Kinetics of CheA autophosphorylation and dephosphorylation reactions.
The protein kinase CheA of Escherichia coli plays a central role in the signal transduction pathway controlling the swimming behavior of the cell in response to extracellular chemical gradients. CheA autophosphorylates at a rate controlled by the ligand binding state of chemotaxis receptor/transducer proteins. CheA directs the activities of CheY and CheB, effector proteins that become phosphorylated as a result of their interaction with phospho-CheA. In this study, we performed a detailed kinetic analysis of CheA's autophosphorylation reaction, and its dephosphorylation by ADP. Our kinetic data are consistent with a three-step mechanism for CheA autophosphorylation/dephosphorylation involving (i) substrate binding, (ii) phospho-transfer, and (iii) product release. We determined the dissociation constant for the kinetically defined CheA.ATP complex to be approximately 300 microM and the limiting rate constant for autophosphorylation to be approximately 0.026 s-1 at saturating ATP concentration. Our results indicate that the apparent dissociation constant of the phospho-CheA.ADP complex is approximately 42 microM and that the limiting rate constant for CheA dephosphorylation is approximately 0.028 s-1 at saturating ADP concentration. We corroborated the kinetically determined Kd values by performing independent ligand binding experiments. In addition, we found that the kinetics of trans-phosphorylation, involving mutant proteins CheA48HQ and CheA470GK, exhibited kinetic properties similar to those observed for autophosphorylation of wild-type CheA, although the limiting rate constant (0.008 s-1) was somewhat slower for this trans-phosphorylation reaction. These results will provide a framework for assessing the effects of various cheA mutations as well as for exploring the nature of CheA regulation by the chemotaxis receptor/transducer proteins.
Tawa
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P
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Stewart
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RC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002633:Chemotaxis; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006639:Histidine / Q000378:metabolism; D000071677:Histidine Kinase; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D006639:Histidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli