Inactivation of cytochrome P450 2E1 by benzyl isothiocyanate.
The cytochrome P450 enzymes constitute a family of phase I enzymes that play a prominent role in the metabolism of a great variety of endogenous and xenobiotic compounds. In this study, the kinetics for the inactivation of cytochrome P450 2E1 by benzyl isothiocyanate (BITC) were elucidated. BITC is a naturally occurring compound found in cruciferous vegetables such as broccoli. BITC inhibited the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified and reconstituted P450 2E1 in a time- and concentration-dependent manner. The concentration of inactivator needed for half-maximal inactivation (K(I)) was 13 microM, and the maximum rate of inactivation at saturation (k(inact)) was 0.09 min-1. The partition ratio for the inactivation of P450 2E1 by BITC was found to have an approximate value of 27. Inactivation of P450 2E1 by BITC was dependent on the presence of NADPH. Following incubation for 5 min with BITC, a 65% loss in enzymatic activity was observed, while approximately 74% of the spectrally detectable enzyme remained. 7-Ethoxycoumarin (7-EC), a substrate of P450 2E1, protected P450 2E1 from BITC inactivation, reducing the loss in 7-EFC O-deethylation activity from 50 to 18% when a 1:20 molar ratio of BITC:7-EC was used. Inactivation of P450 2E1 by BITC was irreversible, and no activity was regained after extensive washes to remove BITC. Addition of cytochrome b(5) to the reconstituted system did not affect the rate of inactivation. Reductase activity was unaffected by BITC. The results reported here indicate that BITC is a mechanism-based inactivator of cytochrome P450 2E1 and that the inactivation was primarily due to a modification of the apoprotein by BITC.
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
R L
RL
Moreno
U M
UM
Kent
K
K
Hodge
P F
PF
Hollenberg
D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry*; D015786:Cytochromes b5 / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006418:Heme / Q000032:analysis; D017879:Isothiocyanates / Q000737:chemistry*; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D011485:Protein Binding
D004791:Enzyme Inhibitors; D017879:Isothiocyanates; D006418:Heme; D009249:NADP; C031403:benzyl isothiocyanate; D015786:Cytochromes b5; D019392:Cytochrome P-450 CYP2E1