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 homodim er that catalyzes the reversible transfer of a gamma-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kin etic studies of rates of autophosphorylation show a second order depen dence on CheA concentrations at submicromolar levels that is consisten t with dissociation of the homodimer into inactive monomers. The disso ciation was confirmed by chemical cross-linking studies. The dissociat ion constant (CheA(2) <-> 2cheA; K-D = 0.2-0.4 mu M) was not affected by nucleotide binding, histidine phosphorylation, or binding of the re sponse regulator, CheY. The turnover number per active site within a d imer (assuming 2 independent sites/dimer) at saturating ATP was approx imately 10/min. The kinetics of autophosphorylation and ATP/ADP exchan ge indicated that the dissociation constants of ATP and ADP bound to C heA were similar (K-D values approximate to 0.2-0.3 mM), whereas ATP h ad a reduced affinity for CheA similar to P (K-D approximate to 0.8 mM ) compared with ADP (K-D approximate to 0.3 mM). The rates of phosphot ransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (k(cat) appr oximate to 10 min(-1)).