The bacterial phosphoenolpyruvate-dependent phosphotransferase system. Isolation of active site peptides by reversed-phase high-performance liquid chromatography and determination of their primary structure.

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.

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.

HPr proteins of different microorganisms studied by hydrogen-1 high-resolution nuclear magnetic resonance: similarities of structures and mechanisms.

The HPr proteins of Streptococcus lactis, Streptococcus faecalis, Bacillus subtilis, and Escherichia coli were studied by 1H NMR at 360 MHz. The "active-center" histidines of all HPr proteins are characterized by a low pK value between 5.6 and 6.1 and similar spectral parameters. Phosphorylation of the histidyl residues leads to an increase of the pK value of 2-3 units and spectral changes characteristic for N-1 phosphorylation of the histidyl ring. The spectra of the HPr proteins of S. lactis, S. Faecalis, B. subtilis, and Staphylococcus aureus reveal many similarities, whereas the spectrum of the E. coli protein is different with exception of the active-center histidine. The HPr protein of S. lactis is formylated at its terminal amino group.