Multiple substrate binding by cytochrome P450 3A4:: Estimation of the number of bound substrate molecules
Multiple substrate binding by cytochrome P450 3A4:: Estimation of the number of bound substrate molecules
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DOI：
10.1124/dmd.108.021733
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发表时间：
2008-10-01
影响因子：
3.9
通讯作者：
Roberts, Gordon C. K.
中科院分区：
文献类型：
--
作者：
Kapelyukh, Yury;Paine, Mark J. I.;Roberts, Gordon C. K.
细胞色素P450 3A4是人体内一种主要的药物代谢酶，它对许多底物具有非米氏稳态动力学，表明多个底物可以同时与酶结合，但事实证明很难获得确切地多少底物分子可以结合的可靠估计。我们使用了一种简单的方法，包括研究大的抑制剂对Hill系数的影响，以从简单的稳态动力学提供更好的底物化学计量比估计。使用一组八种抑制剂，我们证明了广泛使用的CYP3A4底物7-苄氧基喹啉中至少有四个分子可以同时与酶结合。计算对接研究表明，这与最近报道的酶的晶体结构是一致的。在咪达唑仑的情况下，它表现出简单的Michaelis-Menten动力学，抑制剂效应表明两个分子必须同时结合，这与先前的证据一致，而对于地尔硫卓，实验没有提供超过一个分子结合的证据。讨论了这种“抑制剂诱导的协同性”对药代动力学和药物间相互作用预测的影响。
Cytochrome P450 3A4, a major drug-metabolizing enzyme in man, is well known to show non-Michaelis-Menten steady-state kinetics for a number of substrates, indicating that more than one substrate can bind to the enzyme simultaneously, but it has proved difficult to obtain reliable estimates of exactly how many substrate molecules can bind. We have used a simple method involving studies of the effect of large inhibitors on the Hill coefficient to provide improved estimates of substrate stoichiometry from simple steady-state kinetics. Using a panel of eight inhibitors, we show that at least four molecules of the widely used CYP3A4 substrate 7-benzyloxyquinoline can bind simultaneously to the enzyme. Computational docking studies show that this is consistent with the recently reported crystal structures of the enzyme. In the case of midazolam, which shows simple Michaelis-Menten kinetics, the inhibitor effects demonstrate that two molecules must bind simultaneously, consistent with earlier evidence, whereas for diltiazem, the experiments provide no evidence for the binding of more than one molecule. The consequences of this "inhibitor-induced cooperativity" for the prediction of pharmacokinetics and drug-drug interactions are discussed.