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

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

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)965-973
Number of pages9
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1767
Issue number7
DOIs
Publication statusPublished - 2007 Jul

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

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