Steady-state kinetic study of magnesium and ATP effects on ligand affinity and catalytic activity of sheep kidney sodium, potassium-adenosinetriphosphatase

Abstract

A non-linear least squares computer analysis of steady-state kinetic data of ATP hydrolysis catalyzed by sheep kidney (Na +, K +-ATPase (ATP phosphohydrolase, EC 3.6.1.3) gives kinetic parameters of various enzyme intermediates. Chemical models that fit the whole group of experimental data allow an overview of the effects of varying concentrations of Mg 2+ and ATP on the ligand affinity and catalytic activity of the enzyme, and also make possible the following rationale in relation to the active transport of sodium and potassium across a cell membrane: In the presence of 60 mM of sodium and the absence of ATP, potassium binds favorably to the enzyme. This effective binding of potassium is consistent with extracellular binding of potassium prior to transport into a cell, because the extracellular medium consists of high sodium, low potassium and no ATP. Binding of potassium is decreased by free ATP and Mg · ATP complex. This “weakening” of potassium binding may be related to the low affinity sites of the enzyme for potassium inside a cell where ATP and Mg 2+ are present. In the presence of 10 mM potassium, binding of sodium is insignificant, but is stimulated by Mg 2+, ATP, and Mg · ATP. This is consistent with the intracellular event of sodium binding prior to transport out of the cell. The breakdown of the Mg · ATP-containing enzyme complexes may cause release of potassium into cell and put the enzyme in a form that does not bind sodium favorably in the absence of ATP; hence, sodium is released to extracellular medium which contains no ATP.