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

KAUFMAN, SERGIO BENJAMÍN BENJAMIN; PLACENTI, M AGUEDA; GONZÁLEZ-LEBRERO, RODOLFO MARTÍN; GONZALEZ FLECHA, F LUIS

JOURNAL OF PHYSICAL CHEMISTRY B - (Print)

AMER CHEMICAL SOC

Año: 2017 vol. 121 p. 4949 - 4957

1520-6106

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. In all conditions tested, thermal inactivation of the enzyme is concomitant with a structural change involving the ATP binding site and membraneassociated 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 explain the experimental data. We concluded that despite both ligands stabilize the enzyme, the species with one K + and one ATP simultaneously bound is unstable.