Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin

J Biol Chem. 1997 Jun 6;272(23):14769-75. doi: 10.1074/jbc.272.23.14769.

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carboxylesterase
  • Carboxylic Ester Hydrolases / chemistry
  • Carboxylic Ester Hydrolases / isolation & purification
  • Carboxylic Ester Hydrolases / metabolism*
  • Catalysis
  • Chromatography, DEAE-Cellulose
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Cocaine / metabolism*
  • Cricetinae
  • DNA Primers
  • DNA, Complementary
  • Heroin / metabolism*
  • Humans
  • Hydrolysis
  • Kinetics
  • Liver / enzymology*
  • Mice
  • Molecular Sequence Data
  • Phylogeny
  • Rabbits
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • DNA Primers
  • DNA, Complementary
  • Recombinant Proteins
  • Heroin
  • Carboxylic Ester Hydrolases
  • Carboxylesterase
  • Cocaine

Associated data

  • GENBANK/U60553