Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin
Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin
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DOI：
10.1074/jbc.272.23.14769
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发表时间：
1997-06-06
影响因子：
4.8
通讯作者：
Bosron, WF
中科院分区：
文献类型：
--
作者：
Pindel, EV;Kedishvili, NY;Bosron, WF
从人肝中纯化了一种人肝羧酸酯酶（hCE-2），它能催化可卡因的苯甲酰基和4-甲基伞形酮乙酸酯、海洛因和6-单乙酰吗啡的乙酰基水解。纯化的酶在SDS-聚丙烯酰胺凝胶电泳上显示单一条带，亚基质量约为60 kDa。天然酶是单体的。hCE-2的等电点约为4.9。用糖苷内切酶H处理引起电泳迁移率增加，表明肝羧酸酯酶是高甘露糖型的糖蛋白。测定了其cDNA全序列。cDNA的真实性通过来自从人肝脏纯化的hCE-2的78个氨基酸的完全序列匹配来证实。由该cDNA编码的成熟的533个氨基酸的酶与兔肝羧酸酯酶2型（73%）和仓鼠肝羧酸酯酶AT 51 p（67%）具有最高的序列同一性。与hCE-2具有高度序列同一性的羧酸酯酶在小鼠和大鼠肝脏中尚未报道。hCE-2显示出与称为hCE-1的人肝羧酸酯酶不同的药物酯底物特异性，其水解可卡因的甲酯，hCE-2对4-甲基伞形酮乙酸酯、海洛因和6-单乙酰吗啡的水解具有更高的催化效率，并且比hCE-1具有更大的艾司氯胺酮抑制作用。hCE-2可能在人体组织中可卡因和海洛因的降解中起重要作用。
A human liver carboxylesterase (hCE-2) that catalyzes the hydrolysis of the benzoyl group of cocaine and the acetyl groups of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine was purified from human liver. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis with a subunit mass of approximately 60 kDa. The native enzyme was monomeric. The isoelectric point of hCE-2 was approximately 4.9. Treatment with endoglycosidase H caused an increase in electrophoretic mobility indicating that the liver carboxylesterase was a glycoprotein of the high mannose type. The complete cDNA nucleotide sequence was determined. The authenticity of the cDNA was confirmed by a perfect sequence match of 78 amino acids derived from the hCE-2 purified from human liver. The mature 533-amino acid enzyme encoded by this cDNA shared highest sequence identity with the rabbit liver carboxylesterase form 2 (73%) and the hamster liver carboxylesterase AT51p (67%). Carboxylesterases with high sequence identity to hCE-2 have not been reported in mouse and rat liver. hCE-2 exhibited different drug ester substrate specificity from the human liver carboxylesterase called hCE-1, which hydrolyzes the methyl ester of cocaine, hCE-2 had higher catalytic efficiencies for hydrolysis of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine and greater inhibition by eserine than hCE-1. hCE-2 may play an important role in the degradation of cocaine and heroin in human tissues.