congresos y reuniones científicas
Thermodynamic parameters of activation for K+ dephosphorylation of the Na+,K+-ATPase
San Francisco, California, EEUU
Congreso; Biophysical Society 54th Annual Meeting; 2010
<!-- @page { margin: 2cm } P { margin-bottom: 0.21cm } --> The Na+/K+-ATPase couples two processes: the endergonic transport of ions and the exergonic hydrolysis of ATP, which occurs through the formation and breakdown of phosphorylated intermediates, involving an aspartyl-phosphate covalent bond [1]. As the Na+/K+-ATPase shows several exchange modes of ion transport and many of its partial reactions can be measured, including those that involve transport intermediates, it seems a very good system to study the coupling mechanism between transport and ATP hydrolysis. We show here results comparing thermodynamic parameters of activation between the hydrolysis of phosphoenzyme (EP): i) mediated by the Na+-ATPase, ii) enhanced by K+, and those of the dephosphorylation of denatured (unfolded) phosphoenzyme (EPdenat). The thermodynamic parameters of activation were obtained by Arrhenuis plot and applying the transition states theory. All reactions took place in media containing: 25 mM Imidazole-HCl (pH 7.4), 150 mM NaCl, variable o 10 µM ATP, 0.2 mM EDTA-Imidazole (pH 7.4), 700 µM MgCl2, 0 or 500 µM K+. The temperatures tested went from 8.6 to 35.3 °C. All reactions yielded linear Arrhenius plots that allowed to calculate the enthalpy of activation (DH‡). The parameters for the dephosphorylation of EPdenat were: kobs(25 °C) = 1.5 x 10-4 s 1, DH‡ = 25 kcal/mol, and TDS‡ (at 25 °C) = 2.4 kcal/mol. In the case of the Na-ATPase, the parameter values were: kcat(25 °C) = 1.72 s-1, DH‡ = 15 kcal/mol, and TDS‡ (at 25 °C) = -2.13 kcal/mol and, for K+ dephosphorylation of EP were: kobs(25 °C) = 33.42 s 1, DH‡ = 11.77 kcal/mol, and TDS‡ (at 25 °C) = -3.6 kcal/mol. The Na-ATPase increased the rate constant of dephosphorylation of EP respect to the EPdenat by a factor of 11,500 with DDG‡ = -5.47 kcal/mol, DDH‡ = -10 kcal/mol y TDDS‡ = -4.53 kcal/mol. While the K+ (at 500 µM) enhanced the EP hydrolysis by a factor of 220,000 with: DDG‡ = -1.76 kcal/mol, DDH‡ = -3.23 kcal/mol y TDDS‡ = -1.47 kcal/mol. This results are in accordance with the fact that the transition states of dephosphorylation of the Na+,K+-ATPase is stabilized by the same forces in the presence or absence of K +. Both acceleration (Na-ATPase and activation by K+) are achieved by lowering the enthalpy of activation whereas the entropy of activation is unfavorable for the catalytic effect of the pump. The enthalpic and entropic magnitudes of this rate enhancement would seem understandable if polar forces of attraction in an ordered surrounding, rather than hydrophobic effects, were responsible for the stabilization of transition state. [1] Glynn, I.M. (1985). The Na+,K+-Transporting Adenosine Triphosphatase, in: The Enzymes of Biological Membranas (ed. A.N. Martonosi) 3: 35-114. Plenum Press, New Cork, London