Kinetic modeling of the interactions between 4-methylumbelliferone, 1-naphthol, and zidovudine glucuronidation by UDP-glucuronosyltransferase 2B7 (UGT2B7) provides evidence for multiple substrate binding and effector sites
Kinetic modeling of the interactions between 4-methylumbelliferone, 1-naphthol, and zidovudine glucuronidation by UDP-glucuronosyltransferase 2B7 (UGT2B7) provides evidence for multiple substrate binding and effector sites
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DOI：
10.1124/mol.108.048645
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发表时间：
2008-10-01
影响因子：
3.6
通讯作者：
Miners, John O.
中科院分区：
文献类型：
--
作者：
Uchaipichat, Verawan;Galetin, Aleksandra;Miners, John O.
使用多位点和经验动力学模型分析了UGT 2B 7催化的齐多夫定（AZT）、4-甲基伞形酮（4 MU）和1-萘酚（1 NP）的葡萄糖醛酸化之间的相互作用，以探索这种重要药物代谢酶中是否存在多个底物和效应物结合位点。通过UGT 2B 7进行的4 MU和1 NP葡萄糖醛酸化表现出同向协同性（自活化）的S形动力学特征，假设存在两个等效的相互作用底物结合位点，可以对其进行建模。相反，UGT 2B 7催化的AZT葡萄糖醛酸化遵循双曲线（Michaelis-Menten）动力学。虽然4 MU和1 NP降低了AZT的结合亲和力，但在两种修饰剂存在下，AZT葡萄糖醛酸化的动力学从双曲线变为S形。数据进行了很好的描述了一个通用的两个基板结合位点模型，其中有没有在4 MU或1 NP的情况下，网站之间的相互作用，但异向性协同效应的结果从结合的修改器。AZT对4 MU和1 NP葡萄糖醛酸化的抑制以及4 MU和1 NP之间的相互作用需要更复杂的三位点模型，其中修饰剂通过不同的效应位点起作用，以改变底物结合亲和力或V（max），而不影响4 MU和1 NP葡萄糖醛酸化的同向协同特性。值得注意的是，1 NP抑制4 MU葡萄糖醛酸化，而4 MU激活1 NP葡萄糖醛酸化。结果与UGT 2B 7活性位点内每个底物存在两个“催化”位点以及多个效应位点沿着一致。结合和效应位点的多样性导致UGT 2B 7底物之间复杂的动力学相互作用，这可能使抑制筛选研究复杂化。
Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or V(max) without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.