The chemical modification of human liver UDP-glucuronosyltransferase UGT16 reveals the involvement of a carboxyl group in catalysis

• Published in: FEBS letters 1994-06, Vol.346 (2), p.146-150
• Main Authors: Battaglia, Eric, Senay, Claire, Fournel-Gigleux, Sylvie, Herber, Re´gine, Siest, Ge´rard, Magdalou, Jacques
• Format: Article
• Language: English
• Subjects: 4-MU, 4-methylumbelliferone; Car☐yl-specific reagent; Catalysis; Cell Line; Cross-Linking Reagents - pharmacology; DCC, dicyclohexylcarbodiimide; Dicyclohexylcarbodiimide - pharmacology; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; Essential residue; Ethyldimethylaminopropyl Carbodiimide - pharmacology; Glucuronates - metabolism; Glucuronosyltransferase - antagonists & inhibitors; Glucuronosyltransferase - chemistry; Glucuronosyltransferase - metabolism; HPLC, high- performance liquid chromatography; Human liver, Chemical modification; Hydrogen-Ion Concentration; Hymecromone - metabolism; Indicators and Reagents; Isoxazoles - pharmacology; Kinetics; Liver - enzymology; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - metabolism; Structure-Activity Relationship; UDP-GlcU, UDP- glucuronic acid; UDP-glucuronosyltransferase; UGT, UDP-glucuronosyltransferase; WK reagent, N-ethyl-5-phenylisoxazolium-3′-sulfonate
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(94)00453-6

The treatment of UDP-glucuronosyltransferase UGT1*6 stably expressed in V79 cells with three car☐yl-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 p K a 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 V max, without affecting the apparent K m towards UDP-glucuronic acid and 4-methylumbelliferone. The results support the involvement of a car☐yl group in the catalytic process.