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.
Centre du Médicament, URA CNRS 597, Nancy, France.
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E
Battaglia
C
C
Senay
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Fournel-Gigleux
R
R
Herber
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G
Siest
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J
Magdalou
D002384:Catalysis; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005965:Glucuronates / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000378:metabolism; D007202:Indicators and Reagents; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D005965:Glucuronates; D007202:Indicators and Reagents; D007555:Isoxazoles; D011994:Recombinant Proteins; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D006923:Hymecromone; D004024:Dicyclohexylcarbodiimide; D014453:Glucuronosyltransferase; D005022:Ethyldimethylaminopropyl Carbodiimide