Lakaschus G, Löffler M
Department of Physiological Chemistry, School of Medicine, Philipps-University of Marburg, Federal Republic of Germany.
Biochem Pharmacol. 1992 Mar 3;43(5):1025-30. doi: 10.1016/0006-2952(92)90609-m.
To verify the assumption of a specific and potent drug action on de novo pyrimidine biosynthesis, isolated dihydroorotate dehydrogenase (DHO-DH) (EC 1.3.3.1) was exposed to Brequinar Sodium (6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt, NSC 368 390) (Brequinar). The membrane-bound DHO-DH was purified to apparent homogeneity (25,000-fold) from rat liver mitochondria in six steps via detergent extraction and subsequent chromatography using the dye ligand Matrex Gel Orange A. Using molecular mechanistic studies (MM2) this ligand was found to mimic closely the stereochemical conformation of Brequinar. SDS-PAGE revealed two protein bands for the purified enzyme with apparent molecular masses of 58 (major) and 68 kDa (minor). In vitro, two modes of action of the DHO-DH are possible: (i) acting as a dehydrogenase in the presence of ubiquinone as proximal electron acceptor and (ii) direct reaction with oxygen as oxidase. A novel assay for the measurement of the oxidase activity was adapted using leuco-dichlorofluorescein-diacetate. Inhibition experiments revealed a striking difference in the susceptibility of DHO-dehydrogenase/oxidase to Brequinar: apparent Ki = 6.09 +/- 0.05 (SD) nM (DHO; ubiquinone n = 10), but Ki = 3.10 +/- 0.09 (SD) mM (DHO; O2). Analyses of initial velocity experiments showed non-competitive inhibition of Brequinar with respect to the substrate dihydroorotic acid in both assays (dehydrogenase and oxidase). The inhibitory effect of the latter was compared to that of the competitive inhibitor 5-aza-dihydroorotate (apparent Ki = 15 +/- 0.25 (SD) microM). The present kinetic data on the action of the purified rodent DHO-DH with Brequinar and computer-aided analyses provide a better insight into the drug-enzyme interaction.
为验证针对从头嘧啶生物合成存在特异性强效药物作用这一假设，将分离出的二氢乳清酸脱氢酶（DHO - DH）（EC 1.3.3.1）暴露于布喹那钠（6 - 氟 - 2 -（2'-氟 - 1,1'-联苯 - 4 - 基）- 3 - 甲基 - 4 - 喹啉羧酸钠盐，NSC 368390）（布喹那）。通过去污剂提取以及随后使用染料配体Matrex Gel Orange A进行的色谱法，分六步从大鼠肝脏线粒体中纯化膜结合的DHO - DH至表观均一性（25000倍）。利用分子力学研究（MM2）发现该配体紧密模拟布喹那的立体化学构象。SDS - PAGE显示纯化酶有两条蛋白带，表观分子量分别为58 kDa（主要）和68 kDa（次要）。在体外，DHO - DH有两种作用模式：（i）在泛醌作为近端电子受体存在时作为脱氢酶起作用，以及（ii）作为氧化酶与氧气直接反应。采用无色二氯荧光素二乙酸酯改进了一种用于测量氧化酶活性的新测定法。抑制实验揭示了DHO - 脱氢酶/氧化酶对布喹那敏感性的显著差异：表观Ki = 6.09±0.05（SD）nM（DHO；泛醌，n = 10），但Ki = 3.10±0.09（SD）mM（DHO；O2）。初始速度实验分析表明，在两种测定法（脱氢酶和氧化酶）中，布喹那对底物二氢乳清酸均表现为非竞争性抑制。将后者的抑制作用与竞争性抑制剂5 - 氮杂二氢乳清酸的抑制作用（表观Ki = 15±0.25（SD）μM）进行了比较。目前关于纯化的啮齿动物DHO - DH与布喹那作用的动力学数据以及计算机辅助分析为药物 - 酶相互作用提供了更好的见解。
