Sugar transport by the bacterial phosphotransferase system. Structural and thermodynamic domains of enzyme I of Salmonella typhimurium

C. LiCalsi, T. S. Crocenzi, E. Freire, S. Roseman

Research output: Contribution to journalArticle

Abstract

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 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 compete sequence is presented here and compared to Escherichia coli ptsI.

Original languageEnglish (US)
Pages (from-to)19519-19527
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number29
StatePublished - Jan 1 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Sugar transport by the bacterial phosphotransferase system. Structural and thermodynamic domains of enzyme I of Salmonella typhimurium'. Together they form a unique fingerprint.

  • Cite this