Hsiao Yi-Yuong, Pan Yih-Jiuan, Hsu Shen-Hsing, Huang Yun-Tzu, Liu Tseng-Huang, Lee Ching-Hung, Lee Chien-Hsien, Liu Pei-Feng, Chang Wen-Chi, Wang Yung-Kai, Chien Lee-Feng, Pan Rong-Long
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan.
Biochim Biophys Acta. 2007 Jul;1767(7):965-73. doi: 10.1016/j.bbabio.2007.04.007. Epub 2007 May 3.
Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R-->A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R-->A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F- inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+-PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment.
植物液泡H⁺转运无机焦磷酸酶（V-PPase，EC 3.6.1.1）利用无机焦磷酸（PPi）作为能源，产生H⁺梯度电位，用于离子和代谢物跨液泡膜的次级转运。在本研究中，通过定点诱变确定了绿豆V-PPase中精氨酸残基的功能作用。几种生物体中依赖K⁺的V-PPase氨基酸序列比对显示，所有15个精氨酸残基中的11个高度保守。将精氨酸残基逐个替换为丙氨酸残基，产生R→A替换的V-PPase，然后在酵母中进行异源表达。随后对突变体变体的特性进行了仔细研究。结果，除了R242A、R523A和R609A突变体明显丧失PPi水解和相关H⁺转运能力外，大多数R→A替换的V-PPase表现出与野生型相似的酶活性。此外，这三个精氨酸的突变改变了最佳pH值，并显著降低了酶活性的K⁺刺激，这意味着替换后酶反应发生了构象变化或修饰。特别是，R242A对特定的精氨酸修饰剂2,3-丁二酮和苯乙二醛表现出显著抗性，表明Arg242很可能是这两种试剂的主要靶标残基。Arg242处的突变也消除了F⁻抑制作用，推测这是由于干扰了底物复合物Mg²⁺-PPi的形成。因此，我们的结果表明，V-PPase的活性口袋可能包含嵌入更疏水环境中的必需Arg242。
