title: Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids.
abstract: The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.
authors.affiliation: Department of Biomedicine, University of Aarhus, Aarhus C, Denmark. fc@biophys.au.dk
authors.forename: Flemming
authors.initials: F
authors.lastname: Cornelius
authors.forename: Yasser A
authors.initials: YA
authors.lastname: Mahmmoud
authors.forename: Chikashi
authors.initials: C
authors.lastname: Toyoshima
mesh_terms: D000818:Animals; D002316:Cardiotonic Agents / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry* / Q000378:metabolism; D010042:Ouabain / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012754:Sharks; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002316:Cardiotonic Agents; D029941:Fish Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D005459:Fluorides