The role of the Na-ATPase in the thermodynamic parameters of activation for the ATP hydrolysis

RODOLFO MARTIN GONZALEZ-LEBRERO; SERGIO B. KAUFMAN

Rosario, Sante Fe, Argentina.

Congreso; XXXV reunión anual de la Sociedad Argentina de Biofísica (SAB); 2006

Sociedad Argentina de Biofísica (SAB)

The Na/K-ATPase is a membrane protein, which creates and maintains ionic concentration gradients of Na+ and K+ across the cellular membrane [1]. The system 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 aspatyl-phosphate covalent bond. As the Na/K-ATPase shows a variety of exchange modes of the transported ions and many of its partial reactions can be measured, including those that involve transport intermediates, it seems a very good system to study the mechanism of coupling between transport and ATP hydrolysis. With this purpose we here show 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 that of the non-enzymatic reaction. These parameters were obtained from Arrhenius plots for both reactions. Na-ATPase activity and spontaneous ATP hydrolysis in water were measured according to Schwarzbaum et al. [2]. Both reactions took place in media containing: 25 mM Imidazole-HCl, pH 7.4, 150  mM NaCl, 8 µM ATP (i.e. a concentration which, for all the temperatures tested, saturates its sites on the enzyme), 0.2 mM EDTA-Imidazole pH 7.4, 700 µM MgCl2, 0 or 24 µg/ml of enzyme. The temperatures tested went from 14,7 to 28,5 °C for the spontaneous ATP hydrolysis and from 19.7 to 29.8 °C for enzymatic hydrolysis. We constructed Arrhenius plots with the rate of spontaneous hydrolysis of ATP and of the kcat of the Na-ATPase reaction. Both reactions yielded linear Arrhenius plots that allowed us to calculate the activation enthalpy (DH‡). The rate constant at 25 °C for spontaneous ATP hydrolysis was 1.6. 10-8 s-1, the DH‡ = 26.04 kcal/mol and TDS‡ = -2.01 kcal/mol at 25 °C. In the case of the enzymatic ATP hydrolysis, the observed value of kcat at 25 °C was 2.56 s-1, the DH‡ = 12.73 kcal/mol and TDS‡ = -4.15 kcal/mol at 25 °C. Comparison of the values for the catalyzed and spontaneous ATP hydrolysis reactions indicates that Na-ATPase enhances the reaction rate by a factor of 1.56 108 (DDG‡ = 11.17 kcal/mol) by reducing to 1/2 the enthalpy of activation (DDH‡ = 13.31 kcal/mol), whereas the entropy of activation (DDS‡ = 2.14 kcal/mol) is slightly less favorable for the enzyme reaction than in solution. These results can be used to evaluate the role of the aspartyl-phosphate bond of the phosphorylated intermediates as a reservoir of energy during the transduction between the hydrolysis of ATP and the transport of ions. (Con subsidios de ANPCYT y CONICET) [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 [2] Schwarzbaum, P.J., Kaufman, S.B., Rossi, R.C., and Garrahan, P.J., (1995) Biochim. Biophys. Acta, 1233:33-40.