title: Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: equilibrium kinetics and mechanism of enzyme i phosphorylation.
abstract: The phosphorylation of enzyme I from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system was studied by means of isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H--2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate. 14C--12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect (nu V/nu D = 1.9), showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer. Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated. From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse second-order rate constants of the reaction were determined to be 3 X 10(7) and 8 X 10(5) M-1 min-1, respectively, yielding an equilibrium constant of approximately 40 and a delta G degree for enzyme I phosphorylation of --2.3 kcal/mol. The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.
mesh_terms: D001665:Binding Sites; D002250:Carbon Radioisotopes; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011773:Pyruvates
chemical_list: D002250:Carbon Radioisotopes; D011773:Pyruvates; D010728:Phosphoenolpyruvate; D003903:Deuterium; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System