Isolation and characterization of the newly evolved ebg beta-galactosidase of Escherichia coli K-12.
The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.
D000942:Antigens, Bacterial; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology; D005696:Galactosidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D005796:Genes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D009596:Nitrophenols / Q000378:metabolism; D009598:Nitrophenylgalactosides / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D013696:Temperature
D000942:Antigens, Bacterial; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D010710:Phosphates; D009598:Nitrophenylgalactosides; D005696:Galactosidases; D008274:Magnesium