Cytochrome P450 Compound I: Capture, Characterization, and C-H Bond Activation Kinetics
Cytochrome P450 Compound I: Capture, Characterization, and C-H Bond Activation Kinetics
复制标题
DOI：
10.1126/science.1193478
复制
发表时间：
2010-11-12
期刊：
影响因子：
56.9
通讯作者：
Green, Michael T.
中科院分区：
文献类型：
--
作者：
Rittle, Jonathan;Green, Michael T.
细胞色素 P450 酶负责大约 75% 已知药物的 I 相代谢。 P450 通过 C-H 键的受控激活来执行此功能和其他重要的生物学功能。在此，我们报告了该过程中长期寻找的主要中间体 P450 化合物 I (P450-I) 的光谱和动力学表征，我们通过 CYP119 铁与间氯过苯甲酸反应制备了该化合物，收率约为 75%。 CYP119-I的穆斯堡尔谱与氯过氧化物酶化合物I的穆斯堡尔谱相似，尽管其电子顺磁共振谱反映了垂直条J垂直条/D（交换耦合与零场分裂的比率）的增加。 CYP119-I 羟基化月桂酸的未活化 C-H 键 [D(C-H) 类似于每摩尔 100 千卡]，表观二阶速率常数为 k(app) = 1.1 x 10(7) 每摩尔每秒（4 摄氏度）。直接测量对结合底物氧化的速率常数设置了 k >= 210 每秒的下限，而涉及动力学同位素效应的分析则预测了一个值每秒超过 1400 个。
Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mossbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in vertical bar J vertical bar/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) similar to 100 kilocalories per mole], with an apparent second-order rate constant of k(app) = 1.1 x 10(7) per molar per second at 4 degrees C. Direct measurements put a lower limit of k >= 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.