Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process

Arch Biochem Biophys. 1994 Mar;309(2):266-72. doi: 10.1006/abbi.1994.1112.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Animals
  • Catalysis
  • Cell Line
  • Cricetinae
  • Cricetulus
  • Diethyl Pyrocarbonate / pharmacology*
  • Enzyme Activation / drug effects
  • Glucuronates / metabolism
  • Glucuronosyltransferase / antagonists & inhibitors*
  • Glucuronosyltransferase / chemistry
  • Glucuronosyltransferase / metabolism
  • Histidine / chemistry*
  • Hydrogen-Ion Concentration
  • Hydroxylamine
  • Hydroxylamines / pharmacology
  • Hymecromone / metabolism
  • Hymecromone / pharmacology
  • Kinetics
  • Lung
  • Recombinant Proteins
  • Uridine Diphosphate Glucuronic Acid / analogs & derivatives
  • Uridine Diphosphate Glucuronic Acid / metabolism


  • Affinity Labels
  • Glucuronates
  • Hydroxylamines
  • Recombinant Proteins
  • 5-azido-UDP-glucuronic acid
  • Uridine Diphosphate Glucuronic Acid
  • Hydroxylamine
  • Hymecromone
  • Histidine
  • Glucuronosyltransferase
  • Diethyl Pyrocarbonate