Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase.
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.
Hsiao
Yi-Yuong
YY
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan.
Pan
Yih-Jiuan
YJ
Hsu
Shen-Hsing
SH
Huang
Yun-Tzu
YT
Liu
Tseng-Huang
TH
Lee
Ching-Hung
CH
Lee
Chien-Hsien
CH
Liu
Pei-Feng
PF
Chang
Wen-Chi
WC
Wang
Yung-Kai
YK
Chien
Lee-Feng
LF
Pan
Rong-Long
RL
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*
D001120:Arginine; D014357:Trypsin; D043564:Inorganic Pyrophosphatase; D003931:Diacetyl; D010658:Phenylglyoxal