Cho Myung-A, Yoon Jihoon G, Kim Vitchan, Kim Harim, Lee Rowoon, Lee Min Goo, Kim Donghak
Department of Biological Sciences, Konkuk University, Seoul 05029, Republic of Korea.
Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.
Biomol Ther (Seoul). 2019 Nov 1;27(6):577-583. doi: 10.4062/biomolther.2019.112.
Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants-including three novel variants F69S, L310V, and Q324X-that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high values; however, their values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher and lower values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower and values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.
人细胞色素P450 2C9是一种高度多态性的酶，参与药物和外源性物质的代谢。在此，我们研究了11种P450 2C9基因变体，包括在韩国患者中临床鉴定出的3种新变体F69S、L310V和Q324X。P450 2C9变体酶在[具体表达体系未给出]中表达，并制备了它们的双顺反子膜组分。获得了9种酶变体的CO结合光谱，表明存在P450全酶，但M02（L90P）变体未获得。由于早期无义突变，M11（Q324X）变体无法表达。以双氯芬酸为底物，进行LC-MS/MS分析以测定P450 2C9变体的催化活性。稳态动力学分析表明，所有9种P450 2C9变体的催化效率均低于野生型P450 2C9酶。M05（R150L）和M06（P279T）变体显示出高Km值；然而，它们的Vmax值也很高。由于M01（F69S）、M03（R124Q）、M04（R125H）、M08（I359L）、M09（I359T）和M10（A477T）变体表现出比野生型酶更高的Km值和更低的Vmax值，它们的催化效率与野生型酶相比降低了约50倍。此外，新变体M07（L310V）显示出比野生型酶更低的Km和Vmax值，导致其催化效率降低（80%）。P450 2C9的X射线晶体结构显示在底物结合腔周围的残基中存在突变。这些基因变体的功能表征有助于理解药物遗传学结果。
