title: Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.
abstract: Chemical modification with diethyl pyrocarbonate (DEPC) of the recombinant human liver UDP-glucuronosyltransferase UGT1*6 in enriched membrane fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M-1.min-1 at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decreased the apparent Vmax 2.4-fold without significantly affecting the apparent Km toward 4-methylumbelliferone and UDP-glucuronic acid. Similarly, the binding of the photoactivatable cosubstrate analog [beta-32P]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inactivation by 4-methylumbelliferone, suggesting that the modified residue was located in or near the aglycone binding site. In contrast, the cosubstrate UDP-glucuronic acid potentiated the irreversible inhibition, indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modification of an amino acid residue with a pKa of 6.1. On the other hand, analysis of the variation of Vmax and Vmax/Km values of the glucuronidation reaction as a function of the pH revealed the presence of two essential residues with a pKa within the range 5.7-6.0. The data of the chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be the base catalyst of the glucuronidation reaction supported by human UGT1*6.
authors.affiliation: Centre du Médicament, URA CNRS No. 597, Nancy, France.
authors.forename: E
authors.initials: E
authors.lastname: Battaglia
authors.forename: M
authors.initials: M
authors.lastname: Pritchard
authors.forename: M
authors.initials: M
authors.lastname: Ouzzine
authors.forename: S
authors.initials: S
authors.lastname: Fournel-Gigleux
authors.forename: A
authors.initials: A
authors.lastname: Radominska
authors.forename: G
authors.initials: G
authors.lastname: Siest
authors.forename: J
authors.initials: J
authors.lastname: Magdalou
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005965:Glucuronates / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D006923:Hymecromone / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008168:Lung; D011994:Recombinant Proteins; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D005965:Glucuronates; D006898:Hydroxylamines; D011994:Recombinant Proteins; C071932:5-azido-UDP-glucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D019811:Hydroxylamine; D006923:Hymecromone; D006639:Histidine; D014453:Glucuronosyltransferase; D004047:Diethyl Pyrocarbonate