Functional analysis of phenylalanine residues in the active site of cytochrome P450 2C9.
Functional analysis of phenylalanine residues in the active site of cytochrome P450 2C9.
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DOI：
10.1021/bi801231m
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发表时间：
2008-11-11
期刊：
影响因子：
2.9
通讯作者：
Rettie, Allan E.
中科院分区：
文献类型：
--
作者：
Mosher, Carrie M.;Hummel, Matthew A.;Tracy, Timothy S.;Rettie, Allan E.
与（S）-华法林或氟比洛芬复合的CYP 2C 9的两种已发表晶体结构表明，在配体结合中存在一组三个活性位点苯丙氨酸残基（F100、F114、F476）。然而，这三个残基似乎与这两个配体的静态晶体结构的基础上不同的相互作用。为了阐明CYP 2C 9的活性位点苯丙氨酸对底物结合、定位和催化转换的重要性，构建了一系列亮氨酸和色氨酸突变体，并检测了它们与（S）-华法林和（S）-氟比洛芬的相互作用。F100→L突变对底物结合和各底物代谢的影响较小。相比之下，F114 L和F476 L突变体表现出显著降低的（S）-华法林代谢和改变的羟基代谢产物谱，但仅适度降低NSAID的周转，同时保持产品的区域选择性。F114→W和F476→W突变对（S）-华法林与NSAID转换也有相反的影响。值得注意的是，F476 W突变体使（S）-华法林代谢的效率增加5倍，但使（S）-氟比洛芬周转的效率降低20倍。1H-NMR T1弛豫研究表明，相对于野生型酶，（S）-华法林在F476 W突变体中与血红素的位置稍近，化学计量研究表明，（S）-华法林的还原当量与产物形成的偶联增强，再次与（S）-氟比洛芬观察到的效果相反。这些数据表明，F114和F476，而不是F100，影响（S）-华法林的催化取向。F476突变体与两种底物的差异相互作用表明它们的催化生产结合模式是不可重叠的。
The two published crystal structures of CYP2C9, complexed with (S)-warfarin or flurbiprofen, implicate a cluster of three active-site phenylalanine residues (F100, F114, F476) in ligand binding. However, these three residues appear to interact differently with these two ligands based on the static crystal structures. To elucidate the importance of CYP2C9’s active site phenylalanines on substrate binding, orientation and catalytic turnover, a series of leucine and tryptophan mutants was constructed and their interactions with (S)-warfarin and (S)-flurbiprofen examined. The F100→L mutation had minor effects on substrate binding and metabolism of each substrate. In contrast, the F114L and F476L mutants exhibited substantially reduced (S)-warfarin metabolism and altered hydroxy metabolite profiles, but only modestly decreased NSAID turnover, while maintaining product regioselectivity. The F114→W and F476→W mutations also had opposing effects on (S)-warfarin versus NSAID turnover. Notably, the F476W mutant increased the efficiency of (S)-warfarin metabolism 5-fold, yet decreased the efficiency of (S)-flurbiprofen turnover 20-fold. 1H-NMR T1 relaxation studies suggested a slightly closer positioning of (S)-warfarin to the heme in the F476W mutant relative to the wild-type enzyme, and stoichiometry studies indicated enhanced coupling of reducing equivalents to product formation for (S)-warfarin, again in contrast to effects observed with (S)-flurbiprofen. These data demonstrate that F114 and F476, but not F100, influence (S)-warfarin’s catalytic orientation. Differential interactions of F476 mutants with the two substrates suggest that their catalytically productive binding modes are not superimposable.
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