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.