Expression of Na,K-ATPase in yeast allowed targeting of αβ-units with lethal substitutions at the phosphorylation site αl(D369N)β1 and αl(D369A)_/3β1 at the cell surface at the same concentration of α-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 (K_D = 2.8 nM) that was 39-fold higher than for wild type Na,K-ATPase (K_D = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the γ-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 α-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 K_D = 4.9 nM for D369A compared to K_D = 51 nM for D369N and K_D= 133 nM for wild type, and [³H]ouabain binding (K_D = 153 nM) to D369A was detectable even in absence of Mg²⁺. In addition to its function as receptor of the γ-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.