Consequences of mutations to the phosphorylation site of the alpha-subunit of Na,K-ATPase for ATP binding and E(1)-E(2) conformational equilibrium

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 [H-3]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 [H-3]-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 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 Asp(369) of the alpha-subunit caused a large shift in conformational equilibrium toward the E(2)-form. This was accompanied by a large increase in affinity for [H-3]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 [H-3]ouabain binding (K-D = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp(369) has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E(1)-E(2) transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.