Studies of Catalysis by β-Glucuronidase: ACTIVE SITE

Abstract

Abstract In an attempt to understand the structure of the active center of β-glucuronidase (EC 3.2.1.31), the behavior of the kinetic parameters of the liver lysosomal enzyme as a function of pH was examined. The pH optimum of the enzyme in the presence of 0.65 m NaCl is 5.1. This dependence of maximal velocity on pH suggests that two dissociable groups are involved, for which the pK values 4.14 and 6.21 were calculated. These values, and those of the heats of dissociation calculated for these two groups, are suggestive of the participation of a carboxylate group and perhaps a carboxyl group in the catalysis. The inactivation of the enzyme by a carbodiimide and by isoxazolium salts is compatible with this hypothesis. The interaction between β-glucuronidase and phenolphthalein-β-d-glucopyranosiduronic acid was found to involve ionic forces, as suggested by the absence of substrate activity or inhibitory effects of p-nitrophenyl-β-d-glucopyranosiduronamide, by the greater inhibitory activity of an isoxazolium salt bearing a sulfonate group than of one that did not contain this group, and by the effect of ionic strength on the kinetic constant Vmax/Km. It was inferred from these findings that a cationic group at the active center of the enzyme is probably involved in attacking the substrate. That this cationic group is probably an imidazolium group was suggested by the inactivation effected by photooxidation, which was pH-dependent, and by diazonium salts.