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

The kinetics of the E(2) --> E(1) 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 degrees C). The enzyme was preequilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to initially stabilize the E(2) 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/tau, 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 E(2) state with a dissociation constant of 31 (+/-7) mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, 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/tau 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 E(2) --> E(1) conformational transition by interaction with the E(2) state.