ACTIVE-SITE INTERFERENCE AND ASYMMETRIC ACTIVATION IN THE CHEMOTAXIS PROTEIN HISTIDINE KINASE CHEA

The histidine protein kinase CheA is a multidomain protein that mediat es stimulus-response coupling in bacterial chemotaxis, We have previou sly shown that the purified protein exhibits an equilibrium between in active monomer and active dimer (Surette, M., Levit, M., Liu, Y., Luka t, 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 k inetics (K-m = 0.26 mM for ATP and 0.10 mM for phosphoacceptor domain; k(obs) = 17 min(-1)), The catalytic domain exhibits the same equilibr ium between inactive monomers and active dimers as the full-length Che A protein, Thus, CheA dimerization is an intrinsic property of this do main, 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 dimeri zation are localized entirely on the catalytic domain, An analysis of the kinetics of phosphorylation catalyzed by CheA catalytic domain het erodimers indicates half of the sites reactivity, The rate of CheA pho sphorylation 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 r eceptors involves a mechanism that directs catalysis to one active sit e while preventing interference from the other.