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J Biol Chem. 2008 Sep 19;283(38):26228-40. doi: 10.1074/jbc.M804858200. Epub 2008 Jul 15.

A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis.

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1
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA.

Abstract

The F(0)F(1)-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 gamma, epsilon, and c subunits. To understand the gamma 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 F(1)-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, betaD380C and betaE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gammaCys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg.ATP conditions retained the burst of hydrolysis but was not able to release P(i). These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P(i) release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the betaGlu-381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.

PMID:
18628203
PMCID:
PMC2533770
DOI:
10.1074/jbc.M804858200
[Indexed for MEDLINE]
Free PMC Article
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