Rate determination in phosphorylation of shark rectal Na,K-ATPase by ATP: Temperature sensitivity and effects of ADP

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Nat was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E-2-P, which follows formation of E-1-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) similar to 90 s(-1) and a minor slow phase with a k(obs) of similar to 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of similar to 3.1 x 10(7) M-1 and 1.8 x 10(5) M-1, respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E1P followed by a conformational change to E-2-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K+ the highly fluorescent E-2-P is slowly transformed into the "K+-insensitive" E-2-P subconformation forming a side branch of the main cycle.