Functional roles of arginine residues in mung bean vacuolar H⁺-pyrophosphatase

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• 作者：Yi-Yuong Hsiao ; Yih-Jiuan Pan ; Shen-Hsing Hsu ; Yun-Tzu Huang ; Tseng-Huang Liu ; Ching-Hung Lee ; Chien-Hsien Lee ; Pei-Feng Liu ; Wen-Chi Chang ; Yung-Kai Wang ; Lee-Feng Chien ; Rong-Long Pan
• 关键词：Proton translocation ; Tonoplast ; Vacuole ; Site-directed mutagenesis ; Vacuolar H⁺-pyrophosphatase
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2007
• 期刊代码：13_00052728
• 类别：bio
• 出版时间：July 2007
• 卷：1767
• 期：7
• 页码：965-973
• 文件大小：1037 K

摘要

Plant vacuolar H⁺-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PP_i) 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 PP_i 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⁺–PP_i. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg²⁴² which is embedded in a more hydrophobic environment.