The chemical modification of human liver UDP-glucuronosyltransferase UGT1*6 reveals the involvement of a carboxyl group in catalysis.

The treatment of UDP-glucuronosyltransferase UGT1*6 stably expressed in V79 cells with three carboxyl-specific reagents, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K), resulted in a fast, dose-dependent decrease of the 4-methylumbelliferone glucuronidation. The inactivation reactions followed pseudo-first order kinetics. The pKa of the modified residue was close to 5.0. A partial protection against inactivation by Woodward's reagent was observed at pH 7.4 in the presence of UDP-glucuronic acid, UDP, and, to a lesser extent, in the presence of 4-methylumbelliferone. Dicyclohexylcarbodiimide significantly decreased the Vmax, without affecting the apparent Km towards UDP-glucuronic acid and 4-methylumbelliferone. The results support the involvement of a carboxyl group in the catalytic process.