Sugar transport by the bacterial phosphotransferase system. Structural and thermodynamic domains of enzyme I of Salmonella typhimurium.
Enzyme I, the first in the sequence of phosphocarrier proteins of the bacterial phosphoenolpyruvate:glycose phosphotransferase system, is a potential critical point for regulating sugar uptake. The thermal stability of Enzyme I was studied by high sensitivity differential scanning calorimetry. At pH 7.5, thermal unfolding of the protein exhibits two peaks with maxima (Tm) at 47.6 and 55.1 degrees C, indicating that the protein comprises two cooperative unfolding structures. Interaction between the two domains is markedly dependent on pH within the range 6.5-8.5. At pH 7.5, catalytic activity was unaffected by heating through the first transition but was lost by heating through the second. Cleavage of Enzyme I (63.5 kDa) by trypsin, chymotrypsin, or Staphylococcus aureus V8 protease yields a 30-kDa fragment, EI-N, containing the NH2 terminus and the active site, His-189. Protease and differential scanning calorimetry experiments show that EI-N is the structural domain corresponding to the cooperative region in the intact enzyme that unfolds at the higher Tm. EI-N catalyzes one activity of Enzyme I; it accepts a phosphoryl group from phosphohistidine-containing phosphocarrier protein but cannot be phosphorylated by phospho-Enzyme I or phosphoenolpyruvate. The phosphoryl transfer between EI-N and the histidine-containing phosphocarrier protein is reversible. Portions of the Salmonella typhimurium ptsI DNA sequence are known; the complete sequence is presented here and compared to Escherichia coli ptsI.
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McCollum-Pratt Institute, Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Crocenzi
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Freire
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Roseman
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D001483:Base Sequence; D001692:Biological Transport; D002152:Calorimetry, Differential Scanning; D050260:Carbohydrate Metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D017852:Phosphotransferases (Nitrogenous Group Acceptor); D011487:Protein Conformation; D011489:Protein Denaturation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013816:Thermodynamics
D004269:DNA, Bacterial; D010770:Phosphotransferases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase