congresos y reuniones científicas
Thermodynamic parameters of activation for dephosphorylation of the Na+/K+ ATPase
University of Aarhus, Aarhus, Denmark
Conferencia; 12th International ATPase Conference. Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease; 2008
Institución organizadora:
University of Aarhus
<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> 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 ATP mediated by the Na+ ATPase (one of the possible modes of hydrolysis of ATP) with those of the dephosphorylation of denatured (unfolded) phosphoenzyme (EPdenat). Reactions were measured at temperatures ranging from 19,7 to 30,0 °C. Na+-ATPase activity took place in media containing 150 mM NaCl, 8 µM ATP (i.e. a concentration that ensures the achievement of Vmax irrespectively of the temperature tested), 25 mM Imidazole-HCl, 0.2 mM EDTA-Imidazole, 700 µM MgCl2, 24 µg/ml of enzyme (pH was kept at 7.4 for all temperatures). Dephosphorylation of EPdenat was measured as follows: once phosphoenzyme was obtained in steady-state conditions in the aforementioned medium, trichloroacetic acid was added and after 10 seconds a large (80 times) volume of the same reaction medium (t=0 for dephosphorylation) was added. We constructed Arrhenius plots of kobs for the EPdenat rate of dephosphorylation and of the kcat of the Na+-ATPase activity. Both reactions yielded linear Arrhenius plots that allowed to calculate the enthalpy of activation (DH‡). Parameters for the dephosphorylation of EPdenat were: kobs = 1.4 x 10-4 s 1, DH‡ = 34.39 kcal/mol, and TDS‡ (at 25 °C) = 11.68 kcal/mol. In the case of the Na-ATPase, the parameter values were: kcat = 1.72 s-1, DH‡ = 15 kcal/mol, and TDS‡ (at 25 °C) = -2.13 kcal/mol. The comparison between the results obtained for the Na+-ATPase activity and the dephosphorylation of EPdenat indicates that Na+-ATPase enhances the reaction rate 12.3 103 times (DDG‡ = -5.58 kcal/mol) by reducing to 1/2 the enthalpy of activation (DDH‡ = -19.39 kcal/mol), whereas the entropy of activation (D(TDS‡) = -13.81 kcal/mol) is considerably less favorable for the Na+-ATPase than for the rate of dephosphorylation of denatured phosphoenzyme. These results can be used to evaluate the role of the pump on the hydrolysis of aspartyl-phosphate bond; and thus, for understanding the K+ action on the enhancement of intermediates dephosphorylation.   [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 York, London [2] Schwarzbaum, P.J., Kaufman, S.B., Rossi, R.C., and Garrahan, P.J., (1995) Biochim. Biophys. Acta, 1233:33-40.   This work was supported with grants from CONICET, University of Buenos Aires and ANPCyT, Argentina