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

Abstract

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na+ 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 E2-P, which follows formation of E1-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) ~90 s-1 and a minor slow phase with a k(obs) of ~9 s-1 (20°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 ~3.1 X 107 M-1 and 1.8 x 105 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 E1-P followed by a conformational change to E2-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K+ the highly fluorescent E2-P is slowly transformed into the 'K+-insensitive' E'2-P subconformation forming a side branch of the main cycle.