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Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase β subunit

Nucleotide-induced conformational changes of the catalytic β subunits play a crucial role in the rotary mechanism of F1-ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated β subunit from thermophilic Bacillus PS3 (Tβ), the binding of this monomer to Mg(II)-free and Mg(II)-bound adenosine nucleotides was characterized using high-precision isothermal titration calorimetry. The interactions of Mg(II) with free ATP or ADP were also measured calorimetrically. A model that considers simultaneously the interactions of Tβ with Mg·ATP or with ATP and in which ATP is able to bind two Mg(II) atoms sequentially was used to determine the formation parameters of the Tβ-Mg·ATP complex from calorimetric data. This analysis yielded significantly different ΔHb and ΔSb values in relation to those obtained using a single-binding site model, while ΔGb was almost unchanged. Published calorimetric data for the titration of Tβ with Mg·ADP [Perez-Hernandez, G., et al. (2002) Arch. Biochem. Biophys. 408, 177-183] were reanalyzed with the ternary model to determine the corresponding true binding parameters. Interactions of Tβ with Mg·ATP, ATP, Mg·ADP, or ADP were enthalpically driven. Larger differences in thermodynamic properties were observed between Tβ-Mg·ATP and Tβ-ATP complexes than between Tβ-Mg·ADP and Tβ-ADP complexes or between Tβ-Mg·ATP and Tβ-Mg·ADP complexes. These binding data, in conjunction with those for the association of Mg(II) with free nucleotides, allowed for a determination of the energetic effects of the metal ion on the recognition of adenosine nucleotides by Tβ [i.e., Tβ·AT(D)P + Mg(II) ? Tβ·AT(D)P-Mg]. Because of a more favorable binding enthalpy, Mg(II) is recognized more avidly by the Tβ·ATP complex, indicating better stereochemical complementarity than in the Tβ·ADP complex. Furthermore, a structural-energetic analysis suggests that Tβ adopts a more closed conformation when it is bound to Mg·ATP than to ATP or Mg·ADP, in agreement with recently published NMR data [Yagi, H., et al. (2009) J. Biol. Chem. 284, 2374-2382]. Using published binding data, a similar analysis of Mg(II) energetic effects was performed for the free energy change of F1 catalytic sites, in the framework of bi- or tri-site binding models.

SYNTHESIS, STRUCTURE, PROTON-NUCLEAR MAGNETIC RESONANCE, AND FOURIER TRANSFORM INFRARED SPECTROSCOPY OF SEVERAL TRANSITION AND NONTRANSITION METAL-ADENOSINE-5-TRIPHOSPHATE COMPLEXES

Several complexes of adenosine-5-triphosphate disodium salt (Na2H2ATP) with the metal ions, Na(1+), Mg(2+), Ca(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), and cis- and trans-Pt(NH3)2Cl2 and K2PtCl4 at pH = 3.5 and 7.2 have been isolated, identified, and studied.Marked spectral similarities have been observed for the structurally known metal-phosphate bonded compounds, 2.7H2O and 2.4H2O and all the metal-ATP complexes studied here, except the Pt-ATP complexes.The metal binding is through the α, β, and γ phosphate oxygen atoms when the N1-position of adenine is protonated.Spectral changes have also been observed for the Pt-ATP complexes in which there is a Pt-N7 and -N1 coordination.The sugar pucker in the Na2H2ATP.3H2O crystal dimers is C3'-endo-anti (in one) and C2'-endo-anti (in the other) with a characteristic infrared band at 818 cm-1.In the corresponding Cu(2+) and Zn(2+) complexes the sugar has C3'-endo-anti conformation with the marker band at about 814 cm-1.The C2'-endo-anti conformation is observed for all the metal-ATP complexes prepared here with a marker band at 825-822 cm-1.