Differential effects of naturally occurring isothiocyanates on the activities of cytochrome P450 2E1 and the mutant P450 2E1 T303A

The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the ac tivity of a P450 2E1 mutant where the conserved threonine at position 303 w as replaced with an. alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K-I of 1.6 muM. PEITC also inactivated the wildtype P450 2E1 as efficiently with a K-I of 2.7 muM. The inactivati on was entirely dependent on NADPH and followed pseudo-first-order kinetics . Previously we reported the mechanism-based inactivation of wild-type P450 2E1 by BITC with a K-I of 13 muM. In contrast to the wild-type enzyme, the P450 2E1 T303A mutant was not inactivated by BITC but it was inhibited in a competitive manner with a K-i of 3 muM. The binding constants determined by spectral binding studies were similar for both enzymes. The binding of B ITC produced characteristic Type I spectral changes in the wild-type and mu tant enzyme. A radiolabeled BITC metabolite bound to P450 2E1 and to P450 2 E1 T303A when both enzymes were incubated with [C-14]BITC and NADPH. Whole protein electrospray ion trap mass spectrometry indicated that a mass consi stent with one molecule of benzylisocyanate and oxygen was adducted to the wild-type enzyme. The mass adducted to the T303A mutant was consistent with the addition of one hydroxylated BITC or of one benzylisocyanate moiety an d one sulfur molecule. Analysis of the metabolites of BITC indicated that e ach enzyme produced similar metabolites but that the mutant enzyme generate d significantly higher amounts of benzaldehyde and benzoic acid when compar ed to the wild-type enzyme. (C) 2001 Academic Press.