INACTIVATION OF CYTOCHROME-P450 2E1 BY TERT-BUTYLISOTHIOCYANATE

Several naturally occurring and synthethic isothiocyanates were evalua ted for their ability to inactivate the major ethanol-inducible hepati c cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyana te (tBITC) was found to be the most selective inactivator of the 2E1 p -nitrophenol hydroxylation activity. tBITC was more specific for inact ivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacte rially expressed rabbit cytochrome, was inactivated by tBITC in a mech anism-based manner. The loss of activity followed pseudo-first-order k inetics and was NADPH- and tBITC-dependent. The maximal rates for inac tivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min(-1) and the apparent K-I values were 11 and 7.6 mu M, respectively. When cytochrome bg was co-reconstituted with P450 2E1, the apparent K-I for P450 2E1 inactivation by tBITC wa s similar to that seen in microsomes (14 mu M). P450 2E1 T(303)A was a lso inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Go-incubations with an alternate substrate protecte d P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactiva tion by tBITC resulted in a comparable loss of the ability of the enzy me to form a reduced CO complex.