Roberts Arthur G, Atkins William M
Department of Medicinal Chemistry; Box 357610, University of Washington, Seattle, WA 98195-7610, USA.
Arch Biochem Biophys. 2007 Jul 1;463(1):89-101. doi: 10.1016/j.abb.2007.03.006. Epub 2007 Apr 2.
Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, alpha-naphthoflavone (ANF) and the steroid, testosterone (TST). UV-vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: (1) a relatively tight spin-state insensitive binding site (CYP.ANF) and (2) a relatively low affinity spin-state sensitive binding site (CYP.ANF.ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s). The spin-state insensitive binding site of ANF appears to compete with the spin-state insensitive binding site of TST. The formation of the spin-state insensitive CYP.ANF complex is strongly temperature dependent, when compared to the formation of the CYP.TST complex, suggesting that the formation of the CYP3A4.ANF complex leads to long-range conformational changes within the protein. When the CYP.ANF complex is titrated with TST, the formation of CYP.ANF.TST is favored by 3:1 over the formation of CYP.TST.TST, suggesting that there is an allosteric interaction between ANF and TST. A model of heterotropic cooperativity of CYP3A4 is presented, where the spin-state insensitive binding of ANF occurs at the same peripheral binding site of CYP3A4 as TST.
细胞色素P450 3A4（CYP3A4）参与大多数药物的代谢。使用类黄酮α-萘黄酮（ANF）和类固醇睾酮（TST）研究了药物与CYP3A4结合的异源协同作用。CYP3A4的紫外可见光谱和电子顺磁共振光谱表明，ANF与CYP3A4的结合表现出明显的负协同作用，并且至少存在两个结合位点：（1）一个相对紧密的对自旋态不敏感的结合位点（CYP.ANF）和（2）一个相对低亲和力的对自旋态敏感的结合位点（CYP.ANF.ANF）。由于ANF与自旋态不敏感结合位点的结合比对TST的结合要紧密得多，因此自旋态不敏感结合位点可能被ANF占据，同时在其他位点滴定TST。ANF的自旋态不敏感结合位点似乎与TST的自旋态不敏感结合位点竞争。与CYP.TST复合物的形成相比，自旋态不敏感的CYP.ANF复合物的形成强烈依赖于温度，这表明CYP3A4.ANF复合物的形成导致蛋白质内部的长程构象变化。当用TST滴定CYP.ANF复合物时，CYP.ANF.TST的形成比CYP.TST.TST的形成更有利于3:1，这表明ANF和TST之间存在变构相互作用。本文提出了CYP3A4异源协同作用的模型，其中ANF的自旋态不敏感结合与TST在CYP3A4的相同外周结合位点发生。
