Structure-Function of Drug Metabolizing Enzymes

目的：确定人体内β-内酰胺酶的药物和异源生物质特异性的原因。 CYP 2C亚家族在基因水平上高度同源，但在底物特异性上明显不同。CYP 2C 9特异性代谢许多抗炎药物，如布洛芬和双氯芬酸。 相反，密切相关的CYP 2C 19特异性代谢其他药物，如抗惊厥药美芬妥英。 本研究的目的是定义参与底物特异性的氨基酸并解决这些CYP 2C的结构。 结构活性研究将有助于预测哪些药物将被这些多态性酶代谢。成果：我们使用美芬妥英作为人CYP 2C 19的原型底物。CYP 2C 9和CYP 2C 19之间嵌合体的初步实验表明，CYP 2C 19需要多个底物识别位点才能识别和代谢特定底物。初步实验表明，底物识别位点（SRS）（SRS 1）中的I99 H由Gotoh提出的与FG环中的220 Ser Pro和220 Pro Thr以及2C 19的SRS 3和14组合需要将CYP 2C 9转化为高转换美芬妥英羟化酶。 后来的定点突变实验表明，I99 H、S221 P、P221 T和SRS中的三个氨基酸是将CYP 2C 9转化为美芬妥英羟化酶所必需的。 它们是286、2292和295。 因此，CYP 2C 19特异性似乎需要多个区域，尽管相反，将CYP 2C 19转化为2C 9样酶仅需要几个变化。农药和环境化学物质的底物特异性表明，某些有机磷农药（如毒死蜱）至少部分由CYP 2C 19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.