Chemical mechanism of ATP synthase - Magnesium plays a pivotal role in formation of the transition state where ATP is synthesized from ADP and inorganic phosphate
Chemical mechanism of ATP synthase - Magnesium plays a pivotal role in formation of the transition state where ATP is synthesized from ADP and inorganic phosphate
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DOI：
10.1074/jbc.274.41.28853
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发表时间：
1999-10-08
影响因子：
4.8
通讯作者：
Pedersen, PL
中科院分区：
文献类型：
--
作者：
Ko, YH;Hong, SJ;Pedersen, PL
尽管最近已经阐明了两种形式的F-1催化扇区(亚单位化学计量比，α(3)β(3)γβepsilon)的三维结构，但ATP合成酶催化ATP合成的化学机制尚不清楚。缺乏关于处于过渡态的每个P-亚基的催化部位发生的化学事件的关键信息。在较早的报告(Ko，Y.H.，Plancet，M.A.，Amzel，L.M.和Pedersen，P.L，(1997)J.Biol.化学。272,18875-18881)，我们提供了在镁离子，腺苷二磷酸和原钒酸(V-I)存在下形成过渡态的证据，V-I是一种光反应磷酸类似物，具有类似于肌球蛋白等酶的伽马-P在过渡态中的几何构型，在紫外线和O-2的存在下，MgADP.V-I-F-1复合体在丙氨酸158的单个P-亚基的P-环(GG(A)下)内被裂解，这意味着该残基在过渡态ATP的伽马-P的接触距离内。在这里，我们报告说，ADP虽然促进了过渡态的形成，但不是必需的。在单独存在Mg2+和V-I的情况下，生成的MgVi-F-1络合物的催化活性受到的抑制程度与对MgADP.V-I-F-1络合物的抑制程度几乎相同。ADP、Mg~(2+)或V-I单独作用不能观察到抑制作用。值得注意的是，在紫外光和O-2存在下，镁Vi-F-1复合体也在丙氨酸158的单个β亚基的P-环内被切割，这一点被两种不同抗体的Western印迹分析、N末端序列分析和未反应的β亚基的数量的定量所证实。这些新的发现表明，在ATP合成酶催化的ATP合成过程中，镁在过渡态的形成中起着关键作用，这一作用既涉及到它与P-I的优先配位，也涉及到P-环的重新定位，将非极性的丙氨酸158带入催化口袋。本文首次提出了一个描述镁离子在过渡态形成中的作用的三磷酸腺苷合成酶反应方案。
The chemical mechanism by which ATP synthases catalyze the synthesis of ATP remains unknown despite the recent elucidation of the three-dimensional structures of two forms of the F-1 catalytic sector (subunit stoichiometry, alpha(3)beta(3)gamma delta epsilon). Lacking is critical information about the chemical events taking place at the catalytic site of each P-subunit in the transition state. In an earlier report (Ko, Y. H., Planchet, M. A., Amzel, L. M., and Pedersen, P. L, (1997) J. Biol. Chem. 272, 18875-18881), we provided evidence for transition state formation in the presence of Mg2+, ADP, and orthovanadate (V-i), a photoreactive phosphate analog with a trigonal bipyramidal geometry resembling that of the gamma-P of ATP in the transition state of enzymes like myosin, In the presence of ultraviolet light and O-2, the MgADP.V-i-F-1 complex was cleaved within the P-loop (GG (A) under bar GVGKT) of a single P-subunit at alanine 158, implicating this residue as within contact distance of the gamma-P of ATP in the transition state. Here, we report that ADP, although facilitating transition state formation, is not essential. In the presence of Mg2+ and V-i alone the catalytic activity of the resultant MgVi-F-1 complex is inhibited to nearly the same extent as that observed for the MgADP.V-i-F-1 complex. Inhibition is not observed with ADP, Mg2+, or V-i alone. Significantly, in the presence of ultraviolet light and O-2, the MgVi-F-1 complex is cleaved also within the P-loop of a single beta-subunit at alanine 158 as confirmed by Western blot analyses with two different antibodies, by N-terminal sequence analyses, and by quantification of the amount of unreacted beta-subunits. These novel findings indicate that Mg2+ plays a pivotal role in transition state formation during ATP synthesis catalyzed by ATP synthases, a role that involves both its preferential coordination with P-i and the repositioning of the P-loop to bring the nonpolar alanine 158 into the catalytic pocket. A reaction scheme for ATP synthases depicting a role for Mg2+ in transition state formation is proposed here for the first time.