Active-site topologies of human CYP2D6 and its aspartate-301 --> glutamate, asparagine, and glycine mutants.
Active-site topologies of human CYP2D6 and its aspartate-301 --> glutamate, asparagine, and glycine mutants.
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人类 CYP2D6 及其天冬氨酸 301 --> 谷氨酸、天冬酰胺和甘氨酸突变体的活性位点拓扑。
DOI：
10.1006/abbi.1996.0291
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发表时间：
1996
影响因子：
3.9
通讯作者：
OrtizdeMontellano,PR
中科院分区：
文献类型：
--
作者：
Mackman,R;Tschirret-Guth,RA;Smith,G;Hayhurst,GP;Ellis,SW;Lennard,MS;Tucker,GT;Wolf,CR;OrtizdeMontellano,PR
细胞色素P450 2D6(细胞色素P450 2D6)催化底物氧化，底物上有一个带正电的氮原子。用苯基、2-萘基和对-联苯基重氮烯与P450酶反应生成σ键合的芳基铁(Fe-Ar)络合物，研究了其活性中心的拓扑结构。铁氰化物介导的芳基从铁到卟啉氮的迁移生成了随后的芳基原卟啉IX区域异构体(Nb：Na：NC：ND，其中芳基分别结合到吡咯环B、A、C和D的氮上)，其比例如下(零表示<；5%)：苯基，10：90：00：00；2-萘基，09：91：00：00；和对联苯，16：84：00：00。这些结果表明，CYP2D6活性中心在吡咯环A上方开放，在吡咯环B上方略高，但在吡咯环C和D上方封闭。这一几何构型不同于已知晶体结构的P450的几何构型。用保留了羧酸侧链的Glu取代Asp-301，不会引起当时芳基卟啉区域异构体的可检测到的变化，只有微小的催化活性变化。Asp-301被AsN或Gly取代，消除了带负电荷的侧链，抑制了芳基向吡咯环B的迁移，而不影响到吡咯环A的迁移，几乎取消了催化活性。这些结果为CYP2D6的活性部位提供了一个改进的模型。他们进一步证实，当Asp-301被中性残基取代时，活性的丧失是由于失去了与底物正电荷的电荷配对相互作用和/或吡咯环B附近的微妙结构效应，而不是由于活性中心的主要结构重组。
Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5–7 Å from the site of the oxidation. The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, andp-biphenyldiazene, which react with P450 enzymes to form σ-bonded aryl–iron (Fe–Ar) complexes. Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces theN-arylprotoporphyrin IX regioisomers (NB:NA:NC:ND, in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; andp-biphenyl, 16:84:00:00. These results suggest that the CYP2D6 active site is open above pyrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D. This geometry differs from those determined by the same method for P450s for which crystal structures are available. Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in theN-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity. Replacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually abolishes catalytic activity. These results provide a refined model of the active site of CYP2D6. They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ring B, but not to major structural reorganization of the active site.