Transient state kinetics of Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli: equilibrium and second-order rate constants for the phosphotransfer reactions with phosphoenolpyruvate and HPr.
The first two reactions in the phosphotransfer sequence of bacterial phosphoenolpyruvate:glycose phosphotransferase systems are the autophosphorylation of Enzyme I by phosphoenolpyruvate followed by the transfer of the phospho group to the low-molecular weight protein, HPr. Transient state kinetic methods were used to estimate the second-order rate constants for both phosphotransfer reactions. These measurements support previous conclusions that only the dimer of Enzyme I, EI2, is autophosphorylated, and that the rate of formation of dimer is slow compared to the rate of its phosphorylation. The rate constants of the two autophosphorylation reactions of EI2 by PEP are 6.6 x 10(6) M(-1) s(-1), and differ from one another by a factor of less than 3. The rate constant for the transfer reaction between phospho-EI2 and HPr is unusually large for a covalent reaction between two proteins (220 x 10(6) M(-1) s(-1)), while the constant for the reverse reaction is 4.2 x 10(6) M(-1) s(-1). Using the previously reported equilibrium constant for the autophosphorylation reaction, 1.5, the overall equilibrium constant for phosphotransfer from PEP to HPr is 80, somewhat higher than that previously reported. The results also show that EI2 can phosphorylate multiple molecules of HPr without dissociating to a monomer (EI), and that EI can accept a phospho group from phospho-HPr. These results are directly applicable to predicting the rates of phosphoenolpyruvate phosphotransferase system sugar uptake in whole cells.
Meadow
Norman D
ND
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Mattoo
Roshan L
RL
Savtchenko
Regina S
RS
Roseman
Saul
S
D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase