CATALYTIC MECHANISM OF PHOSPHORYLATION AND DEPHOSPHORYLATION OF CHEY - KINETIC CHARACTERIZATION OF IMIDAZOLE PHOSPHATES AS PHOSPHODONORS AND THE ROLE OF ACID CATALYSIS

Kinetic and equilibrium measurements of phosphotransfer events involvi ng CheY carried out over a range of pH conditions elucidated several f eatures of the phosphotransfer mechanism, Using tryptophan fluorescenc e intensity measurements as a monitor of phosphorylation, we showed th at phosphorylation using small molecule phosphodonors occurred by fast association of CheY with the phosphodonor, followed by rate-limiting phosphotransfer. Two previously uncharacterized phosphodonors, monopho sphoimidazole and diphosphoimdazole, were able to phosphorylate CheY a t a concentration about 6-fold lower than that of the previously descr ibed phosphodonors acetyl phosphate and phosphoramidate. This was show n to be due to tighter binding of the imidazole phosphates to CheY and implied the presence of binding interactions between CheY and the imi dazole group. The ability of CheY to autophosphorylate through the pH range of 5-10 differed for various phosphodonors. Acetyl phosphate and diphosphoimidazole were unaffected by pH over this range, whereas pho sphoramidate and monophosphoimidazole showed a steep dependence on pH with a loss of phosphorylation ability al about pH 7.4 (midpoint) for monophosphoimidazole and pH 7.8 (midpoint) for phosphoramidate, This b ehavior correlated with the loss of the positive charge on the nitroge n atom in the nitrogen-phosphorus bond in both monophosphoimidazole an d phosphoramidate and implied that CheY was not capable of donating a proton to the leaving group in phosphotransfer with small molecules, T he rate of phosphotransfer from [P-32]CheA-phosphate to wild type CheY also decreased markedly (>150 times) between pH 7.5 and 10. Because t he mutant CheY proteins K109R and Ts7A showed the same pH dependence a s the wild type, the loss of activity in the alkaline range could not be attributed to deprotonation of either of these active site residues . This observation, combined with the moderate decreases in phosphotra nsfer rates for these mutants relative to that of wild type CheY, indi cated that it is unlikely that either Thr87 or Lys109 plays a direct r ole in the catalysis of phosphotransfer. Finally, we showed that the r ate of autodephosphorylation of CheY was independent of pH over the ra nge of 4.5-11. Together, these studies led to a model with CheY playin g a largely entropic role in its own phosphorylation and dephosphoryla tion.