title: Identification of the critical residues for the function of vacuolar H⁺-pyrophosphatase by mutational analysis based on the 3D structure.
abstract: H(+)-translocating pyrophosphatase (H(+)-PPase) converts energy from hydrolysis of inorganic pyrophosphate (PPi) to active H(+) translocation across membranes. From the 3D structure resolved by crystallography, 17 amino acid residues in several domains of mung bean (Vigna radiata) enzyme were selected and substituted with alanine individually. The mutant enzymes were expressed in yeast cells to evaluate their biochemical role. The highly conserved residues in the substrate-binding site (T249, D269, D507 and N534) were shown to be essential for PPi hydrolysis and H(+) pump. The amino acid substitution of residues in the H(+) translocation pathway (I545, L555, N738, V746 and L749) resulted in mild decrease in the PPase activity and strong suppression of the H(+) pump. These results suggest that the decoupling of PPi hydrolysis and active H(+) translocation occurred in these five mutants including I545A. The alanine substitution of the C124 and C132, which form an intra-molecular disulfide bond, did not affect the enzyme activity. The modifications of the other residues in the vacuolar lumen loop, and M15 had relatively mild effect on the enzyme function. Functional roles of the 17 residues are discussed with consideration of the 3D structural information.
authors: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors: Mariko
authors: M
authors: Asaoka
authors: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors: Shoji
authors: S
authors: Segami
authors: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan maeshima@agr.nagoya-u.ac.jp.
authors: Masayoshi
authors: M
authors: Maeshima
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D007887:Fabaceae / Q000201:enzymology*; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D043564:Inorganic Pyrophosphatase; D000409:Alanine
keywords: H+-translocating pyrophosphatase; energy conversion; site-directed mutagenesis; structure–function relationship; vacuolar membrane