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Bi-site catalysis in F1-ATPase: does it exist?

J Weber1, A E Senior

  • 1Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.

The Journal of Biological Chemistry
|July 14, 2001
PubMed
Summary
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F(1)F(0)-ATP synthase operates via a tri-site mechanism, not a bi-site one. This finding, supported by ITP and ATP hydrolysis studies, clarifies enzyme function and subunit rotation during ATP synthesis and hydrolysis.

Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Molecular biology

Background:

  • The catalytic mechanism of F(1)F(0)-ATP synthase remains debated, with proposed tri-site and bi-site models.
  • Understanding the precise mechanism is crucial for elucidating energy transduction in cells.

Purpose of the Study:

  • To resolve the controversy between tri-site and bi-site mechanisms for F(1)F(0)-ATP synthase.
  • To determine the predominant mechanism driving enzyme activity and subunit rotation.

Main Methods:

  • Utilized inosine triphosphate (ITP) as a hydrolysis substrate due to its lower binding affinity compared to ATP, allowing for precise site occupancy assessment.
  • Calculated and compared the distributions of all eight enzyme species across varying ITP concentrations with measured ITPase activity.

Related Experiment Videos

  • Performed theoretical analyses of proposed bi-site catalytic mechanisms.

    • Main Results:

    • Experimental data using both ITP and ATP strongly support a tri-site mechanism.
    • Bi-site hydrolysis activity was found to be negligible or non-existent.
    • Theoretical analysis revealed significant flaws in proposed bi-site mechanisms, including incorrect subunit rotation predictions and reliance on infrequent enzyme species.

    Conclusions:

    • The F(1)F(0)-ATP synthase predominantly functions via a tri-site hydrolysis mechanism.
    • Tri-site hydrolysis is the driving force for enzyme subunit rotation.
    • Existing bi-site models are inconsistent with experimental and theoretical findings.