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. Mg(2+) 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 Mg(2+) 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).
Highlights
The ars operon of R-factor R773 encodes an arsenite extrusion system that confers resistance in Escherichia coli to the metalloids arsenite (As(III)) and antimonite (Sb(III)) [1]
Kaur [10] has suggested that ArsA exhibits both unisite and multisite catalysis in which only NBD1 participates in unisite catalysis and that a functional A1 NBD is required for NBD2 to participate in multisite catalysis
Characterization of ArsA M446W—Beginning with a derivative of ArsA in which all of the tryptophan residues had been replaced with tyrosine residues and a six-histidine tag had been added to the C terminus [14], Met446 was changed to a tryptophan residue
Summary
The ars operon of R-factor R773 encodes an arsenite extrusion system that confers resistance in Escherichia coli to the metalloids arsenite (As(III)) and antimonite (Sb(III)) [1] This efflux pump has a catalytic subunit, the ArsA ATPase, and a membrane subunit, the ArsB arsenite carrier [2, 3]. Under presteady state conditions the addition of Sb(III) produces two bursts of phosphate liberation, one of which is ϳ250-fold faster than the other (k ϭ 49 and 0.2 sϪ1) [12] From these results, it appears that both NBDs hydrolyze ATP in the activated state. In the absence of metalloid (unisite conditions), both ADP and ATP produced an enhancement of M446W protein fluorescence that was stable with time, indicating binding but not hydrolysis of ATP in NBD2. Genotype/description lacZ53 mutS201::Tn5 thyA36 rha-5 metB1 deoC IN(rrnD-rrnE) recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⌬(lac-proAB) FЈ [traD36
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