pH Dependence of the Activity of β‐Galactosidase from Escherichia coli

Abstract

The “specific reactivity” of β‐galactosidase from Escherichia coli for different substrates has been studied at the optimal pH value. The pH dependence of the enzyme activity has been reinvestigated in highly controlled conditions with respect to Mg2+ and Na+ concentrations and ionic strength. The different kinetic parameters kcat, km and the kcat/km ratio have been determined with o‐nitrophenyl‐β‐d‐galactoside and o‐nitrophenyl‐β‐d‐fucoside, at different pH values between 5.16 and 10, for both Mg2+‐enzyme and Mg2+‐free enzyme, since a residual activity has been found in the absence of Mg2+. The activity of both types of enzyme is controlled by a protonated group which ionizes in the alkaline range and by at least one unprotonated group which ionizes in the acidic range. This latter group has a pK smaller than 6 in both types of enzyme; it is plausible to assume that this group is a carboxylate. In the alkaline range, the pK of the involved group shifts from about 6.5 in Mg2+‐free enzyme to 8.4 in the Mg2+ enzyme. Either the substrate or the Mg binding induces this shift in the ionization of the group. The β‐galactosidase catalyzed reactions proceed via two intermediary complexes. For o‐nitrophenyl‐β‐d‐galactoside substrate the limiting process is not the same for the Mg2+ enzyme and for the Mg2+‐free enzyme. Copyright © 1971, Wiley Blackwell. All rights reserved