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.
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. phollen@umich.edu
Ute M
UM
Kent
Lise
L
Pascual
Rebecca A
RA
Roof
David P
DP
Ballou
Paul F
PF
Hollenberg
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