title: Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.
abstract: The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases.
authors.affiliation: Department of Biology, University of California at San Diego, La Jolla 92093-0116, USA.
authors.forename: S
authors.initials: S
authors.lastname: Koch
authors.forename: S L
authors.initials: SL
authors.lastname: Sutrina
authors.forename: L F
authors.initials: LF
authors.lastname: Wu
authors.forename: J
authors.initials: J
authors.lastname: Reizer
authors.forename: K
authors.initials: K
authors.lastname: Schnetz
authors.forename: B
authors.initials: B
authors.lastname: Rak
authors.forename: M H
authors.initials: MH
authors.lastname: Saier
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; C030182:phosphocarrier protein HPr; C055931:phosphoenolpyruvate-N-acetylglucosamine phosphotransferase; C031629:phosphoenolpyruvate-glucose phosphotransferase; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D005947:Glucose; D000117:Acetylglucosamine