Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.