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 (T_m) at 47.6 and 55.1°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 T_m. EI-N catalyzes one activity of Enzyme I; it accepts a phosphoryl group from phoephohistidine-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.