Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin.
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.
Pindel
E V
EV
Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Kedishvili
N Y
NY
Abraham
T L
TL
Brzezinski
M R
MR
Zhang
J
J
Dean
R A
RA
Bosron
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003042:Cocaine / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D003932:Heroin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D003932:Heroin; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D003042:Cocaine