Calcium ion and sodium and potassium dependent adenosine triphosphatase: its mechanism of inhibition and identification of the E₁-P intermediate

Calcium ion inhibits (Na⁺ + K⁺) ATPase primarily by reducing the rate of the phosphorylation step. Inhibition of this step is competitive with respect to Na⁺ and noncompetitive with respect to Mg⁺⁺. Although Ca⁺⁺ acts to reduce the rate of the phosphorylation step, high concentrations are required to reduce the steady state levels of phospho enzyme, and Ca⁺⁺ alone stimulates a slow phosphorylation of the enzyme. In the presence of Ca⁺⁺ and Mg⁺⁺ the phospho enzyme formed is sensitive to K⁺ and ouabain, and its steady state levels are low when K⁺ is present. If the concentration of Mg⁺⁺ is low, the phospho enzyme formed in the presence of Ca⁺⁺ is insensitive to K⁺ and ouabain, and reacts readily with ADP. The reactivity of the Ca⁺⁺ dependent phospho enzyme with ADP is reduced by the addition of Mg⁺⁺; conversely, the reactivity of the Mg⁺⁺ dependent phospho enzyme with ADP is increased by high concentrations of Na⁺. Ca⁺⁺ also inhibits the Mg⁺⁺ and P(i) dependent pathway of [³H] ouabain binding. The identification of an ADP sensitive intermediate in the reaction cycle of this enzyme is reported. Its transformation to a K⁺ sensitive intermediate is influenced by Na⁺, Mg⁺⁺, and Ca⁺⁺ in a manner consistent with proposed reaction mechanisms for this enzyme.