Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion.
An artificial bifunctional enzyme, beta-galactosidase/galactokinase, has been prepared by gene fusion. The hybrid protein catalyzes the hydrolysis of lactose followed by phosphorylation of galactose. The protein has been purified using DEAE-Sephacel chromatography and gel filtration on Sephacryl S-400 Superfine. The configuration of the hybrid protein is mainly tetrameric but also higher aggregates can be detected. The monomer Mr is 160,000 as judged from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the native Mr has been calculated to be 600,000-650,000 from gel filtration experiments. beta-Galactosidase/galactokinase has different thermostability curves, pH/activity profiles and Km values as compared with the native enzymes. By using a third enzyme, galactose dehydrogenase, which competes with galactokinase for the galactose formed by beta-galactosidase, substrate channeling can be detected. This proximity effect becomes even more pronounced in an assay mixture containing poly(ethylene glycol).
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism*; D005696:Galactosidases / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010957:Plasmids; D011092:Polyethylene Glycols / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D009097:Multienzyme Complexes; D011092:Polyethylene Glycols; D005686:Galactokinase; D005696:Galactosidases; D001616:beta-Galactosidase