Mechanistic studies with N-benzyl-1-aminobenzotriazole-inactivated CYP2B1: differential effects on the metabolism of 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, and benzphetamine.
Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised. Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme. The reduced rate of reduction alone could not account for the loss in 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) O-deethylation or testosterone hydroxylation activity. Surprisingly, the ability of the BBT-modified enzyme to generate formaldehyde from benzphetamine was much less affected. Benzphetamine metabolite analysis by electrospray ionization-mass spectrometry showed that the BBT-modified enzyme had a slightly greater propensity towards aromatic hydroxylation together with reduced levels of N-demethylation and little change in the N-debenzylation of benzphetamine. Orientation of substrates within the active site of the BBT-inactivated enzyme may be affected such that the more flexible benzphetamine can be metabolized, whereas metabolism of rigid, planar molecules such as EFC and testosterone is hindered.
Kent
Ute M
UM
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. phollen@umich.edu
Pascual
Lise
L
Roof
Rebecca A
RA
Ballou
David P
DP
Hollenberg
Paul F
PF
D000693:Anaerobiosis; D000818:Animals; D001589:Benzphetamine / Q000737:chemistry / Q000378:metabolism*; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D020318:Rats, Long-Evans; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism*; D014230:Triazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D003374:Coumarins; D004791:Enzyme Inhibitors; D014230:Triazoles; D001589:Benzphetamine; C067013:N-benzyl-1-aminobenzotriazole; C056033:7-ethoxy-4-trifluoromethylcoumarin; D013739:Testosterone; D009249:NADP; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1; D010089:Oxidoreductases, N-Demethylating