The Journal of biological chemistry, 2010-07, Vol.285 (31), p.23655-23664
2010
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- Autor(en) / Beteiligte
- Titel
- Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer
- Ist Teil von
- The Journal of biological chemistry, 2010-07, Vol.285 (31), p.23655-23664
- Ort / Verlag
- United States: Elsevier Inc
- Erscheinungsjahr
- 2010
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0021-9258eISSN: 1083-351XDOI: 10.1074/jbc.M110.134916Titel-ID: cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2911311
- Format
- –
- Schlagworte
- Amino Acid Motifs, Atomic Force Microscopy, Bioenergetics, Catalytic Domain, Clostridium tetani - enzymology, Dimerization, Enzymology, Escherichia coli - enzymology, Fluorescence Resonance Energy Transfer (FRET), Fluorescence Resonance Energy Transfer - methods, Inorganic Pyrophosphatase - chemistry, Inorganic Pyrophosphatase - physiology, Ligands, Microsomes - metabolism, Mutation, Protein Sorting Signals, Protein Transport, Proton Pumps, Proton Transport, Pyrophosphatases - chemistry, Site-directed Mutagenesis, Spectrometry, Fluorescence