Moreno R L, Kent U M, Hodge K, Hollenberg P F
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Chem Res Toxicol. 1999 Jul;12(7):582-7. doi: 10.1021/tx9900019.
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.
细胞色素P450酶是一类I相酶，在多种内源性和外源性化合物的代谢中发挥着重要作用。在本研究中，阐明了异硫氰酸苄酯（BITC）使细胞色素P450 2E1失活的动力学。BITC是一种天然存在的化合物，存在于西兰花等十字花科蔬菜中。BITC以时间和浓度依赖性方式抑制纯化和重组的P450 2E1的7-乙氧基-4-（三氟甲基）香豆素（7-EFC）O-脱乙基活性。半最大失活所需的失活剂浓度（K（I））为13μM，饱和时的最大失活速率（k（inact））为0.09 min-1。发现BITC使P450 2E1失活的分配比约为27。BITC使P450 2E1失活依赖于NADPH的存在。与BITC孵育5分钟后，观察到酶活性损失65%，而光谱可检测的酶约有74%保留。7-乙氧基香豆素（7-EC）是P450 2E1的底物，可保护P450 2E1免受BITC失活，当使用1:20摩尔比的BITC:7-EC时，7-EFC O-脱乙基活性的损失从50%降至18%。BITC使P450 2E1失活是不可逆的，在大量洗涤以去除BITC后，活性无法恢复。向重组系统中添加细胞色素b（5）不影响失活速率。还原酶活性不受BITC影响。此处报道的结果表明，BITC是细胞色素P450 2E1的一种基于机制的失活剂，失活主要是由于BITC对脱辅基蛋白的修饰。
