Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/67697
標題: Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase
作者: Hsiao, Y.Y.
Pan, Y.J.
Hsu, S.H.
Huang, Y.T.
Liu, T.H.
Lee, C.H.
Liu, P.F.
Chang, W.C.
Wang, Y.K.
Chien, L.F.
Pan, R.L.
關鍵字: proton translocation;tonoplast;vacuole;site-directed mutagenesis;vacuolar H+-pyrophosphatase;translocating inorganic pyrophosphatase;inhibition;site;arabidopsis;mutation;binding;mutagenesis;expression;transport;membranes
Project: Biochimica Et Biophysica Acta-Bioenergetics
期刊/報告no:: Biochimica Et Biophysica Acta-Bioenergetics, Volume 1767, Issue 7, Page(s) 965-973.
摘要: 
Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6. 1. 1) utilizes inorganic pyrophosphate (PP) 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 I I 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 W-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 Arg 242 is most likely the primary target residue for these two reagents. The mutation at Arg 242 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 Arg 242 which is embedded in a more hydrophobic environment. (C) 2007 Elsevier B.V. All rights reserved.
URI: http://hdl.handle.net/11455/67697
ISSN: 0005-2728
DOI: 10.1016/j.bbabio.2007.04.007
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