Dimerization is required for the activity of the protein histidine kinase CheA that mediates signal transduction in bacterial chemotaxis

The histidine protein kinase CheA plays an essential role in stimulus- response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a γ-phosphoryl group from ATP to the N- 3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross- linking studies. The dissociation constant (CheA₂ mutually implies 2CheA; K(D) = 0.2-0.4 μM) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (K(D) values ≃ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheAP (K(D) ≃ 0.8 mM) compared with ADP (K(D) ≃ 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to he essentially equal (kcat ≃ 10 min^-1).