Fluorescent probes for cytochrome P450 structural characterization and inhibitor screening
Fluorescent probes for cytochrome P450 structural characterization and inhibitor screening
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DOI：
10.1021/ja0271678
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发表时间：
2002-09-04
影响因子：
15
通讯作者：
Gray, HB
中科院分区：
文献类型：
--
作者：
Dunn, AR;Hays, AMA;Gray, HB
我们合成了两个以细胞色素P450 cam为靶点的荧光探针（D-4-Ad和D-8-Ad）。D-4-Ad发光在结合时被Förster能量转移猝灭（Kd= 0.83 μM），但当探针被樟脑从活性位点置换时，发光恢复。与此相反，D-8-Ad（Kd = 0.02 μM）不从酶中被取代，即使在大量过量樟脑的存在下也是如此。D-8-Ad：P450 cam复合物的2.2 μ m分辨率的晶体结构揭示了探针和酶之间广泛的疏水接触，这是由B '，F和G螺旋的构象柔性引起的。具有与D-4-Ad类似性质的探针可能用于筛选P450抑制剂。
We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd= 0.83 μM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd≈ 0.02 μM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 Å resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B‘, F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.