Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsin Chu 30013, Taiwan.
J Biol Chem. 2013 Jul 5;288(27):19312-20. doi: 10.1074/jbc.M113.469353. Epub 2013 May 29.
Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.
同源二聚体质子转运焦磷酸酶（H(+)-PPase；EC 3.6.1.1）对于许多维持细胞器 pH 平衡的生物体是必不可少的。这种独特的质子泵将 PPi 的水解与质子跨膜转运偶联。H(+)-PPase 由 14-16 个相对疏水的跨膜结构域组成，推测用于质子转运，以及主要嵌入催化位点的亲水环。在 H(+)-PPase 中，位于转运途径入口处或附近的几个高度保守的极性残基对于质子泵活性至关重要。在这项研究中，单分子 FRET 用于在结合配体时剖析同型二聚体破伤风梭菌 H(+)-PPase 中位于途径入口处的作用。底物类似物，二磷酸咪唑介导两个位于途径入口处的位点相互靠近。此外，在第一个质子携带残基（Arg-169）发生突变后进行的单分子 FRET 分析表明，入口处的构象变化对于 H(+)-PPase 质子转运的初始步骤可能是必不可少的。因此，提出了一个工作模型来阐明在底物结合时 H(+)-PPase 转运途径入口处的挤压。
