Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase.
Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase.
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DOI：
10.1097/fpc.0b013e32833e0cb5
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发表时间：
2010-10
影响因子：
2.6
通讯作者：
Miller WL
中科院分区：
文献类型：
--
作者：
Agrawal V;Choi JH;Giacomini KM;Miller WL
CYP3A4从P450氧化还原酶(POR)获得电子，代谢约50%的临床用药。细胞色素P3A4的催化活性有很大的个体间差异，这是不能用细胞色素P3A4基因变异来解释的。CYP3A4是灵活和可膨胀的，使其能够适应不同形状和大小的底物。为了阐明CYP3A4催化中的可变性机制，我们研究了POR基因变异的影响，并探索了底物诱导的CYP3A4构象变化不同地影响POR变异支持催化的可能性。我们在细菌中表达了人CYP3A4和四个POR突变体(Q153R、A287P、R457H、A503V)，并在体外重组了它们，以睾酮、咪达唑仑、奎尼丁和红霉素为底物，测定了它们的米氏常数和最大运动速度。POR A287P和R457H对所有底物的活性都很低，Q153R对咪达唑仑和红霉素的活性是野生型的76-94%，对睾酮和奎尼丁的活性是129%-150%。在睾酮和咪达唑仑的作用下，A503V多态使细胞色素P3A4的活性降低到野生型的61-77%，而在奎尼丁和红霉素的作用下，细胞色素P3A4的活性接近野生型。POR变异体影响细胞色素P3A4活性。POR变异体对CYP3A4催化的影响是底物特异性的，可能是由于底物诱导的CYP3A4的构象变化。
CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4.