METABOLIC ACTIVITY OF HUMAN RECOMBINANT CYTOCHROME P450 ENZYMES

人重组细胞色素 P450 酶的代谢活性

• 批准号: 6432311
• 负责人: BURHAN I GHANAYEM
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432311
• 关键词: cytochrome P450; drug metabolism; enzyme activity; genetic polymorphism; high performance liquid chromatography; human genetic material tag; human tissue; hydroxylation; molecular cloning; nonsteroidal antiinflammatory agent; recombinant proteins; yeasts

Significant variations in the metabolism of various drugs and environmental chemicals which are metabolized via cytochrome P450 (CYP) enzymes exist between humans. Many of these interindividual variations are attributed to polymorphisms in the CYP2C subfamily of enzymes. In the current work, two new CYP2C8 polymorphisms were identified which contain coding changes. One allele termed CYP2C8*2 had an lle269Phe substitution in exon 5. The second allele termed CYP2C8*3 contained Arg139Lys and Lys399Arg amino acid substitution in exons 3 and 5. An HPLC method was developed to assess the ability of these enzymes to metabolize paclitaxol was developed. When CYP2C8*1 (wild type), CYP2C8*2, and CYP2C8*3 cDNAs were expressed in an E. Coli expression system, the ability of CYP2C8*3 to metabolize paclitaxol was markedly impaired. The CYP2C8*2 allele exhibited a 2 fold higher Km and 1 2-fold lower intrinsic clearance than that of CYP2C8*1. The turnover number for CYP2C8*3 for paclitaxol was only 16% of CYP2C8*1. Thus, the CYP2C8*3 allele is defective in metabolism of CYP2C8 substrates such as paclitaxol. This polymorphism may have important clinical and physiological implications in individuals homozygous for this allele.

通过细胞色素 P450 (CYP) 酶代谢的各种药物和环境化学物质的代谢在人类之间存在显着差异。 许多这些个体差异归因于 CYP2C 酶亚家族的多态性。 在当前的工作中，鉴定了两个新的 CYP2C8 多态性，其中包含编码变化。 一个称为 CYP2C8*2 的等位基因在外显子 5 中具有 lle269Phe 取代。第二个称为 CYP2C8*3 的等位基因在外显子 3 和 5 中包含 Arg139Lys 和 Lys399Arg 氨基酸取代。开发了一种 HPLC 方法来评估这些酶代谢紫杉醇的能力。发达。 当CYP2C8*1（野生型）、CYP2C8*2和CYP2C8*3 cDNA在大肠杆菌表达系统中表达时，CYP2C8*3代谢紫杉醇的能力明显受损。 CYP2C8*2 等位基因表现出比 CYP2C8*1 高 2 倍的 Km 和低 2 倍的内在清除率。 紫杉醇的CYP2C8*3的周转数仅为CYP2C8*1的16%。 因此，CYP2C8*3等位基因在CYP2C8底物如紫杉醇的代谢中存在缺陷。 这种多态性可能对该等位基因纯合的个体具有重要的临床和生理意义。