Identification of amino acid residues participating in the energy coupling and proton transport of Streptomyces coelicolor A3(2) H+-pyrophosphatase.
The H(+)-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14-17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H(+)-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr(409), Val(411), and Gly(414) showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe(388), Thr(389), and Val(396) in cytoplasmic loop i were essential for efficient H(+) translocation. Ala(436) and Pro(560) in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H(+)-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme.
Hirono
Megumi
M
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Nakanishi
Yoichi
Y
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004734:Energy Metabolism / Q000235:genetics; D043564:Inorganic Pyrophosphatase / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism; D011522:Protons; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D008565:Membrane Proteins; D017494:Proton Pumps; D011522:Protons; D043564:Inorganic Pyrophosphatase