Nucleotide/Protein Interaction: Energetic and Structural Features of Na,K-ATPase |
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Authors: | Grell E Lewitzki E Schacht A Stolz M |
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Institution: | (1) Max-Planck-Institut für Biophysik, D-60439 Frankfurt, Germany |
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Abstract: | Microcalorimetric titrations allow to recognize and investigate high-affinity ligand binding to Na,K-ATPase. Titrations with
the cardiac glycoside Ouabain, which acts as a specific inhibitor of the enzyme, have provided not only the thermodynamic
parameters of high-affinity binding with a stoichiometric coefficient of about 0.6 but also evidence for low-affinity binding
to the lipid. The marked enthalpic contribution of -95 kJ mol-1 at 298.2 K is partially compensated by a large negative entropy change, attributed to an increased interaction between water
and the protein. The calorimetric ADP and ATP titrations at 298.2 K are indicative of high-affinity nucleotide binding either
in 3 mM NaCl, 3 mM MgCl2 or at high ionic strength such as 120 mM choline chloride. However, no binding is detected in the buffer solution alone at
low ionic strength. The affinities for ADP and ATP are similar, around 106 M-1 and the stoichiometric coefficients are close to that of Ouabain binding. The exothermic binding of ADP is characterized
by a ΔH and ΔS value of -65 kJ mol-1 and -100 J mol-1 K-1, respectively. TheΔH value for ATP binding is larger than for ADP and is compensated by a larger, unfavorable ΔS value. This
leads to an enthalpy/entropy compensation, which could express that H-bond formation represents the major type of interaction.
As for Ouabain, the negative ΔS values that are also characteristic of nucleotide binding can indicate an increase of solvate
interaction with the protein due to a conformational transition occurring subsequent to the binding process. The resulting
binding constants are discussed with regard to the results of other studies employing different techniques. A molecular interaction
model for nucleotide binding is suggested.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | microcalorimetry titration calorimetry ATP binding cardiac glycoside Na K-ATPase nucleotide binding nucleotide protein interaction Ouabain |
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