A Rotor-Stator Cross-link in the F1-ATPase Blocks theRate-limiting Step of RotationalCatalysis

摘要

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 analysisprovides additional insights into how the enzyme achieves efficient couplingand implicates the βGlu-381 residue for proper formation of therate-limiting transition state involving γ subunit rotation.