Studies of Catalysis by β-Glucuronidase

The relative rates of hydrolysis of monosubstituted phenyl-β-d-glucopyranosiduronic acids (phenyl-β-glucuronides) catalyzed by β-glucuronidase correlate with Hammett values, yielding a biphasic curve with the curve being concave up and with the change-over occurring at the unsubstituted compound. A similar type of plot has not previously been obtained with other enzymes. The relative rates of hydrolysis of p-methoxyphenyl, p-chlorophenyl, and p-nitrophenyl-β-d-glucopyranosiduronic acids, and of the most commonly used substrate, phenolphthalein-β-d-glucopyranosiduronic acid, were compared in D2O and H2O, and it was observed that the pH optimum in D2O shifted towards higher values, with the rate of hydrolysis in D2O decreasing by a factor of 1.7 to 2.0 as compared to that in H2O. These results suggest the involvement of a rate-determining proton transfer in the catalytic steps of the enzymatic reaction. A possible explanation for the change in slope in the Hammett plot is that there is a shift in the relative electronic importance of the general acid-nucleophile portions of the push-pull mechanism.