Interaction of substrate and effector binding sites in the ArsA ATPase.
The ars operon of plasmid R773 confers resistance to antimonials and arsenicals in Escherichia coli by encoding an ATP-dependent extrusion system for the oxyanions. The catalytic subunit, the ArsA protein, is an ATPase with two nucleotide binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA ATPase is allosterically activated by tricoordinate binding of As(3+) or Sb(3+) to three cysteine thiolates. Previous measurements suggested that the intrinsic fluorescence of tryptophans might be useful for examining binding of Mg2+ ATP and antimonite. In the present study an increase in intrinsic tryptophan fluorescence was observed upon addition of Mg2+ ATP. This enhancement was reversed by addition of antimonite. The ArsA protein contains four tryptophan residues: Trp159, Trp253, Trp522, and Trp524. The first two were altered to tyrosine residues by site-directed mutagenesis. Cells expressing both the arsAW159Y and arsAW253Y mutations retained resistance to arsenite, and the purified W159Y and W253Y proteins retained ATPase activity. While the intrinsic tryptophan fluorescence of the W253Y protein responded to addition of Mg2+ ATP, intrinsic tryptophan fluorescence in the purified W159Y protein was no longer enhanced by substrate. These results suggest that Trp159 is conformationally coupled to one or both of the nucleotide binding sites and provides a useful probe for the interaction of effector and substrate binding sites.
Zhou
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State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Academia Sinica, Beijing, China.
Liu
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S
Rosen
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BP
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D053501:Arsenite Transporting ATPases; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D016623:Ion Pumps; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009876:Operon; D011193:Potassium Iodide / Q000494:pharmacology; D011815:R Factors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D016623:Ion Pumps; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D011193:Potassium Iodide; D014364:Tryptophan; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D003545:Cysteine