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 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 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) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 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 approximately 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 E(1)-P 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.
Cornelius
F
F
Department of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark. fc.biophys.au.dk
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D011726:Pyridinium Compounds; D012007:Rectum / Q000201:enzymology; D012754:Sharks; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013343:Styrenes; D013696:Temperature
D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase