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 Am and cations in lethal or partially active mutations of Na,K-ATP ase that were expressed at high yield in yeast cells. ATP binding stud ies allowed estimation of the expense in free energy required to posit ion the gamma-phosphate in proximity of the carboxylate groups of the phosphorylated residue Asp(369) and the role of this residue in govern ing long range E-1-E-2 transitions. An arginine residue (Arg(546)) app earing to be involved in ATP binding has been identified. Wild type ye ast enzyme was capable of occluding two Tl+-ions per ouabain binding s ite or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/ - 2 mu M), like the purified Na,K-ATPase from pig kidney. The substitu tions to Glu(327) (Gln,Asp), Asps(804) (Asn,Glu), Asp(808) (Asn,Glu) a nd Glu(779) (Asp) completely abolished occlusion or severely reduced t he affinity for Tl+ ions. The substitution of Glu(779) for Gln reduced the occlusion capacity to one Tl+ ion per alpha 1 beta 1 unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 mM). These carboxylate groups in transmembrane segments 4, 5, and 6 therefore appear to be essential for high affinity occlusion of K+-io ns.