Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374.
We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.
Crespi
C L
CL
GENTEST Corporation, Woburn, MA 01801, USA.
Steimel
D T
DT
Penman
B W
BW
Korzekwa
K R
KR
Fernandez-Salguero
P
P
Buters
J T
JT
Gelboin
H V
HV
Gonzalez
F J
FJ
Idle
J R
JR
Daly
A K
AK
D001402:B-Lymphocytes; D001483:Base Sequence; D002460:Cell Line; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D003907:Dexamethasone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008715:Methionine; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014162:Transfection; D014633:Valine
D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D008715:Methionine; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; C043872:bufuralol 1'-hydroxylase; D014633:Valine