Inactivation of Cytochrome P450 2B1 by Benzyl Isothiocyanate, a Chemopreventative Agent from Cruciferous Vegetables
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- 作者：Theunis C. Goosen ; Ute M. Kent ; Linda Brand ; and Paul F. Hollenberg
- 刊名：Chemical Research in Toxicology
- 出版年：2000
- 出版时间：December 2000
- 年：2000
- 卷：13
- 期：12
- 页码：1349 - 1359
- 全文大小：107K
- 年卷期：v.13,no.12(December 2000)
- ISSN：1520-5010
文摘
A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purifiedcytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethylisothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibitionof cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities.The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a time-and concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl andphenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitorsof P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized.The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivatedin a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-orderkinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (KI) was 5.8 
M, and the maximal rate constant for inactivation was0.66 min-1 at 23 
C. BITC was a very efficient inactivator of P450 2B1 with a partition ratioof approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was alsoinvestigated. More than 80% of the catalytic activity was lost within 12 min with a concomitantloss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude ofthe UV/visible absorption spectrum of the inactivated protein did not decrease significantly,and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC andprotein precipitation analyses indicated that the P450 apoprotein was covalently modified bya metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 ± 0.16mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggestingthe modification of a single amino acid residue per molecule of enzyme that was inactivated.The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1and that inactivation occurs primarily through protein modification.
M, and the maximal rate constant for inactivation was0.66 min-1 at 23 C. BITC was a very efficient inactivator of P450 2B1 with a partition ratioof approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was alsoinvestigated. More than 80% of the catalytic activity was lost within 12 min with a concomitantloss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude ofthe UV/visible absorption spectrum of the inactivated protein did not decrease significantly,and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC andprotein precipitation analyses indicated that the P450 apoprotein was covalently modified bya metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 ± 0.16mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggestingthe modification of a single amino acid residue per molecule of enzyme that was inactivated.The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1and that inactivation occurs primarily through protein modification.