Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na,K-ATPase.

This work evaluates the results of measurements of equilibrium binding of ATP and cations in lethal or partially active mutations of Na,K-ATPase that were expressed at high yield in yeast cells. ATP binding studies allowed estimation of the expense in free energy required to position the gamma-phosphate in proximity of the carboxylate groups of the phosphorylated residue Asp369 and the role of this residue in governing long range E1-E2 transitions. An arginine residue (Arg546) appearing to be involved in ATP binding has been identified. Wild type yeast enzyme was capable of occluding two T1(+)-ions per ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(T1+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. The substitutions to Glu327(Gln,Asp), Asp804(Asn,Glu), Asp808(Asn,Glu) and Glu779(Asp) completely abolished occlusion or severely reduced the affinity for T1+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to one T1+ ion per alpha 1 beta 1 unit with a 3-fold decrease of the apparent affinity for the ion (Kd(T1+) = 24 +/- 8 mM). These carboxylate groups in transmembrane segments 4, 5, and 6 therefore appear to be essential for high affinity occlusion of K(+)-ions.