CYP2A13-catalysed coumarin metabolism: comparison with CYP2A5 and CYP2A6.
1. We investigated the total metabolism of coumarin by baculovirus (BV)-expressed CYP2A13 and compared it with metabolism by BV-expressed CYP2A6. The major coumarin metabolite formed by CYP2A13 was 7-hydroxycoumarin, which accounted for 43% of the total metabolism. The product of 3,4-epoxidation, o-hydroxyphenylacetaldehyde (o-HPA), accounted for 30% of the total metabolites. 2. The K(m) and V(max) for CYP2A13-mediated coumarin 7-hydroxylation were 0.48+/-0.07 micro m and 0.15+/-0.006 nmol min(-1) nmol(-1) CYP, respectively. The V(max) of coumarin 7-hydroxylation by CYP2A13 was about 16-fold lower than that of CYP2A6, whereas the K(m) was 10-fold lower. 3. In the mouse, there were two orthologues for CYP2A6: CYP2A4 and CYP2A5, which differed by only 11 amino acids. However, CYP2A5 is an efficient coumarin 7-hydroxylase, where as CYP2A4 is not. We report here that BV-expressed CYP2A4 metabolizes coumarin by 3,4-epoxidation. Two products of the 3,4-epoxidation pathway, o-HPA and o-hydroxyphenylacetic acid (o-HPAA), were detected by radioflow HPLC. 4. The K(m) and V(max) for the coumarin 3,4-epoxidation by CYP2A4 were 8.7+/-3.6 micro m and 0.20+/-0.04 nmol min(-1) nmol(-1) CYP, respectively. Coumarin 7-hydroxylation by CYP2A5 was more than 200 times more efficient than 3,4 epoxidation by CYP2A4.
von Weymarn
L B
LB
Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota Cancer Center, Mayo Mail Code 806, 420 Delaware Street SE., Minneapolis, MN 55455, USA.
Murphy
S E
SE
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; D004852:Epoxy Compounds / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism
D000925:Anticoagulants; D003374:Coumarins; D004852:Epoxy Compounds; C030123:coumarin; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; C585710:CYP2A6 protein, human; C460223:Cyp2a4 protein, mouse; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2