The ArsA ATPase is the catalytic subunit of the pump protein, coupling the hydrolysis of ATP to the movement of arsenicals and antimonials through the membrane-spanning ArsB protein. Previously, we have shown the binding and hydrolysis of MgATP to ArsA to be a multi-step process in which the rate-limiting step is an isomerization between different conformational forms of ArsA. This isomerization occurs after product release, at the end of the ATPase reaction, and involves the return of the ArsA to its original conformation, which can then bind MgATP. ArsA possesses an allosteric site for antimonite [Sb(III)], the binding of which elevates the steady-state ATPase activity. We have used a transient kinetics approach to investigate the kinetics of ternary complex formation that lead to an enhancement in the ATPase activity. These studies revealed that ArsA exists in at least two conformational forms that differ in their ligand binding affinities, and that ATP favours one form and Sb(III) the other. Ternary complex formation is rate-limited by a slow transition between these conformational forms, leading to a lag in attaining maximal steady-state activity. Sb(III) enhances the steady-state ATPase activity by inducing rapid product release, allowing ArsA to adopt a conformation that can bind MgATP for the next catalytic cycle. In the presence of Sb(III), ArsA avoids the rate-limiting isomerization at the end of the ATPase reaction and ATP hydrolysis becomes rate-limiting for the reaction. The binding of Sb(III) probably results in more effective pumping of the substrates from the cell by enhancing the rate of efflux.
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December 2001
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Research Article|
December 10 2001
Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump
Adrian R. WALMSLEY;
Adrian R. WALMSLEY
1
∗Division of Infection and Immunity, The Institute of Biomedical and Life Sciences, Robertson Building, The University of Glasgow, Glasgow G11 6NU, Scotland, U.K.
1To whom correspondence should be addressed (e-mail A.Walmsley@bio.gla.ac.uk).
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Tongqing ZHOU;
Tongqing ZHOU
†Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A.
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M. Ines BORGES-WALMSLEY;
M. Ines BORGES-WALMSLEY
∗Division of Infection and Immunity, The Institute of Biomedical and Life Sciences, Robertson Building, The University of Glasgow, Glasgow G11 6NU, Scotland, U.K.
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Barry P. ROSEN
Barry P. ROSEN
†Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A.
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Publisher: Portland Press Ltd
Received:
May 01 2001
Revision Received:
September 09 2001
Accepted:
October 08 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 589–597.
Article history
Received:
May 01 2001
Revision Received:
September 09 2001
Accepted:
October 08 2001
Citation
Adrian R. WALMSLEY, Tongqing ZHOU, M. Ines BORGES-WALMSLEY, Barry P. ROSEN; Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump. Biochem J 15 December 2001; 360 (3): 589–597. doi: https://doi.org/10.1042/bj3600589
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