Kinetic investigations of the mechanism of the rate-determining step of the Na+,K+-ATPase pump cycle.

: The kinetics of the E2 E1 conformational change of unphosphorylated Na+,K+-ATPase from rabbit kidney were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to initially stabilize the E2 conformation. On mixing enzyme with NaCl alone, tris-ATP alone, or NaCl and tris-ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/τ, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris-ATP of 54 (±3) s−1. The experimental behavior could be described by a binding of Na+ to the enzyme in the E2 state with a dissociation constant of 31 (±7) mM, which induces a subsequent rate-limiting conformational change to the E1 state. Similar behavior, but with a decreased saturating value of 1/τ, was found when NaCl was replaced by choline chloride. Experiments performed with enzyme from shark rectal gland showed similar effects, but with a lower amplitude of the fluorescence change and a higher saturating value of 1/τ for both the NaCl and choline chloride titrations. The results suggest that Na+ ions or salt in general play a regulatory role, similar to ATP, in enhancing the rate of the rate-limiting E2 E1 conformational transition by interaction with the E2 state.