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J. Biol. Chem., Vol. 267, Issue 24, 16943-16950, 08, 1992

Hepatic microsomal bilirubin UDP-glucuronosyltransferase. The kinetics of bilirubin mono- and diglucuronide synthesis

JM Crawford, BJ Ransil, JP Narciso and JL Gollan
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115.

Hepatic biotransformation of bilirubin to the hydrophilic species bilirubin mono- (BMG) and diglucuronide (BDG) by microsomal bilirubin UDP-glucuronosyl-transferase (GT) is a prerequisite for its physiologic excretion into bile. The reaction mechanism of bilirubin-GT and the access of bilirubin and BMG (the intermediate substrate) to the active site of bilirubin-GT are undefined. Highly purified [14C]bilirubin and [3H] BMG were coincubated with rat liver microsomes, and the initial rates of radiolabeled bilirubin glucuronide synthesis were measured. Although these substrates differ markedly in their hydrophilicity, no significant differences were observed in [14C]- and [3H]BDG rates of formation from equimolar [14C]bilirubin and [3H] BMG, in the absence or presence of soluble binding proteins (albumin and hepatic cytosol). In further kinetic studies, [14C]bilirubin and [3H]BMG exhibited mutually competitive inhibition of [3H]- and [14C]BDG synthesis, respectively, and [3H]BMG also inhibited [14C]BMG formation. Finally, unlabeled BMG and BDG inhibited the glucuronidation of [14C]bilirubin, with all three pigments yielding virtual Michaelis-Menten dissociation constants in the 10-20 microM range. These findings indicate that: 1) bilirubin-GT follows Michaelis-Menten kinetics for both bilirubin and BMG glucuronidation over the range of substrate concentrations employed; 2) the findings are consistent with a single active site for the enzymatic synthesis of both BMG and BDG; 3) bilirubin, BMG, and BDG bind competitively to this active site with comparable affinities; and 4) access of both bilirubin and BMG substrates to the enzymatic active site is reduced by soluble binding proteins.
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