title: Catalytic mechanism of phosphorylation and dephosphorylation of CheY: kinetic characterization of imidazole phosphates as phosphodonors and the role of acid catalysis.
abstract: Kinetic and equilibrium measurements of phosphotransfer events involving CheY carried out over a range of pH conditions elucidated several features of the phosphotransfer mechanism. Using tryptophan fluorescence intensity measurements as a monitor of phosphorylation, we showed that phosphorylation using small molecule phosphodonors occurred by fast association of CheY with the phosphodonor, followed by rate-limiting phosphotransfer. Two previously uncharacterized phosphodonors, monophosphoimidazole and diphosphoimdazole, were able to phosphorylate CheY at a concentration about 6-fold lower than that of the previously described phosphodonors acetyl phosphate and phosphoramidate. This was shown to be due to tighter binding of the imidazole phosphates to CheY and implied the presence of binding interactions between CheY and the imidazole group. The ability of CheY to autophosphorylate through the pH range of 5-10 differed for various phosphodonors. Acetyl phosphate and diphosphoimidazole were unaffected by pH over this range, whereas phosphoramidate and monophosphoimidazole showed a steep dependence on pH with a loss of phosphorylation ability at about pH 7.4 (midpoint) for monophosphoimidazole and pH 7.8 (midpoint) for phosphoramidate. This behavior correlated with the loss of the positive charge on the nitrogen atom in the nitrogen-phosphorus bond in both monophosphoimidazole and phosphoramidate and implied that CheY was not capable of donating a proton to the leaving group in phosphotransfer with small molecules. The rate of phosphotransfer from [32P]CheA-phosphate to wild type CheY also decreased markedly (> 150 times) between pH 7.5 and 10. Because the mutant CheY proteins K109R and T87A showed the same pH dependence as the wild type, the loss of activity in the alkaline range could not be attributed to deprotonation of either of these active site residues. This observation, combined with the moderate decreases in phosphotransfer rates for these mutants relative to that of wild type CheY, indicated that it is unlikely that either Thr87 or Lys109 plays a direct role in the catalysis of phosphotransfer. Finally, we showed that the rate of autodephosphorylation of CheY was independent of pH over the range of 4.5-11. Together, these studies led to a model with CheY playing a largely entropic role in its own phosphorylation and dephosphorylation.
authors.affiliation: Department of Microbiology and Immunology, Univeristy of North Carolina, Chapel Hill 27599-7290, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Silversmith
authors.forename: J L
authors.initials: JL
authors.lastname: Appleby
authors.forename: R B
authors.initials: RB
authors.lastname: Bourret
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008956:Models, Chemical; D010755:Organophosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007093:Imidazoles; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010755:Organophosphates; D010750:Phosphoproteins; D011522:Protons; D011994:Recombinant Proteins; C000607606:cheY protein, E coli; C109526:diphosphoimidazole; C109525:monophosphoimidazole; D000071677:Histidine Kinase; C000607608:cheA protein, E coli