Autophosphorylation and dephosphorylation by soluble forms of the nitrate-responsive sensors NarX and NarQ from Escherichia coli K-12.
NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control Escherichia coli gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (K(m)) for ADP were similar for both proteins, about 2 microM, whereas the affinity of MBP-NarQ for ATP was lower, about 23 microM. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 x 10(-4) s(-1)) was lower than that observed for MBP-NarQ (about 2.2 x 10(-4) s(-1)). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s(-1) for MBP-NarX and about 0.01 s(-1) for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 microM (KinA) to about 40 microM (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.
Noriega
Chris E
CE
Department of Microbiology, University of California, One Shields Avenue, Davis, CA 95616-8665, USA.
Schmidt
Radomir
R
Gray
Michael J
MJ
Chen
Li-Ling
LL
Stewart
Valley
V
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D048168:Escherichia coli K12 / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009566:Nitrates; D010750:Phosphoproteins; C088708:narQ protein, E coli; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C074787:narX protein, E coli