化学
代谢物
环氧化物
立体化学
电喷雾电离
呋喃香豆素
细胞色素P450
色谱法
甲氧沙林
质谱法
酶
有机化学
生物化学
医学
皮肤病科
银屑病
催化作用
作者
Luke L. Koenigs,William Trager
出处
期刊:Biochemistry
[American Chemical Society]
日期:1998-08-28
卷期号:37 (38): 13184-13193
被引量:105
摘要
Of several furanocoumarins [5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 5-hydroxypsoralen (5-OH-P), 8-hydroxypsoralen (8-OH-P), 4‘,5‘-dihydro-8-MOP (DH-8-MOP), and psoralen (P)] tested as mechanism-based inactivators (MBIs) of purified reconstituted cytochrome P450 (P450) 2B1, 8-MOP was found to be the most potent (KI, kinact, and partition ratio of 2.9 μM, 0.34 min-1, and 1.3, respectively). The inactivation was not prevented by reactive oxygen species scavengers or nucleophilic trapping agents and proceeded with a decrease in P450 spectral content. Liquid chromatography (LC) separation of the reconstituted enzyme mixture, followed by liquid scintillation counting, indicated that [14C]-8-MOP binding was specific to the apoprotein of P450 2B1 with a binding stoichiometry of 0.7:1. The major metabolites formed by P450 2B1 from the furanocoumarins that were MBIs were characterized by LC electrospray ionization tandem mass spectrometry (ESI-MS/MS) as dihydro diols. Results from H218O incorporation experiments supported initial oxidation of 8-MOP and P to an epoxide which can react with some nucleophilic active site residue and inactivate the enzyme or partition to a dihydro diol metabolite by hydrolytic ring opening. On the other hand, 5-MOP was converted to an epoxide or γ-keto enal intermediate prior to inactivation or dihydro diol formation. Comparison of the ESI mass spectra of P450 2B1 and furanocoumarin exposed P450 2B1, indicated a mass difference consistent with the covalent addition of a furanoepoxide to P450 2B1.
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