A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis

• Published in: The Journal of biological chemistry 2008-09, Vol.283 (38), p.26228-26240
• Main Authors: Scanlon, Joanne A. Baylis, Al-Shawi, Marwan K., Nakamoto, Robert K.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Catalysis; Cross-Linking Reagents - pharmacology; Cysteine - chemistry; Electrochemistry - methods; Escherichia coli - enzymology; Histidine - chemistry; Hydrolysis; Kinetics; Metabolism and Bioenergetics; Models, Biological; Molecular Conformation; Mutation; NAD - chemistry; Plasmids - metabolism; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M804858200

The F0F1-ATP synthase couples the functions of H+ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located γ, ϵ, and c subunits. To understand the γ subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F1-ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, βD380C and βE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved γCys-87. In the pre-steady state, the γ-β cross-linked enzyme at high Mg·ATP conditions retained the burst of hydrolysis but was not able to release Pi. These data show that the rate-limiting rotation step, kγ, occurs after hydrolysis and before Pi release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the βGlu-381 residue for proper formation of the rate-limiting transition state involving γ subunit rotation.