Abstract

Morphine elimination involves UDP-glucuronosyltransferase (UGT) catalyzed conjugation with glucuronic acid to form morphine 3- and 6-glucuronides (M3G and M6G, respectively). It has been proposed that UGT2B7 is the major enzyme involved in these reactions, but there is evidence to suggest that other isoforms also catalyze morphine glucuronidation in man. Thus, we have characterized the selectivity and kinetics of M3G and M6G formation by recombinant human UGTs. UGT 1A1, 1A3, 1A6, 1A8, 1A9, 1A10, and 2B7 all catalyzed M3G formation, but only UGT2B7 formed M6G. The kinetics of M3G formation by the UGT1A family isoforms was consistent with a single enzyme Michaelis-Menten model, with apparent K_m values ranging from 2.6 to 37.4 mM. In contrast, M3G and M6G formation by UGT2B7 exhibited atypical kinetics. The atypical kinetics may be described by a model with high- and low-affinity K_m values (0.42 and 8.3 mM for M3G, and 0.97 and 7.4 mM for M6G) from fitting to a biphasic Michaelis-Menten model. However, a multisite model with an interaction between two identical binding sites in a negative cooperative manner provides a more realistic approach to modeling these data. According to this model, the respective binding affinities (K_s) for M3G and M6G were 1.76 and 1.41 mM, respectively. These data suggest that M6G formation may be used as a selective probe for UGT2B7 activity, and morphine glucuronidation by UGT2B7 appears to involve the simultaneous binding of two substrate molecules, highlighting the need for careful analysis of morphine glucuronidation kinetics in vitro.