DEPHOSPHORYLATION KINETICS OF PIG-KIDNEY NA-ATPASE(,K+)

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase w ere 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 RH42 1. Two different mixing procedures were used: (a) premixing with ATP t o allow phosphorylation to go to completion, followed by mixing with K Cl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedu re (a), the dephosphorylation rate constant of enzyme complexed with K + ions could be determined directly to be less than or equal to 366 s( -1) and the rate constant for spontaneous dephosphorylation (without K +) less than or equal to 60 s(-1). The K+ concentration dependence of the observed reciprocal time constant showed half-saturation at a K+ c oncentration of 2.4-2.6 mM with positive cooperativity involved in the occupation of the K+ binding sites on the E2P conformation of the enz yme. Using mixing procedure (b), it was found that at saturating K+ co ncentrations the dephosphorylation of the enzyme is rate-limited by it s phosphorylation, which occurs with a rate constant of approximately 190 s(-1) (1). These results show that all reactions occurring after p hosphorylation and prior to dephosphorylation, i.e., the E1P to E2P co nformational transition as well as Na+ release and K+ binding steps, m ust be fast (>190 s(-1)).