Mechanism of K+ interaction with fluorescein 5'-isothiocyanate-modified Na+,K(+)-ATPase

The rate of K+ binding to fluorescein 5'-isothiocyanate-modified Na+,K(+)-ATPase has been measured by stopped-flow fluorometry and shown to depend sigmoidally on potassium concentration. By assuming two K+ ions must bind to cause a rate-limiting conformational change, analytical expressions for the rate and the amplitude were derived that fit the experimental data and correctly calculate the half-maximum K+ concentration in amplitude titrations from kinetically estimated rate and dissociation constants. This is the first time that sigmoidal dependence of the time constant for K+ reaction with Na+,K(+)-ATPase on potassium concentration has been observed. The conclusion that two K+ ions are required to cause the E1-->E2 conformational change explains discrepancies between equilibrium and kinetic estimates of the half-maximum K+ concentration in published studies of the reaction and reconciles the mechanism of the conformational change in Na+,K(+)-ATPase with the transport stoichiometry of sodium pump. It is strong, additional evidence that fluorescein reports a conformational change in dephosphoenzyme that is involved in monovalent cation transport. The ratio of the K+ dissociation constants is consistent either with ordered binding to two sites with intrinsic constants differing by a factor of 4 or with binding to two independent and identical sites.