Involvement of the carboxy-terminal 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 that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
J W
JW
Anderson
P
P
Bhanot
F
F
Georges
R E
RE
Klevit
E B
EB
Waygood
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011487:Protein Conformation; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr