Department of Chemistry and Biochemistry, Texas Woman's University, Denton, TX, 76204, USA; Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409, USA.
Department of Biology, Texas Woman's University, Denton, TX, 76204, USA.
Arch Biochem Biophys. 2021 Aug 15;707:108899. doi: 10.1016/j.abb.2021.108899. Epub 2021 May 12.
ATP synthase is essential in aerobic energy metabolism, and the rotary catalytic mechanism is one of the core concepts to understand the energetic functions of ATP synthase. Disulfide bonds formed by oxidizing a pair of cysteine mutations halted the rotation of the γ subunit in two critical conformations, the ATP-waiting dwell (αE284C/γQ274C) and the catalytic dwell (αE284C/γL276C). Tryptophan fluorescence was used to measure the nucleotide binding affinities for MgATP, MgADP and MgADP-AlF (a transition state analog) to wild-type and mutant F under reducing and oxidizing conditions. In the reduced state, αE284C/γL276C F showed a wild-type-like nucleotide binding pattern; after oxidation to lock the enzyme in the catalytic dwell state, the nucleotide binding parameters remained unchanged. In contrast, αE284C/γQ274C F showed significant differences in the affinities of the oxidized versus the reduced state. Locking the enzyme in the ATP-waiting dwell reduced nucleotide binding affinities of all three catalytic sites. Most importantly, the affinity of the low affinity site was reduced to such an extent that it could no longer be detected in the binding assay (K > 5 mM). The results of the present study allow to present a model for the catalytic mechanism of ATP synthase under consideration of the nucleotide affinity changes during a 360° cycle of the rotor.
ATP 合酶在需氧能量代谢中必不可少，而旋转催化机制是理解 ATP 合酶能量功能的核心概念之一。一对半胱氨酸突变形成的二硫键氧化后，阻止 γ 亚基在两种关键构象中的旋转，即 ATP 等待停留（αE284C/γQ274C）和催化停留（αE284C/γL276C）。色氨酸荧光用于测量野生型和突变型 F 在还原和氧化条件下结合 MgATP、MgADP 和 MgADP-AlF（过渡态类似物）的核苷酸亲和力。在还原状态下，αE284C/γL276C F 表现出类似于野生型的核苷酸结合模式；在氧化以将酶锁定在催化停留状态后，核苷酸结合参数保持不变。相比之下，αE284C/γQ274C F 在氧化态与还原态之间的亲和力存在显著差异。将酶锁定在 ATP 等待停留状态会降低所有三个催化位点的核苷酸结合亲和力。最重要的是，低亲和力位点的亲和力降低到如此程度，以至于在结合测定中无法检测到（K > 5 mM）。本研究的结果使得在考虑转子 360°循环过程中核苷酸亲和力变化的情况下，能够提出一种 ATP 合酶催化机制模型。
