[D2O as a modifier of ionic specificity of Na, K-ATPase]

Effect of heavy water D2O on the rate of hydrolysis of ATP and pNPP by Na,K-ATPase was studied. Heavy water of high concentration inhibits the rate of ATPase reaction in all the studied ratios of the ions Na/K at constant ionic strength 150 mM. Activation of the enzyme was observed in the solution with low concentration of heavy water (less than 5%). The value of isotope effects depended on the ratio between sodium and potassium ion concentrations in the medium. At low temperature no activation of the enzyme with heavy water in low concentration was observed. Substitution of usual water for the heavy one was accompanied by a decrease of apparent constants of enzyme activation with sodium and potassium ions. During pNPP hydrolysis with Na,K-ATPase an increase of reaction rate in the medium with heavy water was observed. Substitution of potassium ions by cesium resulted in an increase of isotope effects during ATP and pNPP hydrolysis. Analysis of isotope effects in terms of the molecular model of sodium pump proposed permits a conclusion that the isotope effects of heavy water are explained by its influence as a solvent, the binding centres of potassium and sodium ions are localized in different regions of the enzyme differing in physico-chemical properties. The structure of sodium centres is controlled by hydrogen bonds, and of potassium ones--by hydrophobic interactions; the transport of ions by the enzyme is accompanied by dehydration of ions.