Functional properties of beta-galactosidase from mutant strain 13 PO of Escherichia coli.

Publisher Website
Google Scholar
Abstract
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[catalytic constant]/Km ratio, was observed, principally originated by a weaker binding of the substrates. The kcat, 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 2-step mechanism, with a sequential release of the products.