The mechanism-based inactivation of P450 2B4 by tert-butyl 1-methyl-2-propynyl ether

Structural determination of the adducts to the P450 heme

Linda B von Weymarn, Anna L. Blobaum, Paul F. Hollenberg

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)95-105
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume425
Issue number1
DOIs
StatePublished - May 1 2004
Externally publishedYes

Fingerprint

Heme
Carbon Monoxide
Pyrroles
tert-butyl 1-methyl-2-propynyl ether
NADP
Protons
Iron
Nuclear magnetic resonance
Oxygen
Atoms
Molecules

Keywords

  • Cytochrome P450
  • Heme adduct
  • Mechanism-based inactivation
  • NMR spectroscopy
  • tert-Butyl 1-methyl-2-propynyl ether

Cite this

The mechanism-based inactivation of P450 2B4 by tert-butyl 1-methyl-2-propynyl ether : Structural determination of the adducts to the P450 heme. / von Weymarn, Linda B; Blobaum, Anna L.; Hollenberg, Paul F.

In: Archives of Biochemistry and Biophysics, Vol. 425, No. 1, 01.05.2004, p. 95-105.

Research output: Contribution to journalArticle

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AB - 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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