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. 2001 Dec 15;360(Pt 3):589–597. doi: 10.1042/0264-6021:3600589

Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump.

A R Walmsley 1, T Zhou 1, M I Borges-Walmsley 1, B P Rosen 1
PMCID: PMC1222261  PMID: 11736648

Abstract

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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Selected References

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  1. Ambudkar S. V., Dey S., Hrycyna C. A., Ramachandra M., Pastan I., Gottesman M. M. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol. 1999;39:361–398. doi: 10.1146/annurev.pharmtox.39.1.361. [DOI] [PubMed] [Google Scholar]
  2. Bhattacharjee H., Li J., Ksenzenko M. Y., Rosen B. P. Role of cysteinyl residues in metalloactivation of the oxyanion-translocating ArsA ATPase. J Biol Chem. 1995 May 12;270(19):11245–11250. doi: 10.1074/jbc.270.19.11245. [DOI] [PubMed] [Google Scholar]
  3. Bhattacharjee H., Rosen B. P. Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase. J Biol Chem. 1996 Oct 4;271(40):24465–24470. doi: 10.1074/jbc.271.40.24465. [DOI] [PubMed] [Google Scholar]
  4. Borges-Walmsley M. I., Walmsley A. R. The structure and function of drug pumps. Trends Microbiol. 2001 Feb;9(2):71–79. doi: 10.1016/s0966-842x(00)01920-x. [DOI] [PubMed] [Google Scholar]
  5. Borst P., Ouellette M. New mechanisms of drug resistance in parasitic protozoa. Annu Rev Microbiol. 1995;49:427–460. doi: 10.1146/annurev.mi.49.100195.002235. [DOI] [PubMed] [Google Scholar]
  6. Chen C. M., Misra T. K., Silver S., Rosen B. P. Nucleotide sequence of the structural genes for an anion pump. The plasmid-encoded arsenical resistance operon. J Biol Chem. 1986 Nov 15;261(32):15030–15038. [PubMed] [Google Scholar]
  7. Conseil G., Baubichon-Cortay H., Dayan G., Jault J. M., Barron D., Di Pietro A. Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein. Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9831–9836. doi: 10.1073/pnas.95.17.9831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dey S., Ramachandra M., Pastan I., Gottesman M. M., Ambudkar S. V. Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10594–10599. doi: 10.1073/pnas.94.20.10594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hung L. W., Wang I. X., Nikaido K., Liu P. Q., Ames G. F., Kim S. H. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature. 1998 Dec 17;396(6712):703–707. doi: 10.1038/25393. [DOI] [PubMed] [Google Scholar]
  10. Karkaria C. E., Chen C. M., Rosen B. P. Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase. J Biol Chem. 1990 May 15;265(14):7832–7836. [PubMed] [Google Scholar]
  11. Kaur P. Expression and characterization of DrrA and DrrB proteins of Streptomyces peucetius in Escherichia coli: DrrA is an ATP binding protein. J Bacteriol. 1997 Feb;179(3):569–575. doi: 10.1128/jb.179.3.569-575.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaur P., Rosen B. P. Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase. J Biol Chem. 1992 Sep 25;267(27):19272–19277. [PubMed] [Google Scholar]
  13. Kaur P. The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity. J Biol Chem. 1999 Sep 3;274(36):25849–25854. doi: 10.1074/jbc.274.36.25849. [DOI] [PubMed] [Google Scholar]
  14. Li J., Liu S., Rosen B. P. Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump. J Biol Chem. 1996 Oct 11;271(41):25247–25252. doi: 10.1074/jbc.271.41.25247. [DOI] [PubMed] [Google Scholar]
  15. Li J., Rosen B. P. Steric limitations in the interaction of the ATP binding domains of the ArsA ATPase. J Biol Chem. 1998 Mar 20;273(12):6796–6800. doi: 10.1074/jbc.273.12.6796. [DOI] [PubMed] [Google Scholar]
  16. Li J., Rosen B. P. The linker peptide of the ArsA ATPase. Mol Microbiol. 2000 Jan;35(2):361–367. doi: 10.1046/j.1365-2958.2000.01696.x. [DOI] [PubMed] [Google Scholar]
  17. Mobley H. L., Rosen B. P. Energetics of plasmid-mediated arsenate resistance in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6119–6122. doi: 10.1073/pnas.79.20.6119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morbach S., Tebbe S., Schneider E. The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium. Characterization of the ATPase activity associated with the purified MalK subunit. J Biol Chem. 1993 Sep 5;268(25):18617–18621. [PubMed] [Google Scholar]
  19. Rosen B. P., Bhattacharjee H., Zhou T., Walmsley A. R. Mechanism of the ArsA ATPase. Biochim Biophys Acta. 1999 Dec 6;1461(2):207–215. doi: 10.1016/s0005-2736(99)00159-5. [DOI] [PubMed] [Google Scholar]
  20. Vanden Bossche H., Dromer F., Improvisi I., Lozano-Chiu M., Rex J. H., Sanglard D. Antifungal drug resistance in pathogenic fungi. Med Mycol. 1998;36 (Suppl 1):119–128. [PubMed] [Google Scholar]
  21. Walmsley A. R., Zhou T., Borges-Walmsley M. I., Rosen B. P. A kinetic model for the action of a resistance efflux pump. J Biol Chem. 2000 Nov 28;276(9):6378–6391. doi: 10.1074/jbc.M008105200. [DOI] [PubMed] [Google Scholar]
  22. Walmsley A. R., Zhou T., Borges-Walmsley M. I., Rosen B. P. The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump. J Biol Chem. 1999 Jun 4;274(23):16153–16161. doi: 10.1074/jbc.274.23.16153. [DOI] [PubMed] [Google Scholar]
  23. Zhou T., Liu S., Rosen B. P. Interaction of substrate and effector binding sites in the ArsA ATPase. Biochemistry. 1995 Oct 17;34(41):13622–13626. doi: 10.1021/bi00041a042. [DOI] [PubMed] [Google Scholar]
  24. Zhou T., Radaev S., Rosen B. P., Gatti D. L. Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. EMBO J. 2000 Sep 1;19(17):4838–4845. doi: 10.1093/emboj/19.17.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zhou T., Rosen B. P. Tryptophan fluorescence reports nucleotide-induced conformational changes in a domain of the ArsA ATPase. J Biol Chem. 1997 Aug 8;272(32):19731–19737. doi: 10.1074/jbc.272.32.19731. [DOI] [PubMed] [Google Scholar]
  26. van Veen H. W., Konings W. N. The ABC family of multidrug transporters in microorganisms. Biochim Biophys Acta. 1998 Jun 10;1365(1-2):31–36. doi: 10.1016/s0005-2728(98)00039-5. [DOI] [PubMed] [Google Scholar]

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