Phosphoenolpyruvate-dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system.
Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P-IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIglc of S. typhimurium. Here we present 1H-NMR measurements of IIIglc and P-IIIglc from E. coli which further substantiate that the phosphoryl group in P-IIIglc is linked to the N-3 position of a histidyl residue. After phosphorylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate-dependent phosphorylation.
D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000302:isolation & purification; D010728:Phosphoenolpyruvate / Q000502:physiology*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation
D029968:Escherichia coli Proteins; D010455:Peptides; C032242:crr protein, E coli; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System