Datensatz

Zusammenfassung

The kinetics of K⁺-stimulated dephosphorylation of the Na⁺,K⁺-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K⁺ ions could be determined directly to be </=366 s^-1 and the rate constant for spontaneous dephosphorylation (without K⁺) </=60 s^-1. The K⁺ concentration dependence of the observed reciprocal time constant showed half-saturation at a K⁺ concentration of 2.4-2.6 mM with positive cooperativity involved in the occupation of the K⁺ binding sites on the E₂P conformation of the enzyme. Using mixing procedure (b), it was found that at saturating K⁺ concentrations the dephosphorylation of the enzyme is rate-limited by its phosphorylation, which occurs with a rate constant of approximately 190 s^-1 (1). These results show that all reactions occurring after phosphorylation and prior to dephosphorylation, i.e., the E₁P to E₂P conformational transition as well as Na⁺ release and K⁺ binding steps, must be fast (>190 s^-1).