Mechanism-based inactivation of cytochrome P450 2B1 by N-benzyl-1-aminobenzotriazole.
The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite.
Kent
U M
UM
Department of Pharmacology, The University of Michigan, Ann Arbor 48109, USA.
Bend
J R
JR
Chamberlin
B A
BA
Gage
D A
DA
Hollenberg
P F
PF
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007462:Iodobenzenes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008297:Male; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014230:Triazoles / Q000378:metabolism* / Q000633:toxicity*
D001059:Apoproteins; D003374:Coumarins; D007462:Iodobenzenes; D014230:Triazoles; C067013:N-benzyl-1-aminobenzotriazole; C056033:7-ethoxy-4-trifluoromethylcoumarin; D019362:Cytochrome P-450 CYP2B1; C037276:iodosobenzene