MUTATIONAL ACTIVATION OF CHEA, THE PROTEIN-KINASE IN THE CHEMOTAXIS SYSTEM OF ESCHERICHIA-COLI

In Escherichia coli and Salmonella typhimurium, appropriate changes of cell swimming patterns are mediated by CheA, an autophosphorylating h istidine protein kinase whose activity is regulated by receptor/transd ucer proteins. The molecular mechanism underlying this regulation rema ins unelucidated but may involve CheA shifting between high-activity a nd low-activity conformations. We devised an in vivo screen to search for potential hyperkinase variants of CheA and used this screen to ide ntify two cheA point mutations that cause the CheA protein to have ele vated autokinase activity. Each point mutation resulted in alteration of proline 337. In vitro, CheA337PL and CheA337PS autophosphorylated s ignificantly more rapidly than did wild-type CheA. This rate enhanceme nt reflected the higher affinities of the mutant proteins for ATP and an increased rate constant for acquisition by CheA of the gamma-phosph oryl group of ATP within a kinetically defined CheA ATP complex. In ad dition, the mutant proteins reacted with ADP more rapidly than did wil d-type CheA. We considered the possibility that the mutations served t o lock CheA into an activated signaling conformation; however, we foun d that both mutant proteins were regulated in a normal fashion by the transducer Tsr in the presence of CheW. We exploited the activated pro perties of one of these mutants to investigate whether the CheA subuni ts within a CheA dimer make equivalent contributions to the mechanism of trans phosphorylation. Our results indicate that CheA trans phospho rylation may involve active-site residues that are located both in cis and in trans to the autophosphorylation site and that the two protome rs of a CheA dimer make nonequivalent contributions in determining the affinity of the ATP-binding site(s) of CheA.