title: The bacterial phosphoenolpyruvate-dependent phosphotransferase system. Isolation of active site peptides by reversed-phase high-performance liquid chromatography and determination of their primary structure.
abstract: Using reversed-phase high-performance liquid chromatography (HPLC) it was possible to isolate 32P-labelled active-site regions of various proteins from the bacterial phosphoenolpyruvate-dependent phosphotransferase system. The purified peptides obtained by proteolytic cleavage with Lys-C protease and trypsin were sequenced by the gas phase method. The fragments derived from enzyme I (MW 70 000) of two streptococcal species show 100% homology. The analogous peptide of Staphylococcus aureus Enzyme I differs in the N-terminal region. A labelled peptide from the glucose-specific enzyme III protein of Escherichia coli obtained by cleavage with alkaline protease was isolated and sequenced. It could be fitted into the primary structure of this protein, which was derived from DNA sequence data. The active-site histidine residue of this protein is therefore localized at position 91. The HPLC separation method described is suitable for the isolation of peptides derived from active sites containing labile amino acid derivatives such as phosphohistidines.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D013293:Enterococcus faecalis / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013294:Lactococcus lactis / Q000201:enzymology; D010447:Peptide Hydrolases; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000032:analysis*; D010766:Phosphorylation; D013211:Staphylococcus aureus / Q000201:enzymology; D014357:Trypsin
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D010455:Peptides; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D010447:Peptide Hydrolases; D014357:Trypsin