The mechanism-based inactivation of p450 2B4 by tert-butyl 1-methyl-2-propynyl ether: structural determination of the adducts to the p450 heme.
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.
von Weymarn
Linda B
LB
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA.
Blobaum
Anna L
AL
Hollenberg
Paul F
PF
D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D004987:Ethers / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011817:Rabbits
D065607:Cytochrome P-450 Enzyme Inhibitors; D004987:Ethers; D006418:Heme; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System