The mechanism-based inactivation of p450 2B4 by tert-butyl 1-methyl-2-propynyl ether: structural determination of the adducts to the p450 heme |
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Authors: | von Weymarn Linda B Blobaum Anna L Hollenberg Paul F |
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Affiliation: | Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. |
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Abstract: | tert-Butyl 1-methyl-2-propynyl ether (tBMP) was analyzed for its ability to act as a mechanism-based inactivator of p450 2B4. tBMP inactivated p450 2B4 in a time-, concentration-, and NADPH-dependent manner. Losses in activity occurred with concurrent losses in the reduced CO spectrum and native p450 heme; however, there was a greater loss in activity than could be accounted for by reduced CO spectra or native heme loss. LC/MS analysis demonstrated that the losses in native heme were accompanied by the appearance of two modified hemes with m/z values of 705Da, consistent with tBMP adducted hemes. Both adducts had identical fragmentation patterns when analyzed by LC/MS/MS. The spectra were consistent with a tBMP molecule and an oxygen atom attached to iron-depleted heme. Proton NMR studies suggest that the two modified hemes in p450 2B1 are N-alkylated on pyrrole rings A and D. |
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Keywords: | Cytochrome P450 Mechanism-based inactivation tert-Butyl 1-methyl-2-propynyl ether Heme adduct NMR spectroscopy |
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