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首页> 外文期刊>Biochemistry >Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations
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Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations

机译：香豆素衍生物作为哺乳动物细胞色素 P450 2B4 和 2B6 的底物探针：评估 7-烷氧基链长、卤素取代和非活性位点突变的重要性

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Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the cournarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-alpha-pinene was solved at 2.2 angstrom and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 angstrom with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.
机译：使用结构和生化相结合的方法，探讨了最近描述的 CYP2B4 和 2B6 中由 E、F、G- 和 I 螺旋包围的外周口袋的功能重要性。最初使用三个系列的 4-取代-7-烷氧基香豆素衍生物，其中 -H、-CH3 或 -CF3 位于 cournarin 核心的 4 位，用于监测 CYP2B4 和 2B6 之间的功能差异。7-乙氧基-4-（三氟甲基）香豆素（7-EFC）在这些底物中表现出最高的催化效率。突变体被制成改变侧链极性（V/E194Q）或本体（F/Y244W）以改变对外周口袋的访问。通过对底物乙氧基链的氯化或溴化，突变体对7-EFC O-脱乙基反应的催化效率的适度提高被显著放大。在2.2埃处求解了CYP2B6 Y244W与（+）-α-蒎烯复合物的结构，发现外周口袋中没有CYMAL-5。CYP2B4 F244W的无配体结构在3.0埃处用CYMAL-5在外周口袋中溶解。在这两种情况下，对各自野生型和突变型 CYP2B 酶的比较表明，尽管外周口袋和活性位点位于 I 螺旋的相对两侧，但外周口袋的 CYMAL-5 占据对活性位点残基侧链的拓扑结构影响不大。对可用 CYP2B 结构的分析表明，外周口袋内氨基酸取代的影响源于 F 和 G 螺旋之间相互作用的改变。

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