Kinetic characterization of CheY phosphorylation reactions: Comparison of P-CheA and small-molecule phosphodonors

In the chemotaxis system of Escherichia coli, phosphorylation of the CheY p rotein plays an important role in regulating the swimming pattern of the ce ll. In vitro, CheY can be phosphorylated either by phosphotransfer from pho spho-CheA or by acquiring a phosphoryl group from any of a variety of small , high-energy phosphodonor molecules such as acetyl phosphate. Previous wor k explored the rapid kinetics of CheY phosphorylation by CheA. Here we exte nd that work and examine the kinetics of CheY phosphorylation by several sm all-molecule phosphodonors, including acetyl phosphate, benzoyl phosphate, carbamoyl phosphate, 2-methoxybenzoyl phosphate, and phosphoramidate. Our r esults indicate that these phosphodonors bind to CheY with relatively low a ffinity (K-s values ranging from 10 to 600 mM) and that the rate constant ( k(phos)) for phosphotransfer at saturating phosphodonor concentrations is r elatively slow (values ranging from 0.05 to 0.5 s(-1)). By contrast, under identical conditions, phosphorylation of CheY by phospho-CheA occurs much m ore rapidly (k(phos) similar to 800 s(-1)) and reflects CheY binding to pho spho-CheA considerably more tightly (K-s similar to 60 mu M) than it does t o the small-molecule phosphodonors. In comparing CheA-mediated phosphorylat ion of CheY to small-molecule-mediated phosphorylation of CheY, the large d ifference in k(phos) values suggests that phospho-CheA makes significant co ntributions to the catalysis of CheY phosphorylation. The effects of pH and ionic strength on CheY phosphorylation kinetics were also investigated. Fo r CheA-->CheY phosphotransfer, increasing ionic strength resulted in increa sed K-s values while k(phos) was unaffected. For CheY phosphorylation by sm all-molecule phosphodonors, increasing ionic strength resulted in decreasin g K-s values and increasing k(phos) values. The significance of these effec ts is discussed in relation to the catalytic mechanism of CheY phosphorylat ion by phospho-CheA and small-molecule phosphodonors.