KINETICS OF CHEA AUTOPHOSPHORYLATION AND DEPHOSPHORYLATION REACTIONS

The protein kinase CheA of Escherichia coli plays a central role in th e signal transduction pathway controlling the swimming behavior of the cell in response to extracellular chemical gradients. CheA autophosph orylates at a rate controlled by the ligand binding state of chemotaxi s receptor/transducer proteins. CheA directs the activities of CheY an d CheB, effector proteins that become phosphorylated as a result of th eir interaction with phospho-CheA. In this study, we performed a detai led kinetic analysis of CheA's autophosphorylation reaction, and its d ephosphorylation by ADP. Our kinetic data are consistent with a three- step mechanism for CheA autophosphorylation/dephosphorylation involvin g (i) substrate binding, (ii) phospho-transfer, and (iii) product rele ase. We determined the dissociation constant for the kinetically defin ed CheA ATP complex to be approximately 300 mu M and the limiting rate constant for autophosphorylation to be approximately 0.026 s(-1) at s aturating ATP concentration. Our results indicate that the apparent di ssociation constant of the phospho-CheA.ADP complex is approximately 4 2 mu M and that the limiting rate constant for CheA dephosphorylation is approximately 0.028 s(-1) at saturating ADP concentration. We corro borated the kinetically determined K-d values by performing independen t ligand binding experiments. In addition, we found that the kinetics of trans-phosphorylation, involving mutant proteins CheA48HQ and CheA4 70GK, exhibited kinetic properties similar to those observed for autop hosphorylation of wild-type CheA, although the limiting rate constant (0.008 s(-1)) was somewhat slower for this trans-phosphorylation react ion. 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.