title: The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb(+).
abstract: The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K2)Mg-MgF4, an E2∙Pi-like state capable to occlude K(+) (or Rb(+)). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb(+) with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1-E2 conformational transition and the kinetics of Rb(+) exchange between the medium and the E2(Rb2)Mg-MgF4 state. Our results show that both in the absence and in the presence of Rb(+), simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg-MgF4 complex, that is unable to shift to E1 upon addition of Na(+). The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg-MgF4 formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb(+) caused by Mg(2+). Na(+)-induced release of Rb(+) from E2(Rb2)Mg-MgF4 occurs at the same rate as from E2(Rb2) but is insensitive to ADP. The rate of Rb(+) occlusion into the E2Mg-MgF4 state is 5-8 times lower than that described for the E2Mg-vanadate complex. Since the E2Mg-MgF4 and E2Mg-vanadate complexes represent different intermediates in the E2-P→E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina. Electronic address: mmontes@qb.ffyb.uba.ar.
authors.forename: Mónica R
authors.initials: MR
authors.lastname: Montes
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
authors.forename: Mariela S
authors.initials: MS
authors.lastname: Ferreira-Gomes
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Centeno
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
authors.forename: Rolando C
authors.initials: RC
authors.lastname: Rossi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004795:Enzyme Stability; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D017616:Magnesium Compounds / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012413:Rubidium / Q000737:chemistry / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine; D013997:Time Factors
chemical_list: D017616:Magnesium Compounds; C031288:magnesium fluoride; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D005459:Fluorides; D011188:Potassium
keywords: Crystallized Na; E2P-like states; K-ATPase; Magnesium fluoride complex; P-type ATPases; Rb(+) occlusion