Purification and Cloning of a Broad Substrate Specificity Human Liver Carboxylesterase That Catalyzes the Hydrolysis of Cocaine and Heroin

Abstract

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.Carboxylesterases hydrolyze ester groups of drugs and toxins, and thus play an important role in their metabolism and detoxication. In humans, cocaine and heroin are metabolized in serum and liver tissue to de-esterified metabolites that are excreted in urine. Two different human liver carboxylesterases (here called hCE-1 and hCE-2) 1 were identified and shown to hydrolyze cocaine (1). hCE-1 catalyzes the hydrolysis of the methyl ester of cocaine producing benzoylecgonine and methanol, and the ethyl transesterification of cocaine with ethanol to form cocaethylene and methanol. Benzoyl ester hydrolysis of cocaine is catalyzed by liver hCE-2 (1, 2) and serum cholinesterase (3, 4) and produces ecgonine methyl ester and benzoic acid. Hydrolysis of cocaine by serum cholinesterase and nonenzymatic hydrolysis of the methyl ester of cocaine (1) have complicated the assessment of the relative roles of tissue carboxylesterases involved in cocaine metabolism. Heroin is metabolized by liver hCE-2 and hCE-1 carboxylesterases and serum cholinesterase (5). All three esterases cleave the 3-acetyl group of heroin producing 6-monoacetylmorphine. However, only hCE-2 hydrolyzes 6-monoacetylmorphine to morphine with high catalytic efficiency (5). Hence, the contribution of hCE-2 to the metabolism of cocaine or heroin is only recently recognized and its relative role in drug ester clearance has not been fully elucidated.Human liver hCE-2 and hCE-1 enzymes belong to the family of carboxylesterases with a subunit size of about 60 kDa that exhibit broad subst...

Identification, Expression, and Purification of a Pyrethroidhydrolyzing Carboxylesterase from Mouse Liver Microsomes

“…Employing the carboxylesterase classification system proposed by Satoh and Hosokawa (12) this carboxylesterase belongs to the CES 2 family. It is strongly homologous with carboxylesterases AT41 (NCBI accession number BAA23605; 83% identity, 89% similarity) found in golden hamster (Mesocricetus auratus) (27) and human carboxylesterase 2 (71% identity, 82% similarity) (28).…”

Mechanisms of Acute Cocaine Toxicity

“…In plasma, cocaine is metabolized by butylcholinesterase (pseudocholinesterase) to ecgonine methyl ester [47]. The affinity (Km) of butylcholinesterase for cocaine is approximately 11 microM, whereas the affinity of hChE1 is 116 microM [45] and the affinity of hChE2 is 390 microM [48]. The 10-fold difference in affinity suggests that at commonly encountered serum concentrations, more cocaine will be metabolized by butylcholinesterase than by hChE, and one study found that more than 50% of cocaine is metabolized by butylcholinesterase [49].…”

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

“…Other significant functions of the carboxylesterases include metabolism and subsequent detoxification of many agrochemicals, pharmaceuticals (Redinbo and Potter, 2005;Potter and Wadkins, 2006), metabolism of a number of therapeutics (Williams, 1985), including the cholesterol-lowering drug, lovastatin (Tang and Kalow, 1995), the antiinfluenza drug, Oseltamivir (Tamiflu) (Shi et al, 2006), the narcotic analgesic meperidine (Demerol) (Zhang et al, 1999), cocaine and heroin (Pindel et al, 1997), and resolution of racemic mixtures by transesterification, or the enantioselective hydrolysis of esters for obtaining optically pure compounds (Bornscheuer, 2002). Carboxylesterase activity is also used extensively in soft-and pro-drug design (Bodor and Buchwald, 2000, 2003.…”