INVESTIGADORES
GONZALEZ LEBRERO Rodolfo Martin
congresos y reuniones científicas
Título:
Thermodynamic parameters of activation for dephosphorylation of the Na+/K+ ATPase
Autor/es:
RM. GONZÁLEZ-LEBRERO; SB. KAUFMAN
Lugar:
University of Aarhus, Aarhus, Denmark
Reunión:
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
Resumen:
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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