Expression, purification, biochemical characterization, and comparative function of human cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 allelic isoforms.
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 holo-enzymes 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 the most impaired, exhibiting an estimated enzyme efficiency (as V(max)/K(m)) 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.
Yu
Aiming
A
Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia 26506-9530, USA.
Kneller
Byron M
BM
Rettie
Allan E
AE
Haining
Robert L
RL
D000483:Alleles; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018076:DNA, Complementary / Q000096:biosynthesis; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D009036:Moths / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D018076:DNA, Complementary; D007527:Isoenzymes; D019389:Cytochrome P-450 CYP2D6