The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump

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 Trp(159) ArsA was used to elu cidate the elementary steps of the ATPase mechanism by fluorescence stopped -flow experiments. The binding and hydrolysis of MgATP is a multistep proce ss with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluore scence of ArsA, which is independent of the ATP concentration, indicative o f the build-up of a pre-steady state intermediate, This finding, coupled wi th 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. k(cat) is fa ster than the phosphate burst, indicating that both nucleotide binding site s of ArsA are catalytic, Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burs t. [GRAPHICS] ion is enhanced by the addition of ligand. Thus, ARA70 can function as a li gand-enhanced coactivator of PPAR gamma. Finally, we show that AR can squel ch PPAR gamma-ARA70 transactivation, which suggests that cross-talk may occ ur between PPAR gamma- and AR-mediated responses in adipocytes.