Related Experiment Videos
Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase
Y M Milgrom1, M B Murataliev, P D Boyer
1Department of Biochemistry and Molecular Biology, State University of New York Health Science Center at Syracuse, 750 E. Adams St., Syracuse, NY 13210, USA.
The Biochemical Journal
|April 18, 1998
Summary
Bovine heart mitochondrial F1-ATPase exhibits uni-site catalysis and transitions to multi-site catalysis. Bi-site activation drives near maximal hydrolysis rates, invalidating claims of no catalytic site cooperativity.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Mitochondrial function
Background:
- Bovine heart mitochondrial F1-ATPase is a key enzyme in ATP synthesis.
- Understanding its catalytic mechanisms, including uni-site and multi-site hydrolysis, is crucial.
- Previous studies suggested specific kinetic parameters for F1-ATPase, which are re-evaluated here.
Purpose of the Study:
- To investigate the transition from uni-site to multi-site catalysis in bovine heart mitochondrial F1-ATPase.
- To determine the kinetic parameters governing F1-ATPase activity.
- To address conflicting reports on catalytic site cooperativity in this enzyme.
Main Methods:
- Enzyme kinetic measurements, including initial velocity studies.
- ATP hydrolysis assays under varying substrate and enzyme concentrations.
- Competition assays using ATP analogs like TNP-ATP.
Main Results:
- Uni-site ATP hydrolysis occurs slowly without tightly bound nucleotides or Pi.
- Bi-site activation significantly increases hydrolysis rates, with a Km of ~130 microM and Vmax of ~700 s-1.
- Evidence for a third catalytic site in the millimolar range and invalidation of claims against catalytic cooperativity.
Conclusions:
- Mitochondrial F1-ATPase can achieve near maximal activity through bi-site catalysis.
- The enzyme demonstrates catalytic site cooperativity, contrary to recent assertions.
- Kinetic parameters differ from previously proposed values, highlighting complex regulatory mechanisms.