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Bi-site catalysis in F1-ATPase: does it exist?
1Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
The Journal of Biological Chemistry
|July 14, 2001
Summary
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.
- 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.