Specificity and Structure-Function Studies of Human 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。本研究的目的是确定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代谢这些多态性enzymes.AccomplishMENTS：我们使用布洛芬和双氯芬酸作为特定的底物CYP 2C 9和美芬妥英作为原型底物CYP 2C 19。在细菌的cDNA表达系统中构建嵌合体和定点突变体。底物识别位点（SRS）3和4（Gotoh针对哺乳动物P450提出）似乎赋予了对特定CYP 2C 9底物（如布洛芬和双氯芬酸）的特异性。SRS 3和4似乎都是活性或适当表达所必需的。定点诱变实验表明，286（N S和289 IN）处的突变赋予2C 19对布洛芬和双氯芬酸的高比活性。然而，匡威就不对了。单独的CYP 2C 19的SRS 3和4不会将美芬妥英羟化酶赋予CYP 2C 9。初步实验表明，需要I99 H（SRS 1）与FG环中的220 Ser Pro和220 Pro Thr以及2C 19的SRS 3和14组合将CYP 2C 9转化为高转换率美芬妥英羟化酶。因此，CYP 2C 19特异性似乎需要多个区域，但CYP 2C 9特异性需要的变化要少得多。- 构效关系，CYP 2C 19，CYP 2C 8，CYP 2C 9，P-450

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 ibuprofen and diclofenac as specific substrates for CYP2C9 and mephenytoin as a prototype substrate for CYP2C19. Chimeras and site directed mutants were constructed in a cDNA expression system in bacteria. Substrate recognition sites (SRS) 3 and 4 (proposed by Gotoh for mammalian P450s) appeared to confer specificity for specific CYP2C9 substrates such as ibuprofen and diclofenac. Both SRS 3 and 4 appeared necessary for activity or proper expression. Site directed mutagenesis experiments showed that mutations at 286(N S and 289 I N) conferred high specific activity toward ibuprofen and diclofenac to 2C19. However, the converse is not true. SRS 3 and 4 of CYP2C19 alone do not confer mephenytoin hydroxylase to CYP2C9. Preliminary experiments suggest that I99H (SRS1) 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. Therefore multiple regions appear to be required for CYP2C19 specificity, but much fewer changes are required for CYP2C9 specificity. - Structure-activity, CYP, P-450, CYP2C19, CYP2C8, CYP2C9