Kinetic Characterization of Phosphotransfer between CheA and CheY in the Bacterial Chemotaxis Signal Transduction Pathway

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Abstract
Phosphorylation of the CheY protein is a crucial step in the chemotaxis signal transduction pathway of Escherichia coli. CheY becomes phosphorylated by acquiring a phosphoryl group from CheA, an autophosphorylating protein kinase. In this study, we utilized a rapid-quench instrument to investigate the kinetics of phosphotransfer in single-turnover experiments. Our results are consistent with a three-step mechanism for the CheA-to-CheY phosphotransfer reaction: (i) reversible binding of CheY to P-CheA; (ii) rapid, reversible phosphotransfer to CheY; (iii) reversible dissociation of the resulting CheA·CheY-P complex. Investigation of the effect of CheY concentration on the observed rate of phosphotransfer demonstrated saturation kinetics; the extrapolated limiting rate constant for phosphotransfer was 650 ± 200 s-1, while the Km value indicated from this work was 6.5 ± 2 μM. We demonstrated that the CheA−CheY phosphotransfer reaction was reversible by observing partial transfer of [32P]phosphate from CheY-P to CheA and by observing the effect of high concentrations of unphosphorylated CheA on the equilibrium: P-CheA + CheY ↔ CheA + CheY-P. We found that the rate of phosphotransfer from P-CheA to CheY can be inhibited by unphosphorylated CheA as well as by a fragment of CheA (CheA124-257) that contains the CheY binding site; these results suggest that the unphosphorylated form of CheA can effectively compete with P-CheA for available CheY (Kd ∼ 1.5 ± 0.6 μM for the CheY·CheA124-257 complex and for the CheY·CheA complex).