Metabolic Activity Of Human Recombinant Cytochrome P450

人重组细胞色素 P450 的代谢活性

• 批准号: 6535043
• 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/6535043
• 关键词: anticonvulsants; binding sites; chemical binding; cytochrome P450; drug metabolism; enzyme activity; genetic polymorphism; human tissue; hydroxylation; molecular shape; omeprazole; paclitaxel; recombinant proteins; site directed mutagenesis; tolbutamide

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. CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids related to the specificity of human CYP2C19 for S-mephenytoin 4 ' -hydroxylation, chimeras were constructed by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1-4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4 ' hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4 '-hydroxylation activity with a KM similar to that of CYP2C19 but a 3-fold lower K-cat. Three residues in helix I, S286N, V292A, and F2951, were essential for S-mephenytoin 4 '-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.

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。 CYP2C19 对 S-美芬妥英的 4'-羟基化具有选择性，而高度相似的 CYP2C9 对此底物几乎没有活性。为了鉴定与人 CYP2C19 对 S-美苯妥英 4'-羟基化的特异性相关的关键氨基酸，通过用 CYP2C19 的部分替换含有各种拟议底物识别位点 (SRS) 的 CYP2C9 部分并通过定点突变单个残基来构建嵌合体诱变。只有包含 SRS 1-4 区域的嵌合体具有活性（野生型 CYP2C19 的 30%），表明需要多个区域才能赋予 S-美芬妥英特异性。诱变研究确定了将 CYP2C9 转化为 S-美芬妥英 4' 羟化酶所需的蛋白质的三个拓扑成分中的 6 个残基（野生型 CYP2C19 活性的 6%）。其中，只有位于螺旋 B 和 C 之间的 SRS 1 中的 I99H 差异反映了侧链的变化，该侧链预计位于血红素假体基团上方形成的底物结合腔中。两个额外的取代，S220P 和 P221T 位于螺旋 F 和 G 之间，但不靠近底物结合位点，加上螺旋 I N 端部分的五个差异，赋予了 S-美芬妥英 4'-羟基化活性，其 KM 类似于与 CYP2C19 相比，但 K-cat 低 3 倍。螺旋 I 中的三个残基，S286N、V292A 和 F2951，对于 S-美芬妥英 4'-羟基化活性至关重要。根据密切相关的酶 CYP2C5 的结构，这些残基在催化过程中不太可能直接接触底物，但其位置会影响底物结合位点残基的堆积以及酶中可能的底物进入通道。