Requirements for cytochrome b5 in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline, and N-nitrosodimethylamine by recombinant cytochrome P450 2E1 and by human liver microsomes

• Published in: Biochemical pharmacology 1996-07, Vol.52 (2), p.301-309
• Main Authors: Yamazaki, Hiroshi, Nakano, Masami, Gillam, Elizabeth M.J., Bell, L.Chastine, Guengerich, F.Peter, Shimada, Tsutomu
• Format: Article
• Language: English
• Subjects: 7-ethoxycoumarin; aniline; Aniline Compounds - metabolism; Biological and medical sciences; chlorzoxazone; Chlorzoxazone - metabolism; Coumarins - metabolism; cytochrome b5; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - metabolism; Cytochromes b5 - antagonists & inhibitors; Cytochromes b5 - metabolism; Dimethylnitrosamine - metabolism; electron transfer; Enzymes. Coenzymes. Vitamins. Pigments; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; Metabolisms and neurohumoral controls; Microsomes, Liver - metabolism; N-nitrosodimethylamine; NADH, NADPH Oxidoreductases - metabolism; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxidoreductases - metabolism; Oxidoreductases, N-Demethylating - antagonists & inhibitors; Oxidoreductases, N-Demethylating - metabolism; P450 2E1; Recombinant Proteins - metabolism; reconstitution; Vertebrates: anatomy and physiology, studies on body, several organs or systems
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(96)00208-0

NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b 5 ( b 5), NADH- b 5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH- b 5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (1̃.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b 5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. K m values were increased and V max values were decreased for 7-ethoxycoumarin O-deethylation when b 5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b 5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b 5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b 5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b 5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.