Characterization of the E. coli glucose permease fused to the maltose-binding protein.
The ptsG gene that encodes the major glucose transporter of Escherichia coli, II Glc, was inserted into a pMALE-amp r expression vector down-stream of the malE gene which encodes the E. coli maltose-binding protein (MBP). II Glc-MBP in the 2 h high speed supernatant of cell lysates eluted from a gel filtration column showing two activity peaks. The glucose-6-phosphate-dependent transphosphorylation (TP) activity of the membrane bound oligomeric peak 1 showed substrate inhibition while that of the soluble monomeric peak 2 did not. Purification of peak 2 yielded activity with weak substrate inhibition, and further gel filtration analyses showed that upon purification, some of the monomeric II Glc-MBP associated to higher molecular size forms. Assays of the phosphoenolpyruvate-dependent and transphosphorylation reactions showed that the specific activity of the purified enzyme from peak 1 was approximately double that from peak 2. The results show that the monomeric II Glc-MBP exhibits no substrate inhibition although the oligomeric form does. Purification promotes subunit association, an increase in catalytic activity, and restoration of substrate inhibition.
Aboulwafa
Mohammad
M
Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA.
Saier
Milton H
MH
D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008757:Methylglucosides / Q000378:metabolism; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008757:Methylglucosides; D011993:Recombinant Fusion Proteins; C027020:methylglucoside; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D005947:Glucose