Site-directed mutagenesis of the phosphocarrier protein. IIIGlc, a major signal-transducing protein in Escherichia coli.
The glucose-specific phosphocarrier protein (IIIGlc) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is a major signal transducer that mediates the intricate interplay among extracellular signals (PTS and non-PTS sugars), cytoplasmic and membrane proteins (PTS and non-PTS transporters), and adenylate cyclase. To further define the central role of IIIGlc in these multiplex signaling mechanisms, we have used site-directed mutagenesis to construct three mutant IIIGlc proteins containing single amino acid changes; Phe-3 was replaced with tryptophan [( Trp3]IIIGlc), and His-75 and the active-site His-90 were replaced with glutamine [( Gln75]IIIGlc and [Gln90]IIIGlc, respectively). [Trp3]IIIGlc resembles the wild-type protein in most properties and should be valuable for spectrophotometric experiments. In contrast, clear differences between mutant and wild-type proteins were observed with both [Gln75]IIIGlc and [Gln90]IIIGlc in in vitro sugar phosphorylation assays. As predicted, [Gln90]IIIGlc with a modified active site cannot be phosphorylated. Unexpectedly, [Gln75]IIIGlc accepts but cannot transfer phosphoryl groups, suggesting His-75 may also be a critical amino acid for IIIGlc-mediated signaling mechanisms. The physiological effects of these mutations are briefly described.
Presper
K A
KA
McCollum-Pratt Institute, Johns Hopkins University, Baltimore, MD 21218.
Wong
C Y
CY
Liu
L
L
Meadow
N D
ND
Roseman
S
S
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D007700:Kinetics; D009154:Mutation; D010641:Phenotype; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D015398:Signal Transduction; D013045:Species Specificity
D029968:Escherichia coli Proteins; C032242:crr protein, E coli; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System