Instituto de Quimica, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Mexico.
Biochemistry. 2010 Jun 29;49(25):5258-68. doi: 10.1021/bi1006767.
Nucleotide-induced conformational changes of the catalytic beta subunits play a crucial role in the rotary mechanism of F(1)-ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated beta subunit from thermophilic Bacillus PS3 (Tbeta), 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 Tbeta 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 Tbeta-Mg.ATP complex from calorimetric data. This analysis yielded significantly different DeltaH(b) and DeltaS(b) values in relation to those obtained using a single-binding site model, while DeltaG(b) was almost unchanged. Published calorimetric data for the titration of Tbeta 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 Tbeta with Mg.ATP, ATP, Mg.ADP, or ADP were enthalpically driven. Larger differences in thermodynamic properties were observed between Tbeta-Mg.ATP and Tbeta-ATP complexes than between Tbeta-Mg.ADP and Tbeta-ADP complexes or between Tbeta-Mg.ATP and Tbeta-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 Tbeta [i.e., Tbeta.AT(D)P + Mg(II) right harpoon over left harpoon Tbeta.AT(D)P-Mg]. Because of a more favorable binding enthalpy, Mg(II) is recognized more avidly by the Tbeta.ATP complex, indicating better stereochemical complementarity than in the Tbeta.ADP complex. Furthermore, a structural-energetic analysis suggests that Tbeta 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 F(1) catalytic sites, in the framework of bi- or tri-site binding models.
核苷酸诱导的催化β亚基构象变化在 F(1)-ATP 酶的旋转机制中起着关键作用。为了深入了解控制分离的嗜热芽孢杆菌 PS3（Tbeta）β亚基识别核苷酸的能量基础，使用高精度等温滴定量热法研究了该单体与无镁（Mg(II)-free）和镁结合的腺苷核苷酸的结合。还通过量热法测量了 Mg(II)与游离 ATP 或 ADP 的相互作用。使用同时考虑 Tbeta 与 Mg.ATP 或与 ATP 相互作用的模型，其中 ATP 能够顺序结合两个 Mg(II)原子，从量热数据确定 Tbeta-Mg.ATP 配合物的形成参数。与使用单结合位点模型获得的结果相比，该分析导致 DeltaH(b)和 DeltaS(b)值有明显差异，而 DeltaG(b)几乎不变。用三元模型重新分析了用于 Tbeta 与 Mg.ADP 滴定的已发表的量热数据[Perez-Hernandez，G.，等。（2002）Arch. Biochem. Biophys. 408, 177-183]，以确定相应的真实结合参数。Tbeta 与 Mg.ATP、ATP、Mg.ADP 或 ADP 的相互作用是由焓驱动的。在 Tbeta-Mg.ATP 和 Tbeta-ATP 配合物之间观察到的热力学性质差异大于 Tbeta-Mg.ADP 和 Tbeta-ADP 配合物或 Tbeta-Mg.ATP 和 Tbeta-Mg.ADP 配合物之间的差异。这些结合数据与游离核苷酸与 Mg(II)结合的数据相结合，确定了金属离子对 Tbeta 识别腺苷核苷酸的能量效应[即 Tbeta.AT(D)P + Mg(II)right harpoon over left harpoon Tbeta.AT(D)P-Mg]。由于结合焓更有利，Mg(II)与 Tbeta.ATP 配合物的识别更强烈，表明与 Tbeta.ADP 配合物相比具有更好的立体化学互补性。此外，结构能量分析表明，当 Tbeta 与 Mg.ATP 结合时，它采用更封闭的构象，而与 ATP 或 Mg.ADP 结合时则不是，这与最近发表的 NMR 数据一致[Yagi，H.，等。（2009）J. Biol. Chem. 284, 2374-2382]。使用已发表的结合数据，在双或三结合位点模型的框架内，对 F(1)催化位点的自由能变化进行了类似的 Mg(II)能量效应分析。
