Amino acid replacement of Asp369 in the sheep alpha 1 isoform eliminates ATP and phosphate stimulation of [3H]ouabain binding to the Na+, K(+)-ATPase without altering the cation binding properties of the enzyme.

Modification of aspartic acid 369 in the sheep alpha 1 Na+,K(+)-ATPase to asparagine results in a membrane-associated form of Na+,K(+)-ATPase that can bind [3H]ouabain with high affinity in the presence of Mg2+ alone (KD = 20.4 +/- 2.6 nM). Ouabain binding to the D369N mutant is not stimulated by inorganic phosphate, confirming that Asp369 is both the catalytic phosphorylation site and the only Pi interaction site which stimulates ouabain binding. Cation inhibition of Mg(2+)-stimulated ouabain binding to the D369N mutant demonstrated that three Na+ and two K+ ions inhibit [3H]ouabain binding and suggests that this inhibition must occur via a cation-sensitive conformational change which does not directly involve dephosphorylation of the enzyme. In the presence of 10 mM Mg2+, ATP stimulates ouabain binding to the wild type protein, (AC50 = 21.4 +/- 2.7 microM) but inhibits the binding to the D369N mutant (IC50 = 2.52 +/- 0.17 microM) indicating that the mutation does not destroy the high affinity site for MgATP but does change the nature of the protein conformation normally induced by a nucleotide-Na+,K(+)-ATPase interaction. Increasing the Mg2+ from 1 to 10 mM did not alter the AC50 or IC50 values for ATP and reveals that the Mg2+ interaction which stimulates ouabain binding in the absence of nucleotide involves a distinct divalent cation site not associated with the binding of the magnesium-nucleotide complex. Thus, altering the catalytic phosphorylation site of Na+,K(+)-ATPase does not affect the expression of the ouabain-sensitive protein in the membrane fraction of NIH 3T3 cells and does not disrupt the binding of Na+, K+, Mg2+, ouabain, or ATP to the enzyme. However, the D369N substitution does inhibit the formation of a nucleotide-protein complex with high affinity for ouabain.