Distance variations between active sites of H+-pyrophosphatase determined by fluorescence resonance energy transfer

Yun Tzu Huang, Tseng Huang Liu, Yen Wei Chen, Chien Hsien Lee, Hsueh Hua Chen, Tsu Wei Huang, Shen Hsing Hsu, Shih Ming Lin, Yih Jiuan Pan, Ching Hung Lee, Ian C. Hsu, Fan Gang Tseng, Chien Chung Fu, Rong Long Pan

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11 Citations (Scopus)


Homodimeric H+-pyrophosphatase (H+-PPase; EC is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H + translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.

Original languageEnglish
Pages (from-to)23655-23664
Number of pages10
JournalJournal of Biological Chemistry
Issue number31
Publication statusPublished - 2010 Jul 30

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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