KINETICS OF CHEA AUTOPHOSPHORYLATION AND DEPHOSPHORYLATION REACTIONS

摘要: The protein kinase CheA of Escherichia coli plays a central role in the signal transduction pathway controlling swimming behavior cell response to extracellular chemical gradients. autophosphorylates at rate controlled by ligand binding state chemotaxis receptor/transducer proteins. directs activities CheY and CheB, effector proteins that become phosphorylated as result their interaction with phospho-CheA. In this study, we performed detailed kinetic analysis CheA's autophosphorylation reaction, its dephosphorylation ADP. Our data are consistent three-step mechanism for autophosphorylation/dephosphorylation involving (i) substrate binding, (ii) phospho-transfer, (iii) product release. We determined dissociation constant kinetically defined CheA.ATP complex be approximately 300 microM limiting 0.026 s-1 saturating ATP concentration. results indicate apparent phospho-CheA.ADP is 42 0.028 ADP corroborated Kd values performing independent experiments. addition, found kinetics trans-phosphorylation, mutant CheA48HQ CheA470GK, exhibited properties similar those observed wild-type CheA, although (0.008 s-1) was somewhat slower trans-phosphorylation reaction. These will provide framework assessing effects various cheA mutations well exploring nature regulation