title: Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase.
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.
authors.affiliation: Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan.
authors.forename: Yi-Yuong
authors.initials: YY
authors.lastname: Hsiao
authors.forename: Yih-Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.forename: Shen-Hsing
authors.initials: SH
authors.lastname: Hsu
authors.forename: Yun-Tzu
authors.initials: YT
authors.lastname: Huang
authors.forename: Tseng-Huang
authors.initials: TH
authors.lastname: Liu
authors.forename: Ching-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Chien-Hsien
authors.initials: CH
authors.lastname: Lee
authors.forename: Pei-Feng
authors.initials: PF
authors.lastname: Liu
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Chang
authors.forename: Yung-Kai
authors.initials: YK
authors.lastname: Wang
authors.forename: Lee-Feng
authors.initials: LF
authors.lastname: Chien
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D003931:Diacetyl / Q000737:chemistry; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics; D007425:Intracellular Membranes / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010658:Phenylglyoxal / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D001120:Arginine; D014357:Trypsin; D043564:Inorganic Pyrophosphatase; D003931:Diacetyl; D010658:Phenylglyoxal