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 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.
Walmsley
A R
AR
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, United Kingdom. A.Walmsel@bio.gla.ac.uk
Zhou
T
T
Borges-Walmsley
M I
MI
Rosen
B P
BP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli; D016623:Ion Pumps; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008956:Models, Chemical; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D018053:Arsenites; D016623:Ion Pumps; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; C015001:arsenite