Direct electrochemistry of immobilized human cytochrome P450 2E1
Direct electrochemistry of immobilized human cytochrome P450 2E1
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DOI：
10.1021/ja049855s
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发表时间：
2004-04-28
影响因子：
15
通讯作者：
Gilardi, G
中科院分区：
文献类型：
--
作者：
Fantuzzi, A;Fairhead, M;Gilardi, G
该通信报告了第一次对人类P450 2 E1的电化学研究，该研究是吸收或共价连接到不同的电极表面。碳电极和金电极给出了活性P450 2 E1的可逆电化学信号。酶的分子建模有助于使结果合理化。在用胱胺/马来酰亚胺修饰的金上获得单层覆盖，所述胱胺/马来酰亚胺共价连接P450 2 E1的表面可接近的半胱氨酸。定向P450 2 E1的中点电位为−177 ± 5 mV，与其他P450酶的Fe III/Fe II相当。该电极的观察到的电子转移速率为10 s-1。用P450 2 E1特异性底物对硝基苯酚测量活性酶的周转率，在施加−300 mV偏压时，aKM为130 ± 3 μM，并形成2.2 μM的对硝基儿茶酚产物。
This communication reports the first electrochemical study of the human P450 2E1 either absorbed or covalently linked to different electrode surfaces. Glassy-carbon and gold electrodes gave reversible electrochemical signals of an active P450 2E1. Molecular modeling of the enzyme helped to rationalize the results. A monolayer coverage was obtained on gold modified with cystamine/maleimide that covalently linked surface accessible cysteines of P450 2E1. The midpoint potential measured for the oriented P450 2E1 was −177 ± 5 mV comparable to that of the FeIII/FeIIof other P450 enzymes. The observed electron-transfer rate for this electrode was 10 s-1. The turnover of the active enzyme was measured with the P450 2E1 specific substratep-nitrophenol, resulting in aKMof 130 ± 3 μM and the formation of 2.2 μM of thep-nitrocatechol product upon application of a −300 mV bias.