ABSTRACT
Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.
Subject(s)
Cytochrome P-450 CYP2E1 Inhibitors , Cytochrome P-450 CYP2E1/chemistry , Enzyme Inhibitors/pharmacology , Isothiocyanates/pharmacology , Animals , Binding Sites/drug effects , Carbon Monoxide/metabolism , Chromatography, Liquid , Cytochrome P-450 CYP2E1/metabolism , Dithionite/pharmacology , Electron Spin Resonance Spectroscopy , Enzyme Activation/drug effects , Fomepizole , Heme/metabolism , Iodobenzenes/pharmacology , NADP/metabolism , Pyrazoles/metabolism , Rabbits , Rats , Spectrometry, Mass, Electrospray IonizationABSTRACT
The metabolism of genistein (4',5,7-trihydroxyisoflavone), a phytoestrogen derived from soy products, was investigated using rat and human liver microsomes and recombinant human cytochrome P450 enzymes. Metabolism of genistein by microsomes obtained from rats treated with pyridine, phenobarbital, beta-naphthoflavone, isosafrole, pregnenolone-16alpha-carbonitrile, or 3-methylcholanthrene resulted in very different product profiles consisting of five different NADPH- and time-dependent metabolites as observed by HPLC reverse-phase analysis at 260 nm. The metabolism of genistein was also investigated with recombinant human cytochrome P450 1A1, 1A2, 1B1, 2B6, 2C8, 2E1, or 3A4. P450s 1A1, 1A2, 1B1, and 2E1 metabolized genistein to form predominantly one product (peak 3) with smaller amounts of peaks 1 and 2. P450 3A4 produced two different products (peaks 4 and 5). Product peaks 1-3 eluted off the HPLC column prior to the parent compound genistein, and the UV/vis spectra, GC/MS, and ESI/MS/MS analyses support the conclusion that these products result from hydroxylation of genistein. The product peak 3 has been identified by tandem mass spectrometry as 3',4',5, 7-tetrahydroxyisoflavone, also known as orobol, and peaks 1 and 2 appear to be hydroxylated at position 6 or 8.
