Identification of the βTP site in the x-ray structure of F1-ATPase as the high-affinity catalytic site

Hui Z. Mao, Joachim Weber

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Abstract

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each β subunit, with widely differing affinities for MgATP or MgADP. During rotational catalysis, the sites switch their affinities. The affinity of each site is determined by the position of the central γ subunit. The site with the highest nucleotide binding affinity is catalytically active. From the available x-ray structures, it is not possible to discern the high-affinity site. Using fluorescence resonance energy transfer between tryptophan residues engineered into γ and trinitrophenyl nucleotide analogs on the catalytic sites, we were able to determine that the high-affinity site is close to the C-terminal helix of γ, but at considerable distance from its N terminus. Thus, the βTP site in the x-ray structure [Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Nature 370:621-628] is the high-affinity site, in agreement with the prediction of Yang et al. [Yang W, Gao YQ, Cui Q, Ma J, Karplus M (2003) Proc Natl Acad Sci USA 100:874-879]. Taking into account the known direction of rotation, the findings establish the sequence of affinities through which each catalytic site cycles during MgATP hydrolysis as low → high → medium → low.

Original languageEnglish
Pages (from-to)18478-18483
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number47
DOIs
StatePublished - Nov 20 2007

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Keywords

  • ATP synthase
  • Catalytic mechanism
  • Rotation

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