title: The N-terminal domain of Escherichia coli enzyme I of the phosphoenolpyruvate/glycose phosphotransferase system: molecular cloning and characterization.
abstract: The bacterial phosphoenolpyruvate/glycose phosphotransferase system (PTS) comprises a group of proteins that catalyze the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to sugars concomitant with their translocation. The first two steps of the phosphotransfer sequence are PEP <--> Enzyme I (EI) <--> HPr (the histidine-containing phosphocarrier protein). We have proposed that many functions of the PTS are regulated by EI, which undergoes a monomer/dimer transition. EI monomer (63.5 kDa) comprises two major domains: a flexible C-terminal domain (EI-C) and a protease-resistant, structurally stable N-terminal domain (EI-N) containing the active site His. Trypsin treatment of Salmonella typhimurium EI yielded EI-N, designated EI-N(t). Homogeneous recombinant Escherichia coli EI-N [i.e., EI-N(r)], has now been prepared in quantity, shows the expected thermodynamic unfolding properties and, similarly to EI-N(t), is phosphorylated by phospho-HPr, but not by PEP. In addition, binding of EI-N(r) to HPr was studied by isothermal titration calorimetry: K/a = 1.4 x 10(5) M(-1) and delta H = +8.8 kcal x mol(-1). Both values are comparable to those for HPr binding to intact EI. Fluorescence anisotropy [dansyl-EI-N(r)] and gel filtration of EI-N(r) show that it does not dimerize. These results emphasize the role of EI-C in dimerization and the regulation of intact EI.
authors.affiliation: Department of Biology, The McCollum-Prat Institute, Baltimore, MD 21218, USA.
authors.forename: F
authors.initials: F
authors.lastname: Chauvin
authors.forename: A
authors.initials: A
authors.lastname: Fomenkov
authors.forename: C R
authors.initials: CR
authors.lastname: Johnson
authors.forename: S
authors.initials: S
authors.lastname: Roseman
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D002151:Calorimetry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase