Expression, purification, biochemical characterization, and comparative function of human cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 Allelic isoforms

Aiming Yu, Byron M. Kneller, Allan E. Rettie, Robert L. Haining

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

Polymorphism at the cytochrome P450 2D6 (CYP2D6) locus is one of the most widely known causes of pharmacogenetic variability in humans. Our goal is to investigate the intrinsic enzymatic differences that exist among active CYP2D6 isoforms to test the hypothesis that these enzymatic differences are substrate-dependent. Active CYP2D6.1,2, 10, and 17 holoenzymes were expressed in vitro and purified to a high degree of homogeneity as confirmed with SDS-polyacrylamide gel electrophoresis, CO-difference spectroscopy, and mass spectral analysis. Purified enzyme was reconstituted with lipid and cytochrome P450 reductase in a 2:1 ratio before kinetic analysis. The reaction rate for dextromethorphan (DXM) O-demethylation, DXM N-demethylation, codeine O-demethylation, and fluoxetine N-demethylation catalyzed by each of the variants was determined. The CYP2D6.10 enzyme was paired, exhibiting an estimated enzyme efficiency (as Vmax/Km) 50-fold lower for DXM O-demethylation and 100-fold lower for fluoxetine N-demethylation when compared with CYP2D6.1, whereas no measurable catalytic activity was observed for this variant toward codeine. The atypical DXM N-demethylation pathway catalyzed by this variant decreased only 2-fold in comparison. In the case of CYPD6.17, estimated clearances for each metabolite were decreased 6 to 33%. Likewise, the intrinsic clearance of CYP2D6.2 enzyme was consistently decreased for each reaction examined, indicating that the ultra-rapid metabolizer phenotype sometimes associated with this genotype is not a function of the underlying amino acid substitutions. Overall enzyme efficiencies for the metabolism of each substrate therefore decreased in the order of 2D6.1 > 2D6.2 > 2D6.17 > 2D6.10.

Original languageEnglish (US)
Pages (from-to)1291-1300
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume303
Issue number3
DOIs
StatePublished - Dec 1 2002
Externally publishedYes

Fingerprint

Dextromethorphan
Cytochrome P-450 Enzyme System
Protein Isoforms
Enzymes
Codeine
Cytochrome P-450 CYP2D6
Fluoxetine
NADPH-Ferrihemoprotein Reductase
Holoenzymes
Pharmacogenetics
Amino Acid Substitution
Carbon Monoxide
Polyacrylamide Gel Electrophoresis
Mass Spectrometry
Genotype
Phenotype
Lipids

ASJC Scopus subject areas

  • Pharmacology

Cite this

Expression, purification, biochemical characterization, and comparative function of human cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 Allelic isoforms. / Yu, Aiming; Kneller, Byron M.; Rettie, Allan E.; Haining, Robert L.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 303, No. 3, 01.12.2002, p. 1291-1300.

Research output: Contribution to journalArticle

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abstract = "Polymorphism at the cytochrome P450 2D6 (CYP2D6) locus is one of the most widely known causes of pharmacogenetic variability in humans. Our goal is to investigate the intrinsic enzymatic differences that exist among active CYP2D6 isoforms to test the hypothesis that these enzymatic differences are substrate-dependent. Active CYP2D6.1,2, 10, and 17 holoenzymes were expressed in vitro and purified to a high degree of homogeneity as confirmed with SDS-polyacrylamide gel electrophoresis, CO-difference spectroscopy, and mass spectral analysis. Purified enzyme was reconstituted with lipid and cytochrome P450 reductase in a 2:1 ratio before kinetic analysis. The reaction rate for dextromethorphan (DXM) O-demethylation, DXM N-demethylation, codeine O-demethylation, and fluoxetine N-demethylation catalyzed by each of the variants was determined. The CYP2D6.10 enzyme was paired, exhibiting an estimated enzyme efficiency (as Vmax/Km) 50-fold lower for DXM O-demethylation and 100-fold lower for fluoxetine N-demethylation when compared with CYP2D6.1, whereas no measurable catalytic activity was observed for this variant toward codeine. The atypical DXM N-demethylation pathway catalyzed by this variant decreased only 2-fold in comparison. In the case of CYPD6.17, estimated clearances for each metabolite were decreased 6 to 33{\%}. Likewise, the intrinsic clearance of CYP2D6.2 enzyme was consistently decreased for each reaction examined, indicating that the ultra-rapid metabolizer phenotype sometimes associated with this genotype is not a function of the underlying amino acid substitutions. Overall enzyme efficiencies for the metabolism of each substrate therefore decreased in the order of 2D6.1 > 2D6.2 > 2D6.17 > 2D6.10.",
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