Characterization of the E. coli glucose permease fused to the maltose-binding protein

✍ Scribed by Mohammad Aboulwafa; Milton H. Saier Jr.

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 280 KB
• Volume: 48
• Category: Article
• ISSN: 0233-111X
• DOI: 10.1002/jobm.200700263

✦ Synopsis

Abstract

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. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)