Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195-7610, USA.
Biochemistry. 2011 Nov 22;50(46):10041-51. doi: 10.1021/bi2013454. Epub 2011 Oct 28.
Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic metabolizing CYP. Despite great interest in CYP enzymology, two in vitro aspects of CYP3A4 catalysis are still not well understood, namely, sequential metabolism and allosteric activation. We have therefore investigated such a system in which both phenomena are present. Here we report that the sequential metabolism of Nile Red (NR) is accelerated by the heterotropic allosteric effector α-naphthoflavone (ANF). ANF increases the rates of formation for NR metabolites M1 and M2 and also perturbs the metabolite ratio in favor of M2. Thus, ANF has as an allosteric effect on a kinetic branch point. Co-incubating deuterium-labeled NR and unlabeled M1, we show that ANF increases k(cat)/k(off) ~1.8-fold in favor of the k(cat) of M2 production. Steady-state metabolic experiments are analyzed using a kinetic model in which the enzyme and substrates are not in rapid equilibrium, and this distinction allows for the estimation of rates of catalysis for the formation of both the primary (M1) and secondary (M2) products, as well as the partitioning of enzyme between these states. These results are compared with those of earlier spectroscopic investigations of NR and ANF cooperativity, and a mechanism of ANF heteroactivation is presented that involves effects on substrate off rate and coupling efficiency.
细胞色素 P450 3A4（CYP3A4）是主要的异源生物代谢 CYP。尽管人们对 CYP 酶学非常感兴趣，但 CYP3A4 催化的两个体外方面仍未得到很好的理解，即顺序代谢和变构激活。因此，我们研究了存在这两种现象的系统。在这里，我们报告说尼罗红（NR）的顺序代谢被异种变构效应物α-萘黄酮（ANF）加速。ANF 增加了 NR 代谢物 M1 和 M2 的形成速率，并且还改变了代谢物的比例，有利于 M2。因此，ANF 对动力学分支点具有变构效应。共孵育氘标记的 NR 和未标记的 M1，我们表明 ANF 使有利于 M2 产生的 k(cat)/k(off)增加约 1.8 倍。使用酶和底物未处于快速平衡的动力学模型分析稳态代谢实验，这种区别允许估计形成主要（M1）和次要（M2）产物的催化速率，以及酶在这些状态之间的分配。将这些结果与早期对 NR 和 ANF 协同作用的光谱研究进行比较，并提出了一种 ANF 异激活的机制，该机制涉及对底物脱落率和偶联效率的影响。
