Structure-Function of Drug Metabolizing Enzymes

目的：确定人体内细胞色素P450酶的药物和异物专一性的原因。细胞色素P450 2 C亚家族在基因水平上高度同源，但在底物特异性上存在显著差异。CYP2C9能特异性代谢布洛芬、双氯芬酸等多种抗炎药。相比之下，密切相关的CYP2C19专门代谢其他药物，如抗癫痫药美苯妥因。本研究的目的是确定参与底物专一性的氨基酸，并解决这些CYP2Cs的结构问题。结构活性研究将有助于预测哪些药物将被这些多态酶代谢。研究成果：我们使用美苯妥因作为人细胞色素P4502C19的原型底物。对CYP2C9和CYP2C19之间嵌合体的初步实验表明，CYP2C19能够识别和代谢特定底物需要多个底物识别位点。初步实验表明，Gotoh提出的底物识别位点(SRS)(SRS1)上的I99H与FG环中的220Ser Pro和220Pro Thr以及2C19的SRS 3和14需要将CYP2C9转化为高周转率的苯妥英羟基酶。后来的定点突变实验表明，需要I99H、S221P、P221T和SRS中的三个氨基酸才能将CYP2C9转化为美苯妥因羟基酶。它们是286、2292和295。因此，CYP2C19的特异性似乎需要多个区域，尽管相反，只需要几个变化就可以将CYP2C19转化为类似2C9的酶。农药和环境化学物质的底物专一性表明，某些有机磷农药，如毒死蜱，至少部分由CYP2C19代谢。

AIMS: To determine the reason for drug and xenobiotic specificity of the CYP enzymes in humans. The CYP2C subfamily is highly homologous at the gene level, but differ markedly in their substrate specificity. CYP2C9 specifically metabolizes a number of anti-inflammatory drugs such as ibuprofen and diclofenac. In contrast, the closely related CYP2C19 specifically metabolizes other drugs such as the anticonvulsant mephenytoin. The aims of this study are to define the amino acids involved in substrate specificity and solve the structure of these CYP2Cs. Structure activity studies will help predict which drugs will be metabolized by these polymorphic enzymes. ACCOMPLISHMENTS: We used mephenytoin as a prototype substrate for human CYP2C19. Preliminary experiments with chimeras between CYP2C9 and CYP2C19 indicated that multiple substrate recognition sites are necessary for CYP2C19 to be able to recognize and metabolize specific substrates Preliminary experiments suggest that I99H in a substrate recognition site (SRS) (SRS1) proposed by Gotoh in combination with 220Ser Pro and 220Pro Thr in the FG loop and SRS 3 and 14 of 2C19 are required to convert CYP2C9 to a high turnover mephenytoin hydroxylase. Later site directed mutagenesis experiments indicate that I99H, S221P, P221T, and three amino acids in SRS are required to convert CYP2C9 to a mephenytoin hydroxylase. These are 286, 2292, and 295. Therefore multiple regions appear to be required for CYP2C19 specificity, although conversely, only a few changes are required for to convert CYP2C19 to a 2C9 like enzyme. Substrate specificity with pesticides and environmental chemicals indicate that certain organophosphorus pesticides such as chlorpyrofos are metabolized at least in part by CYP2C19.