the fluorescence change observed in stopped-flow experiments to an array of data. The estimated rate constants for the conformational change at 15 degrees C are kf = 150 +/- 19 s-1 and kr = 0.13 +/- 0.03 s-1, and the estimated dissociation constants for K+ and Na+ from the E1 conformation of the enzyme are KK = 4.8 +/- 1.2 mM and KNa = 0.38 +/- 0.12 mM, in good agreement with estimates from published binding measurements. Although the possibility of ordered and anticooperative binding cannot be excluded, the equivalent site interpretation is supported by quasi-independent estimates of the macroscopic Na+ dissociation constants that give the ratio 4 predicted for binding to identical, noninteracting sites and by equivalent site interpretations of published transport experiments. Six alternative mechanisms are tested. Noncompetitive binding and mutually exclusive binding of the transported ions can be ruled out. Comparable fits to the data are obtained by three-equivalent-site mechanisms in which enzyme with either two or three K+ bound can undergo the conformational change. Therefore, this study quantitatively supports the conformational hypothesis by showing that a conformational change in sodium pump has the right properties to explain transport.