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Volume 271, Number 50, Issue of December 13, 1996 pp. 32057-32063
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Active Site Interference and Asymmetric Activation in the Chemotaxis Protein Histidine Kinase CheA

(Received for publication, August 8, 1996, and in revised form, September 12, 1996)

From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

The histidine protein kinase CheA is a multidomain protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem. 271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the protein. The reaction fits Michaelis-Menten kinetics (K_m = 0.26 mM for ATP and 0.10 mM for phosphoacceptor domain; k_obs = 17 min¹). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.

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