IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Thermodynamic parameters of activation for K+ dephosphorylation of the Na+,K+-ATPase
Autor/es:
RM GONZÁLEZ-LEBRERO; SB. KAUFMAN
Lugar:
San Francisco, California, EEUU
Reunión:
Congreso; Biophysical Society 54th Annual Meeting; 2010
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 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