Functional properties of beta-galactosidase from mutant strain 13 PO of Escherichia coli.
The functional properties of CZP protein, a mutant deriving from wild-type beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) by a point mutation, were investigated. A large decrease of the specificity, as evaluated by the kcat/Km ratio, was observed, principally originated by a weaker binding of the substrates. The catalytic constants, whose values are strongly affected by the presence of divalent cations, were smaller or larger for mutant enzyme than for wild-type enzyme, depending upon the experimental conditions. Analysis of the kinetic pathway indicates, with some substrates, a change in the limiting step for the mutant enzyme compared to the wild type. Because the k'3 step is rate limiting for hydrolysis of p-nitrophenyl-beta-D-galactoside by the mutant enzyme in the absence of Mg2+ and its value is relatively small, it is possible to observe a burst of p-nitrophenol during hydrolysis. This provides conclusive evidence for the occurrence of a two-step mechanism, with a sequential release of the products.
D002413:Cations, Divalent / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005690:Galactose / Q000494:pharmacology; D005696:Galactosidases / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000494:pharmacology; D009154:Mutation; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D005696:Galactosidases; D001616:beta-Galactosidase; D005947:Glucose; D007785:Lactose; D005690:Galactose