Langeland B T, McKinley-McKee J S
Biokjemisk Institutt, Universitetet i Oslo, Norway.
Arch Biochem Biophys. 1994 Feb 1;308(2):367-73. doi: 10.1006/abbi.1994.1052.
The imidazobenzodiazepine ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine -3-carboxylate (Ro 15-4513) is important as a potential "drink and drive" drug due to effects on receptors in brain neurones, resulting in alcohol intoxication-antagonistic properties. Because of the molecule's importance its effect on alcohol metabolism in liver has been investigated. Ro 15-4513 was found to be, like its parent compound the 8-fluoro analogue flumazenil, a reversible alcohol competitive inhibitor of horse liver alcohol dehydrogenase (EC 1.1.1.1) with a dissociation constant of 345 microM at pH 7.0. Due to its azido group Ro 15-4513 was developed as a potential photoaffinity-labeling reagent for benzodiazepine receptors. Used with horse liver alcohol dehydrogenase, the enzyme is chemically modified and inactivated in a Michaelis-Menten type reaction via a reversible enzyme-Ro 15-4513 complex with a dissociation constant of 8.6 mM at pH 7.0. The inactivation reaction has been studied over the pH 6.0-10.0 range. The dissociation constants for the binding of Ro 15-4513 to the enzyme and the first-order rate constants for inactivation have been determined as a function of pH. These give pKa values of 7.2 and 8.8 for the free enzyme, the latter being assigned to the zinc-water ionization. The enzyme is protected from inactivation in a competitive manner by flumazenil and by many heterocyclic and thiol compounds which combine with the active-site zinc. Flumazenil has a similar binding affinity as Ro 15-4513 with an enzyme-flumazenil dissociation constant of 6.0 mM at pH 7.0. Ro 15-4513 may also have potential as a photoaffinity-labeling reagent for other metallo enzymes. Whether the effects of Ro 15-4513 on alcohol-metabolizing enzymes are also of clinical significance remains to be determined.
咪唑并苯二氮䓬8-叠氮基-5,6-二氢-5-甲基-6-氧代-4H-咪唑并[1,5-a][1,4]苯二氮䓬-3-羧酸乙酯（Ro 15-4513）作为一种潜在的“酒驾”药物具有重要意义，因为它对脑神经元中的受体有作用，从而产生抗酒精中毒特性。由于该分子的重要性，已对其在肝脏中对酒精代谢的影响进行了研究。结果发现，Ro 15-4513与其母体化合物8-氟类似物氟马西尼一样，是马肝醇脱氢酶（EC 1.1.1.1）的可逆酒精竞争性抑制剂，在pH 7.0时的解离常数为345微摩尔。由于其叠氮基，Ro 15-4513被开发为一种潜在的苯二氮䓬受体光亲和标记试剂。与马肝醇脱氢酶一起使用时，该酶在pH 7.0时通过解离常数为8.6毫摩尔的可逆酶-Ro 15-4513复合物在米氏反应中被化学修饰并失活。已在pH 6.0 - 10.0范围内研究了失活反应。已确定Ro 15-4513与酶结合的解离常数以及失活的一级速率常数作为pH的函数。这些给出了游离酶的pKa值为7.2和8.8，后者归因于锌-水离子化。氟马西尼和许多与活性位点锌结合的杂环和硫醇化合物以竞争方式保护酶不被失活。氟马西尼与Ro 15-4513具有相似的结合亲和力，在pH 7.0时酶-氟马西尼解离常数为6.0毫摩尔。Ro 15-4513也可能具有作为其他金属酶光亲和标记试剂的潜力。Ro 15-4513对酒精代谢酶的影响是否也具有临床意义仍有待确定。
