Characterization and reaction mechanism of E. coli α-D-galactosidase

Abstract

$\alpha$-D-Galactosidase (E.C. 3.2.1.22) of $\mbox{\underline{E}}$. $\mbox{\underline{coli}}$ was partially purified by DEAE - cellulose and Sephadex G-100 column chromatography and characterized for the further study of the mode of the enzyme action. The partially purified enzyme preparation showed a specific activity of 65.3 units per mg of protein based on PNPG as a substrate and was purified 28-fold over the crude enzyme, and it was also shown that the general characteristics were in accord with the results of the other reports : substrate specificity, optimum pH 8.0-8.3, optimum temperature 37$^\circ$C, 0.71mM for Km, inhibitory effects of several metal ions, and inactivation by dilution and reactivation by $NAD^+$. The role of $NAD^+$ was proposed as a positive modulator and $\alpha$-galactosidase from $\mbox{\underline{E}}$. $\mbox{\underline{coli}}$ may be an allosteric enzyme. Further studies on the mechanism of enzyme action, i.e., inhibition study by substrate analogues, determination of the ionization constant of the enzyme and the enzyme substrate complex, and inhibition of the enzyme activity by functional group blocking, agents, were carried out. As follows, the evidence for the involvement of functional groups was considered. The implication of the essential function of an imidazolium group was suggested by pKa(6.8 - 6.9) value and inhibition by photo-oxidation. The presence of a thiol group could be postulated from the results of inhibition studies by SH - blocking agents (i.e. PCMPS and N- ethylmaleimide) and the effects of heavy metal ions (i.e. $Ag^+,\; Hg^+$, and $Cu^{+2}$). Moreover with DIFP and hydroxylamine, it was approved that the hydroxyl group and carboxyl group would be involved in the active center. Therefore, based on overall results a model mechanism for the action of $\alpha$ -galactosidase was proposed. In this model mechanism, the sulfhydryl group may act as a general acid and the hydroxyl group as a nucleophile. After being ...