Abstract
The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.
Highlights
The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein
The Kinetics of ATP Binding and Turnover—Previously we established that the tryptophan fluorescence of ArsA (W159H6) was sensitive to the binding of MgATP [13]
A potentially plausible explanation for the fluorescence profile of the reaction is that the binding of MgATP to the ArsA induces an enhancement in the tryptophan fluorescence, which remains constant until the ATP has been depleted, at which point the fluorescence decays as the products are released
Summary
The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. The single tryptophan-containing W159H6 ArsA gave approximately a 4-fold increase in the signal-tonoise ratio in response to MgATP/mol of tryptophan residue when compared with the wild type enzyme [13]. We have used this ArsA derivative for the transient kinetic studies reported . We propose a model for the mechanism of MgATP binding and hydrolysis
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