Reconstitution of recombinant cytochrome P450 2C10(2C9) and comparison with cytochrome P450 3A4 and other forms: effects of cytochrome P450-P450 and cytochrome P450-b5 interactions.
Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.
Yamazaki
H
H
Osaka Prefectural Institute of Public Health, Japan.
Gillam
E M
EM
Dong
M S
MS
Johnson
W W
WW
Guengerich
F P
FP
Shimada
T
T
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002753:Chlorzoxazone / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D004983:Ethanolamines / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009154:Mutation; D009249:NADP / Q000378:metabolism; D009543:Nifedipine / Q000378:metabolism; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D013806:Theophylline / Q000378:metabolism; D014044:Tolbutamide / Q000378:metabolism; D014859:Warfarin / Q000378:metabolism
D004983:Ethanolamines; D010743:Phospholipids; D011994:Recombinant Proteins; D013739:Testosterone; D009249:NADP; D014859:Warfarin; C010831:bufuralol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D013806:Theophylline; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D002753:Chlorzoxazone; D009543:Nifedipine