A Combination of Mutations in theCYP2D6*17(CYP2D6Z) Allele Causes Alterations in Enzyme Function

Abstract

In many black African populations, the capacity for CYP2D6-dependent drug metabolism is generally reduced. A specific variant of the CYP2D6 gene (CYP2D6*17) that carries three functional mutations (T107I, R296C, and S486T) has been found to be present in Zimbabwean subjects with impaired CYP2D6-dependent hydroxylase activity. To evaluate whether the CYP2D6*17 allele was the major cause behind the decreased rate of drug metabolism and to examine the role of the different mutations, CYP2D6 cDNAs containing all eight combinations of the mutations were created. Expression of the cDNAs in COS-1 cells revealed that the CYP2D6 17 enzyme displayed only 20% of the wild-type (CYP2D6 1) activity, whereas the T107I substitution on its own had no significant effect on enzyme function. Expression in yeast showed that the three possible single amino-acid mutant CYP2D6 variants all had properties similar to CYP2D6 1 when the kinetics of bufuralol hydroxylation was examined. However, enzymes containing both the T107I and R296C mutations exhibited a more than 5-fold higher K(m) for bufuralol than the wild-type enzyme, whereas the S486T mutation was of little importance. In contrast, when codeine was used as a substrate, the T107I substitution alone was sufficient to cause a significant increase in the apparent K(m), indicating a differential effect for this substitution depending on the CYP2D6 substrate. In conclusion, the CYP2D6*17 allele represents the first human cytochrome P450 polymorphic variant in which a combination of substitutions is required to alter the enzyme's catalytic properties and is the first case in which a decreased CYP2D6 activity, as monitored in vivo, has been documented to be caused by an enzyme with altered affinity for CYP2D6 substrates.

Identification of CYP4F8 in Human Seminal Vesicles as a Prominent 19-Hydroxylase of Prostaglandin Endoperoxides

“…U-44069 was converted to 19-and 18-hydroxy metabolites at a ratio of ϳ6:1, with a K m of ϳ7 M and a V max of ϳ260 pmol min Ϫ1 pmol P450 Ϫ1 . This V max value is remarkably high for a mammalian cytochrome P450 (20,22). 9,11-Diazo-15-deoxy-PGH 2 (U-51605), PGH 1 , PGH 2 , and 11,9-epoxymethano-PGH 2 (U-46619) were also good substrates, although there was a remarkable difference between U-44069 and U-46619 (Table I).…”

“…The cells were then diluted to ϳ2.5 ϫ 10 6 cells/ml and grown for 22-24 h in YPGE medium (containing per liter: yeast extract 10 g, bactopeptone 10 g, glucose 5 g, ethanol 16 g). Galactose (2%) was added, and the cells were harvested 16 h later by centrifugation (22). The cell walls were disrupted with glass beads, and the microsomal fraction was obtained by differential centrifugations at ϩ4°C (20,000 ϫ g for 10 min and 100,000 ϫ g for 60 min).…”

“…The pelleted microsomes were homogenized in 0.05 M Tris-HCl, 20% glycerol, and 1 mM EDTA (pH 7.4) and stored at Ϫ80°C. Cytochrome P450 and cytochrome P450 reductase were measured as described (22). Control microsomes were prepared from yeast transfected with the V60 plasmid without an insert.…”

“…Control microsomes were prepared from yeast transfected with the V60 plasmid without an insert. Protein was determined as described (22,23).…”

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

“…It encodes in addition to the two missense mutations seen in CYP2D6*2 also T107I, which apparently makes an altered active site structure. This creates an altered substrate specificity, 33 which is evident in vitro 33 but also when subjects of the CYP2D6*17 genotype are phenotyped in vivo. 34,35 In general, the activity of the CYP2D6.17 enzyme is lower than the wild-type enzyme.…”