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.
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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.