We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, γM23K, caused increased energy of interaction between γ- and β-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawi, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F1-ATPase [Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628] γM23K is believed to form an ionized hydrogen bond with βGlu-381 in the conserved β380DELSEED386 segment. In this report, we further test the role of γ-β-subunit interactions by introducing a series of substitutions for βGlu-381 and γArg-242, the residue which forms a hydrogen bond with βGlu-381 in the wild-type enzyme. βE381A, D, and Q were able to restore efficient coupling when co-expressed with γM23K. All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by γM23K. βE381K by itself caused inefficient coupling, but opposite from the effect of γM23K, the transition state thermodynamic parameters were lower than wild-type. These results suggest that the βE381K mutation perturbs the γ-β-subunit interaction and the local conformation of the β380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis. βE381A, L, K, and R, and γR242L and E mutations perturbed enzyme assembly and stability to varying degrees. These results provide functional evidence that the β380DELSEED386 segment and its interactions with the γ-subunit are involved in the mechanism of coupling.
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Research Article|
March 01 1998
Intergenic suppression of the γM23K uncoupling mutation in F0F1 ATP synthase by βGlu-381 substitutions: the role of the β380DELSEED386 segment in energy coupling Available to Purchase
J. Christian KETCHUM;
J. Christian KETCHUM
1Department of Molecular Physiology and Biological Physics, University of Virginia, P.O. Box 10011, Charlottesville, VA 22906-0011, U.S.A.
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Marwan K. AL-SHAWI;
Marwan K. AL-SHAWI
1Department of Molecular Physiology and Biological Physics, University of Virginia, P.O. Box 10011, Charlottesville, VA 22906-0011, U.S.A.
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K. Robert NAKAMOTO
K. Robert NAKAMOTO
1
1Department of Molecular Physiology and Biological Physics, University of Virginia, P.O. Box 10011, Charlottesville, VA 22906-0011, U.S.A.
1To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
May 21 1997
Revision Received:
October 23 1997
Accepted:
November 11 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 330 (2): 707–712.
Article history
Received:
May 21 1997
Revision Received:
October 23 1997
Accepted:
November 11 1997
Citation
J. Christian KETCHUM, Marwan K. AL-SHAWI, K. Robert NAKAMOTO; Intergenic suppression of the γM23K uncoupling mutation in F0F1 ATP synthase by βGlu-381 substitutions: the role of the β380DELSEED386 segment in energy coupling. Biochem J 1 March 1998; 330 (2): 707–712. doi: https://doi.org/10.1042/bj3300707
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