Unexpected Effects of K+ and Adenosine Triphosphate on the Thermal Stability of Na+,K+-ATPase.

M Agueda Placenti,F Luis González Flecha,Rodolfo M González Lebrero,Sergio B Kaufman

doi:10.1021/acs.jpcb.7b00629

Abstract

Na+,K+-ATPase is an integral membrane protein which couples ATP hydrolysis to the transport of three Na+ out and two K+ into the cell. The aim of this work is to characterize the effect of K+, ATP, and Mg2+ (essential activator) on the Na+,K+-ATPase thermal stability. Under all conditions tested, thermal inactivation of the enzyme is concomitant with a structural change involving the ATP binding site and membrane-associated regions. Both ligands exert a clear stabilizing effect due to both enthalpic and entropic contributions. Competition experiments between ATP and K+ showed that, when ATP is present, the inactivation rate coefficient exhibits a biphasic dependence on K+ concentration. At low [K+], destabilization of the enzyme is observed, while stabilization occurred at larger cation concentrations. This is not expected for a simple competition between the enzyme and two ligands that individually protect the enzyme. A model that includes enzyme species with none, one, or two K+ and/or one molecule of ATP bound explains the experimental data. We concluded that, despite both ligands stabilizing the enzyme, the species with one K+ and one ATP simultaneously bound is unstable.

Highlights

Na+,K+-ATPase is an integral membrane protein that couples ATP hydrolysis to the active transport of three Na+ ions out and two K+ ions into the cell.[1]
Structural changes on the Na+,K+-ATPase due to thermal inactivation were evaluated by measuring tryptophan fluorescence according to the previously described procedure (Figure 1B).[29]
To complete a quantitative comparison between the stabilizing properties of different ligands, we evaluated the effect of the addition of 40 mM K+ to the preincubation media used in this work (Figure 4 and Table 1), knowing that this cation protects the enzyme against thermal inactivation. 28,29,46

Summary

Introduction

Na+,K+-ATPase is an integral membrane protein that couples ATP hydrolysis to the active transport of three Na+ ions out and two K+ ions into the cell.[1]. The working cycle of the enzyme includes: i) binding of ATP ii) formation and breakdown of a phosphoenzyme intermediary, iii) conformational changes between two main conformational states -the so called E1 and E2 conformers- and iv) occlusion/ deocclusion of Na+ and K+ ions.[9,10]. The E1 conformer is the main enzyme specie in the presence of Na+, ATP or Mg2+ whereas K+ To other ATPases, enzyme activity requires the presence of Mg2+ as the complex ATP.Mg is postulated to be the true substrate of the enzyme.[2,3] The minimum functional unit is a heterodimer composed of a large catalytic α subunit (1016 residues) and a smaller β subunit (302 residues) with regulatory functions.[4,5] The α subunit has three characteristic cytoplasmic domains -actuator (A), nucleotide-binding (N) and phosphorylation (P) domains- together with a transmembrane domain, formed by 10 helical segments, which includes the cation transport sites.[6,7,8]

Objectives

Methods

Results

Conclusion