A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis.
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.
Scanlon
Joanne A Baylis
JA
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA.
Al-Shawi
Marwan K
MK
Nakamoto
Robert K
RK
D000255:Adenosine Triphosphate / Q000737:chemistry; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008968:Molecular Conformation; D009154:Mutation; D009243:NAD / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*
D003432:Cross-Linking Reagents; D009243:NAD; C033223:polyhistidine; D006639:Histidine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine