Stone Andrew N, Mackenzie Peter I, Galetin Aleksandra, Houston J Brian, Miners John O
Department of Clinical Pharmacology, Flinders Medical Centre, Adelaide, Australia.
Drug Metab Dispos. 2003 Sep;31(9):1086-9. doi: 10.1124/dmd.31.9.1086.
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 Km 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 Km 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 (Ks) 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.
吗啡的消除涉及尿苷二磷酸葡萄糖醛酸基转移酶（UGT）催化与葡萄糖醛酸结合，形成吗啡3-葡萄糖醛酸苷和6-葡萄糖醛酸苷（分别为M3G和M6G）。有人提出UGT2B7是参与这些反应的主要酶，但有证据表明其他同工型也催化人体中的吗啡葡萄糖醛酸化。因此，我们已对重组人UGT形成M3G和M6G的选择性和动力学进行了表征。UGT 1A1、1A3、1A6、1A8、1A9、1A10和2B7均催化M3G的形成，但只有UGT2B7形成M6G。UGT1A家族同工型形成M3G的动力学与单一酶米氏模型一致，表观Km值范围为2.6至37.4 mM。相比之下，UGT2B7形成M3G和M6G表现出非典型动力学。非典型动力学可以通过拟合双相米氏模型的具有高亲和力和低亲和力Km值（M3G为0.42和8.3 mM，M6G为0.97和7.4 mM）的模型来描述。然而，具有两个相同结合位点以负协同方式相互作用的多位点模型为模拟这些数据提供了更现实的方法。根据该模型，M3G和M6G各自的结合亲和力（Ks）分别为1.76和1.41 mM。这些数据表明，M6G的形成可作为UGT2B7活性的选择性探针，并且UGT2B7催化的吗啡葡萄糖醛酸化似乎涉及两个底物分子的同时结合，这突出了在体外仔细分析吗啡葡萄糖醛酸化动力学的必要性。
