Kleingeist B, Böcker R, Geisslinger G, Brugger R
Department of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nürnberg, Universitätsstr. 22, D-91054 Erlangen, Germany.
J Pharm Pharm Sci. 1998 Jan-Apr;1(1):38-46.
In vivo the biotransformation of the imidazobenzodiazepine antagonist flumazenil leads to the formation of two metabolites, flumazenil acid and N-demethylated flumazenil. In the present study we investigated the role of carboxylesterases for the metabolism of flumazenil.
We purified a non-specific carboxylesterase (EC 3.1.1.1) from human liver microsomes that catalyzes the hydrolysis of flumazenil to flumazenil acid and, in presence of methanol the formation of flumazenil methyl ester an in vivo unknown metabolite. The purification procedure included solubilization of the microsomes obtained from human livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, DEAE-sepharose, hydroxyapatite and final chromatofocusing.
The purified esterase isozyme exhibited an apparent subunit molecular weight of 59 kDa as estimated by SDS gelelectrophoresis, a native molecular weight of 170 kDa determined by a calibrated gel filtration column suggesting that the active enzyme is a trimer. The isoelectric point of the enzyme was approximately 5.4. The specific activities of the purified enzyme were 5.8 nmol/(minmg protein) protein for the formation of flumazenil acid and 31 nmol/(minmg protein) for the synthesis of the flumazenil methylester. The purified enzyme obeys simple Michaelis-Menten kinetics with K(M) values of 665 microM for flumazenil acid, 1011 mM for methanol and 900 microM for the flumazenil methylester. PMSF, a specific inhibitor for serine proteases and mammalian acetylcholinesterase, completely inhibited the formation of flumazenil -acid and the flumazenil methylester at a concentration of 100 microM. No synthesis of the flumazenil -methylester could be observed by incubation of the purified esterase with flumazenil acid in the presence of methanol leading to the conclusion that the enzymatically catalyzed reaction is a transesterification. The purified esterase was digested with endoproteinase LysC. A 15 amino acid long peptide was isolated and showed identical matches to carboxylesterase cDNAs from human liver and lung.
Our results show that carboxylesterase isozymes play an important role in the detoxification and metabolism of flumazenil. Because of enzymatic, catalytic and structural properties a similarity of the characterized flumazenil carboxylesterase with human liver cocaine carboxylesterase is possible.
在体内，咪唑并苯二氮䓬拮抗剂氟马西尼的生物转化会产生两种代谢产物，即氟马西尼酸和N - 去甲基氟马西尼。在本研究中，我们调查了羧酸酯酶在氟马西尼代谢中的作用。
我们从人肝微粒体中纯化了一种非特异性羧酸酯酶（EC 3.1.1.1），该酶催化氟马西尼水解为氟马西尼酸，并且在甲醇存在的情况下，会生成一种体内未知的代谢产物氟马西尼甲酯。纯化过程包括用Triton X - 100溶解从人肝脏获得的微粒体，随后将100,000×g上清液在蓝色琼脂糖凝胶、DEAE - 琼脂糖凝胶、羟基磷灰石上进行层析，最后进行色谱聚焦。
通过SDS凝胶电泳估计，纯化的酯酶同工酶的表观亚基分子量为59 kDa，通过校准的凝胶过滤柱测定其天然分子量为170 kDa，这表明活性酶是三聚体。该酶的等电点约为5.4。纯化酶对于氟马西尼酸形成的比活性为5.8 nmol/（min·mg蛋白质），对于氟马西尼甲酯合成的比活性为31 nmol/（min·mg蛋白质）。纯化酶遵循简单的米氏动力学，对于氟马西尼酸的K(M)值为665 μM，对于甲醇为1011 mM，对于氟马西尼甲酯为900 μM。苯甲基磺酰氟（PMSF）是丝氨酸蛋白酶和哺乳动物乙酰胆碱酯酶的特异性抑制剂，在浓度为100 μM时完全抑制了氟马西尼酸和氟马西尼甲酯的形成。在甲醇存在的情况下，将纯化的酯酶与氟马西尼酸一起孵育时，未观察到氟马西尼甲酯的合成，由此得出结论，酶催化反应是转酯反应。用内肽酶LysC消化纯化的酯酶。分离出一个15个氨基酸长的肽段，它与人肝脏和肺中的羧酸酯酶cDNA显示出完全匹配。
我们的结果表明，羧酸酯酶同工酶在氟马西尼的解毒和代谢中起重要作用。由于酶学、催化和结构特性，所鉴定的氟马西尼羧酸酯酶与人肝脏可卡因羧酸酯酶可能存在相似性。
