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The E2P-like State Induced by Magnesium Fluoride Complexes in the Na,K-ATPase. Kinetics of Formation and Interaction With Rb(+)

The E2P-like State Induced by Magnesium Fluoride Complexes in the Na,K-ATPase. Kinetics of Formation and Interaction With Rb(+)

Mónica R Montes, Mariela S Ferreira-Gomes, Mercedes Centeno, Rolando C Rossi

Biochim Biophys Acta. 2015 Jul;1848(7):1514-23.

PMID: 25838127

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP7783406	Magnesium fluoride		7783-40-6	Price

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