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).