Unisite and multisite catalysis in the ArsA ATPase

Tongqing Zhou, Jian Shen, Ye Liu, Barry P. Rosen

Research output: Contribution to journalArticle

Abstract

The ars operon of plasmid R773 encodes an As(III)/ Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg2+ and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg2+ and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis).

Original languageEnglish (US)
Pages (from-to)23815-23820
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number26
DOIs
StatePublished - Jun 28 2002
Externally publishedYes

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Catalysis
Adenosine Triphosphatases
Nucleotides
Adenosine Triphosphate
varespladib methyl
Quenching
Metalloids
Adenosine Diphosphate
Fluorescence
Binding Sites
Tryptophan
Hydrolysis
Rapid quenching
Operon
Extrusion
Catalytic Domain
Plasmids
Pumps

ASJC Scopus subject areas

  • Biochemistry

Cite this

Unisite and multisite catalysis in the ArsA ATPase. / Zhou, Tongqing; Shen, Jian; Liu, Ye; Rosen, Barry P.

In: Journal of Biological Chemistry, Vol. 277, No. 26, 28.06.2002, p. 23815-23820.

Research output: Contribution to journalArticle

Zhou, T, Shen, J, Liu, Y & Rosen, BP 2002, 'Unisite and multisite catalysis in the ArsA ATPase', Journal of Biological Chemistry, vol. 277, no. 26, pp. 23815-23820. https://doi.org/10.1074/jbc.M203432200
Zhou, Tongqing ; Shen, Jian ; Liu, Ye ; Rosen, Barry P. / Unisite and multisite catalysis in the ArsA ATPase. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 26. pp. 23815-23820.
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