MECHANISM OF K-ISOTHIOCYANATE-MODIFIED NA+,K+-ATPASE( INTERACTION WITH FLUORESCEIN 5')

The rate of K+ binding to fluorescein 5'-isothiocyanate-modified Na+,K +-ATPase has been measured by stopped-flow fluorometry and shown to de pend sigmoidally on potassium concentration. By assuming two K+ ions m ust bind to cause a rate-limiting conformational change, analytical ex pressions for the rate and the amplitude were derived that fit the exp erimental data and correctly calculate the half-maximum K+ concentrati on in amplitude titrations from kinetically estimated rate and dissoci ation constants. This is the first time that sigmoidal dependence of t he time constant for K+ reaction with Na+,K+-ATPase on potassium conce ntration has been observed. The conclusion that two K+ ions are requir ed to cause the E1 --> E2 conformational change explains discrepancies between equilibrium and kinetic estimates of the half-maximum K+ conc entration in published studies of the reaction and reconciles the mech anism 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+ dissocia tion 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.