title: Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na,K-ATPase.
abstract: 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.
authors.affiliation: Biomembrane Research Center, August Krogh Institute, University of Copenhagen, Denmark. PLJorgensen@AKI.KU.DK
authors.forename: P L
authors.initials: PL
authors.lastname: Jorgensen
authors.forename: J M
authors.initials: JM
authors.lastname: Nielsen
authors.forename: J H
authors.initials: JH
authors.lastname: Rasmussen
authors.forename: P A
authors.initials: PA
authors.lastname: Pedersen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000235:genetics; D002412:Cations / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000166:cytology / Q000201:enzymology
chemical_list: D002412:Cations; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase