Crespi C L, Steimel D T, Penman B W, Korzekwa K R, Fernandez-Salguero P, Buters J T, Gelboin H V, Gonzalez F J, Idle J R, Daly A K
GENTEST Corporation, Woburn, MA 01801, USA.
Pharmacogenetics. 1995 Aug;5(4):234-43. doi: 10.1097/00008571-199508000-00007.
We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.
我们分析了源自原始CYP2D6 cDNA分离株（Gonzalez FJ等人，《自然》1988年：331, 442 - 446）的cDNA衍生CYP2D6蛋白的动力学参数，该分离株在374位含有甲硫氨酸（CYP2D6 - Met），以及一个在374位含有缬氨酸的修饰cDNA（CYP2D6 - Val）。后一种蛋白是根据CYP2D6基因组序列预测得到的。观察到了一些定量差异，但在代谢方面没有定性差异。与CYP2D6 - Val相比，发现CYP2D6 - Met对（R）（ + ） - 布非洛尔1'-羟化反应的Km值低两倍，周转速率低三倍。相比之下，CYP2D6 - Met和CYP2D6 - Val对去甲异喹胍4 - 羟化反应的Km值相似，而CYP2D6 - Val的周转速率高18倍。CYP2D6 - Val和CYP2D6 - Met对美托洛尔的Km值相似，但与α - 羟化反应相比，CYP2D6 - Val对O - 去甲基化反应的能力高两倍，而α - 羟化反应更类似于在人肝脏中观察到的情况。对于司巴丁，CYP2D6 - Val和CYP2D6 - Met形成2 - 脱氢司巴丁代谢物的能力相似，但CYP2D6 - Met的Km值比CYP2D6 - Val高六倍。通过检测抑制（R,S）（±） - 布非洛尔1'-羟化反应的表观Ki，进一步探究了CYP2D6 - Met和CYP2D6 - Val之间的动力学差异。对于哌克昔林和（R,S） - 普萘洛尔，观察到相似的Ki值（相差三倍以内），而奎尼丁和右美沙芬对CYP2D6 - Val的抑制作用相对于CYP2D6 - Met分别强8.5倍和21倍。针对CYP2D6 - Met等位基因开发了一种等位基因特异性聚合酶链反应检测方法。在83名个体中未发现CYP2D6 - Met等位基因。总体而言，这些数据表明CYP2D6 - Val等位基因在人群中更为常见。这两种酶之间的定量动力学差异对于具有刚性结构的底物/抑制剂最为明显。CYP2D6 - Val通常对那些底物具有显著更低的Km值和/或更高的代谢能力。
