Brzezinski M R, Abraham T L, Stone C L, Dean R A, Bosron W F
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Biochem Pharmacol. 1994 Nov 1;48(9):1747-55. doi: 10.1016/0006-2952(94)90461-8.
The psychomotor stimulant cocaine is inactivated primarily by hydrolysis to benzoylecgonine, the major urinary metabolite of the drug. A non-specific carboxylesterase was purified from human liver that catalyzes the hydrolysis of the methyl ester group of cocaine to form benzoylecgonine. In the presence of ethanol, the enzyme also catalyzes the transesterification of cocaine producing the pharmacologically active metabolite cocaethylene (benzoylecgonine ethyl ester). The carboxylesterase obeys simple Michaelis-Menten kinetics with Km values of 116 microM for cocaine and 43 mM for ethanol. The enzymatic activity suggests that it may play an important role in regulating the detoxication of cocaine and in the formation of the active metabolite cocaethylene. Additionally, the enzyme catalyzes the formation of ethyloleate from oleic acid and ethanol. The carboxylesterase was purified from autopsy liver by gel filtration, chromatofocusing, ion-exchange, and hydrophobic interaction chromatography to purity by SDS-PAGE and agarose gel isoelectric focusing. The subunit molecular weight was determined to be 59,000 and the native molecular weight was estimated to be 170,000 from a calibrated gel filtration column, suggesting that the active enzyme is a trimer. The isoelectric point was approximately 5.8. Digestion of carbohydrate residues on the protein with an acetylglucosaminidase plus binding to several lectins indicates that the enzyme is glycosylated. The esterase was cleaved with two proteases, and the amino acid sequences from fourteen peptides were used to search GenBank. Two identical matches were found corresponding to carboxylesterase cDNAs from human liver and lung.
精神运动兴奋剂可卡因主要通过水解作用转化为苯甲酰爱康宁而失活，苯甲酰爱康宁是该药物的主要尿液代谢产物。从人肝脏中纯化出一种非特异性羧酸酯酶，它能催化可卡因甲酯基团的水解，形成苯甲酰爱康宁。在乙醇存在的情况下，该酶还能催化可卡因的酯交换反应，生成具有药理活性的代谢产物可口乙酮（苯甲酰爱康宁乙酯）。这种羧酸酯酶遵循简单的米氏动力学，对可卡因的Km值为116微摩尔，对乙醇的Km值为43毫摩尔。该酶的活性表明它可能在调节可卡因的解毒以及活性代谢产物可口乙酮的形成中发挥重要作用。此外，该酶还能催化油酸和乙醇生成油酸乙酯。通过凝胶过滤、色谱聚焦、离子交换和疏水相互作用色谱从尸检肝脏中纯化出该羧酸酯酶，经SDS - PAGE和琼脂糖凝胶等电聚焦分析达到纯度。通过校准的凝胶过滤柱测定亚基分子量为59,000，天然分子量估计为170,000，这表明活性酶是三聚体。其等电点约为5.8。用乙酰葡糖胺酶消化蛋白质上的碳水化合物残基并与几种凝集素结合表明该酶是糖基化的。用两种蛋白酶切割该酯酶，并用来自14个肽段的氨基酸序列搜索GenBank。发现了两个与来自人肝脏和肺的羧酸酯酶cDNA相同的匹配序列。
