Activation by Adenosine Triphosphate in the Phosphorylation Kinetics of Sodium and Potassium Ion Transport Adenosine Triphosphatase

The steady state kinetics of sodium and potassium ion transport adenosine triphosphatase (EC 3.6.1.3) suggests an activating action of adenosine triphosphate in addition to its action as a phosphate donor. This action might be an acceleration of dissociation of K+ or its congeners from the enzyme. To test this possibility transient kinetics of the phosphoenzyme was investigated. In membranes from guinea pig kidney the enzyme was phosphorylated in the presence of magnesium ion and sodium ion with adenosine triphosphate which was radioactive in the terminal phosphate group. To this phosphoenzyme were added congeners of potassium ion, namely, rubidium ion or lithium ion at concentrations which produced an equal acceleration of dephosphorylation. At these same concentrations in the steady state, the level of dephosphoenzyme was higher in the presence of rubidium ion than in the presence of lithium ion. Rephosphorylation was relatively inhibited in the presence of rubidium ion. Rubidium ion appeared to combine with the phosphoenzyme, split off the phosphate group, and remain with the dephosphoenzyme in a relatively stable complex. This complex was isolated kinetically by addition of a high concentration of sodium ion with oligomycin. The isolated complex was less stable following an increase in the concentration of adenosine triphosphate. It was concluded that adenosine triphosphate was activating the enzyme in a fashion functionally distinct from its action as a phosphate donor, since adenosine triphosphate does not phosphorylate the enzyme in the presence of rubidium ion without sodium ion and since the concentration of adenosine triphosphate required for activation was much higher than that required for phosphorylation. The relatively stable complex of rubidium ion with the dephosphoenzyme was interpreted as evidence for a closed or occluded conformation of the active site for translocation of potassium ion across the membrane. Toxic agents altered this complex of the dephosphoenzyme; phlorizin increased and tetraphenyl boron decreased its stability. This kinetic mechanism provides an interpretation for a paradoxical feature in the p-nitrophenyl phosphatase activity of this enzyme system.