Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme.
Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism*
D014639:Vanadium; D014638:Vanadates; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium