The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.
Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
J W
JW
Anderson
K
K
Pullen
F
F
Georges
R E
RE
Klevit
E B
EB
Waygood
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D010748:Phosphopeptides; D011994:Recombinant Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D007930:Leucine