Studies on (Na+ + K+)-activated ATPase. XLIV. Single phosphate incorporation during dual phosphorylation by inorganic phosphate and adenosine triphosphate

$\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can be phosphorylated by its substrate ATP as well as by its product inorganic phosphate. The maximal capacity for phosphorylation by either of these two substances is one mol phosphate per mol enzyme. In order to investigate whether the enzyme molecule possesses only one phosphorylation site common to ATP and P_i, or two phosphorylation sites, one for ATP and one for P_i, dual phosphorylation of the enzyme has been carried out. Under conditions, which are maximally favourable for each type of phosphorylation, successive phosphorylation by P_i and ATP leads to a maximal incorporation of only one mol phosphate per mol enzyme. The phosphorylation capacity for ATP decreases by the same amount as the P_i-phosphorylation level increases, without an effect on the apparent affinity for ATP.The results can be explained by assuming either a single common phosphorylation site for P_i and ATP, or a conformational change of the enzyme following phosphorylation by P_i, which excludes phosphorylation by ATP.