Glutamic acid 472 and lysine 480 of the sodium pump alpha 1 subunit are essential for activity. their conservation in pyrophosphatases suggests theirinvolvement in recognition of ATP phosphates

P-type ATPases such as the Na+,K+-ATPase (sodium pump) hydrolyze ATP to pum p ions through biological membranes against their electrochemical gradients . The mechanisms that couple ATP hydrolysis to the vectorial ion transport are not yet understood, but unveiling structures that participate in ATP bi nding and in the formation of the ionophore might help to gain insight into this process. Looking at the alpha- and beta-phosphates of ATP as a pyroph osphate molecule, we found that peptides highly conserved among all soluble inorganic pyrophosphatases are also present in ion-transporting ATPases. I ncluded therein are Glu48 and Lys56 of the Saccharomyces cerevisiae pyropho sphatase (SCE1-PPase) that are essential for the activity of this enzyme an d have been shown in crystallographic analysis to interact with phosphate m olecules. To test the hypothesis that equivalent amino acids are also essen tial for the activity of ion-transporting ATPases, Glu472 and Lys480 of the sodium pump alpha 1 subunit corresponding to Glu48 and Lys56 of SCE1-PPase were mutated to various amino acids. Mutants of the sodium pump al subunit were expressed in yeast and analyzed for their ATPase activity and their a bility to bind ouabain in the presence of either ATP, Mg2+, and Na+ or phos phate and Mg2+. All four mutants investigated, Glu472Ala; Glu472Asp, Lys480 Ala, and Lys480Arg, display only a fraction of the ATPase activity obtained with the wild-type enzyme. The same applies with respect to their ability to bind ouabain, where maximum ouabain binding to the mutants accounts for only about 10% of the binding obtained with the wild-type enzyme. On the ba sis of our results, we conclude that Glu472 and Lys480 are essential for th e activity of the sodium pump. Their function is probably to arrest the alp ha- and beta-phosphate groups of ATP in a proper position prior to hydrolys is of the gamma-phosphate group. The identification of these amino acids as essential components of the ATP-recognizing mechanism of the pump has resu lted in a testable hypothesis for the initial interactions of the sodium pu mp, and possibly of other P-type ATPases, with ATP.