Resolution of multiple substrate binding sites in cytochrome P450 3A4: The stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration
Resolution of multiple substrate binding sites in cytochrome P450 3A4: The stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration
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DOI：
10.1021/bi052491b
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发表时间：
2006-04-04
期刊：
影响因子：
2.9
通讯作者：
Davydov, DR
中科院分区：
文献类型：
--
作者：
Fernando, H;Halpert, JR;Davydov, DR
为探讨人细胞色素P450 3A 4（CYP 3A 4）同向性协同作用的机制，我们采用荧光共振能量转移（FRET）技术研究了该酶与1-芘丁醇（1-PB）、1-芘乙胺（PMA）和溴隐亭（溴隐亭）的相互作用。这些方法结合了一个创新的设置滴定稀释和连续变化（工作滴定）实验允许LIS探测基板结合和随后的自旋跃迁所造成的1-PB或溴隐亭或II型光谱变化引起的PMA之间的关系。1-PB诱导的CYP 3A 4自旋位移显示出显著的同向协同性，其特征在于希尔系数为1.8 +/- 0.3（S-50 = 8-0 +/- 1.1 μ M）。相反，CYP 3A 4与溴隐亭或PMA的相互作用显示无协同性，K-D值分别为0.31 +/- 0.08 μ M和7.1 +/- 2.3 μ M。在酶：底物比为I时，通过FRET内滴定稀释实验监测的所有三种底物的结合揭示了1-PB、PMA和溴隐亭的简单双分子相互作用，K-D值分别为0.16 +/- 0.09、4.8 +/- 1.4和0.18 +/- 0.09 μ M。相应地，Job的滴定实验表明，1-PB诱导的自旋位移反映了酶与两个底物分子形成复合物，而溴隐亭和PMA表现出1：1的结合化学计量。结合我们的FRET实验中获得的KD值的工作滴定的结果，我们证明，CYP 3A 4与1-PB的相互作用服从顺序结合机制，其中的自旋跃迁是由1-PB的结合低亲和力网站，这成为可能，只有在饱和的高亲和力网站。
To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed Lis to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S-50 = 8-0 +/- 1.1 mu M). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting K-D values of 0.31 +/- 0.08 mu M and 7.1 +/- 2.3 mu M, respectively. The binding of all three substrates monitored by FRET intitration-by-dilution experiments at an enzyme: substrate ratio of I reveals a simple bimolecular interaction with K-D values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 mu M for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in Our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.