Inactivation of cytochrome P450 2E1 by benzyl isothiocyanate

The cytochrome P450 enzymes constitute a family of phase I enzymes that pla y a prominent role in the metabolism of a great variety of endogenous and x enobiotic compounds. In this study, the kinetics for the inactivation of cy tochrome P450 2E1 by benzyl isothiocyanate (BITC) were elucidated. BITC is a naturally occurring compound found in cruciferous vegetables such as broc coli. BITC inhibited the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-dee thylation activity of purified and reconstituted P450 2E1 in a time- and co ncentration-dependent manner. The concentration of inactivator needed for h alf-maximal inactivation (K-I) was 13 mu M, and the maximum rate of inactiv ation at saturation (k(inact)) was 0.09 min(-1). The partition ratio for th e inactivation of P450 2E1 by BITC was found to have an approximate value o f 27. Inactivation of P450 2E1 by BITC was dependent on the presence of NAD PH. Following incubation for 5 min with BITC, a 65% loss in enzymatic activ ity was observed, while approximately 74% of the spectrally detectable enzy me remained. 7-Ethoxycoumarin (7-EC), a substrate of P450 2E1, protected P4 50 2E1 from BITC inactivation, reducing the loss in 7-EFC O-deethylation ac tivity from 50 to 18% when a 1:20 molar ratio of BITC:7-EC was used. Inacti vation of P450 2E1 by BITC was irreversible, and no activity was regained a fter extensive washes to remove BITC. Addition of cytochrome bs to the reco nstituted system did not affect the rate of inactivation. Reductase activit y was unaffected by BITC. The results reported here indicate that BITC is a mechanism-based inactivator of cytochrome P450 2E1 and that the inactivati on was primarily due to a modification of the apoprotein by BITC.