Tew D G
SmithKline Beecham Research, Welwyn, Hertfordshire, U.K.
Biochemistry. 1993 Sep 28;32(38):10209-15. doi: 10.1021/bi00089a042.
Diphenyliodonium has been shown to be an irreversible, time-dependent inhibitor of NADPH cytochrome P450 oxidoreductase (EC 1.6.2.4) with the Ki for diphenyliodonium chloride being 2.8 mM. Kinetic studies have indicated that diphenyliodonium interacts with the reduced enzyme and NADPH is essential for inactivation to take place. Cytochrome c acts as a competitive substrate. The use of radiolabeled diphenyliodonium has enabled two sites of covalent modification to be identified. Isolation of radiolabeled cofactor followed by mass spectrometry has shown that a phenyl group is added to FMN while the FMN is effectively trapped in the reduced state. Trypsin digestion of S-carboxymethylated P450 reductase after inhibition with radiolabeled inhibitor shows covalent modification of the protein. Purification of a single radiolabelled peptide followed by automated Edman degradation has enabled identification of the second site of covalent attachment as Trp 419.
二苯基碘鎓已被证明是烟酰胺腺嘌呤二核苷酸磷酸细胞色素P450氧化还原酶（EC 1.6.2.4）的一种不可逆的、时间依赖性抑制剂，氯化二苯基碘鎓的Ki为2.8 mM。动力学研究表明，二苯基碘鎓与还原态的酶相互作用，烟酰胺腺嘌呤二核苷酸磷酸对失活的发生至关重要。细胞色素c作为竞争性底物。使用放射性标记的二苯基碘鎓能够鉴定出两个共价修饰位点。通过质谱法分离放射性标记的辅因子后表明，一个苯基被添加到黄素单核苷酸上，同时黄素单核苷酸有效地被困在还原态。用放射性标记的抑制剂抑制后，对S-羧甲基化的P450还原酶进行胰蛋白酶消化，显示该蛋白质发生了共价修饰。纯化单个放射性标记的肽段，然后进行自动埃德曼降解，已能够鉴定出共价连接的第二个位点为色氨酸419。
