Properties of the C-terminal domain of enzyme I of the Escherichia coli phosphotransferase system.
The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Himatkumar V
HV
Patel
Kavita A
KA
Vyas
Roshan L
RL
Mattoo
Maurice
M
Southworth
Francine B
FB
Perler
Donald
D
Comb
Saul
S
Roseman
D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature
D008024:Ligands; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D008274:Magnesium