In the chemotaxis system of Escherichia coli, phosphorylation of the CheY protein plays an important role in regulating the swimming pattern of the cell. In vitro, CheY can be phosphorylated either by phosphotransfer from phospho-CheA or by acquiring a phosphoryl group from any of a variety of small, high-energy phosphodonor molecules such as acetyl phosphate. Previous work explored the rapid kinetics of CheY phosphorylation by CheA. Here we extend that work and examine the kinetics of CheY phosphorylation by several small-molecule phosphodonors, including acetyl phosphate, benzoyl phosphate, carbamoyl phosphate, 2-methoxybenzoyl phosphate, and phosphoramidate. Our results indicate that these phosphodonors bind to CheY with relatively low affinity (Ks values ranging from 10 to 600 mM) and that the rate constant (kphos) for phosphotransfer at saturating phosphodonor concentrations is relatively slow (values ranging from 0.05 to 0.5 s-1). By contrast, under identical conditions, phosphorylation of CheY by phospho-CheA occurs much more rapidly (kphos approximately 800 s-1) and reflects CheY binding to phospho-CheA considerably more tightly (Ks approximately 60 microM) than it does to the small-molecule phosphodonors. In comparing CheA-mediated phosphorylation of CheY to small-molecule-mediated phosphorylation of CheY, the large difference in kphos values suggests that phospho-CheA makes significant contributions to the catalysis of CheY phosphorylation. The effects of pH and ionic strength on CheY phosphorylation kinetics were also investigated. For CheA-->CheY phosphotransfer, increasing ionic strength resulted in increased Ks values while kphos was unaffected. For CheY phosphorylation by small-molecule phosphodonors, increasing ionic strength resulted in decreasing Ks values and increasing kphos values. The significance of these effects is discussed in relation to the catalytic mechanism of CheY phosphorylation by phospho-CheA and small-molecule phosphodonors.

The N delta 1 proton of His 64 forms a hydrogen bond with Asp 32, as part of the catalytic triad in serine proteases of the subtilisin family. His 64 in subtilisin has been studied by 1H and 15N NMR spectroscopy in the presence and absence of peptidyl trifluoromethyl ketones (TFMKs) that are transition state analog inhibitors. For subtilisin Carlsberg, the downfield resonance of the imidazolium N delta 1 proton is approximately 18.3 ppm and the D/H fractionation factor is 0.55 +/- 0.04 at pH 5.5 (11 degrees C), and 0.63 +/- 0.04 (5 degrees C) and 0.68 +/- 0.04 at pH 6 (11 degrees C). In the complex between subtilisin Carlsberg and Z-L-leucyl-L-leucyl-L-phenylalanyltrifluoromethyl ketone (Z-LLF-CF3) at pH values between 6.5 and 10.6, His 64 remains positively charged, and the D/H fractionation factor of its N delta 1 proton is 0.85 +/- 0.05. In the complex between a subtilisin variant from Bacillus lentus and Z-LLF-CF3, the proton resonance at 18.8 ppm is correlated with a 15N resonance at 197.6 ppm downfield from liquid NH3 with a 1JNH of 81 Hz. The chemical shifts of subtilisin complexes with peptidyl TFMKs are among the most downfield shifts reported for any protein. At pH 9.5, His 64 is neutral and the D/H fractionation factor increases to 1.2 with a chemical shift of 15.0. His 64 is positively charged in the free enzyme at low pH, the inhibitor hemiketal complex at neutral pH, and the transition state for amide bond hydrolysis. These data thus provide indirect evidence for the presence of a low-barrier hydrogen bond in the catalytic mechanism of subtilisin proteases.

Selectively labeled samples of human Hor N-rar p21 ligated to MnnGDP or MnnGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated [4-13 *C]-labeled Asx into p21-MnnGDP and found that the distance from the carboxyl13C of Asp57 to Mn11 is ~4.1Á. Our result is consistent with indirect coordination of this residue to the metal. From a [2-2H]Thr-labeled sample, we estimate that the distance from the Mn11 ion to the 2H of Thr35 is at least 5.8 Á. Thus, the only protein or nucleotide ligands to the metal appear to be Ser 17 and the 3-phosphate of GDP, as previously reported [Larsen, R. G., Halkides, C. J., Redfield, A. G., & Singel, D. J. (1992b) J. Am. Chem. Soc. 114, 9608-9611], In the S'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been reported by X-ray crystallography to be a ligand of the metal

To structurally characterize the activated state of the transiently phosphorylated signal transduction protein CheY, we have constructed an alpha-thiophosphonate derivative of the CheY D57C point mutant and determined its three-dimensional structure at 1.85 A resolution. We have also characterized this analogue with high-resolution NMR and studied its binding to a peptide derived from FliM, CheY's target component of the flagellar motor. The chemically modified derivative, phosphono-CheY, exhibits many of the chemical properties of phosphorylated wild-type CheY, except that it is indefinitely stable. Electron density for the alpha-thiophosphonate substitution is clear and readily interpretable; omit refinement density at the phosphorus atom is greater than 10sigma. The molecule shows a number of localized conformational changes that are believed to constitute the postphosphorylation activation events. The most obvious of these changes include movement of the side chain of the active site base, Lys 109, and a predominately buried conformation of the side chain of Tyr 106. In addition, there are a number of more subtle changes more distant from the active site involving the alpha4 and alpha5 helices. These results are consistent with our previous structural interpretations of other CheY activation mutants, and with our earlier hypotheses concerning CheY activation through propagation of structural changes away from the active site.