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

doi:10.1016/j.abb.2004.03.014

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 journal › Article › peer-review

19 Scopus citations

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 language	English (US)
Pages (from-to)	95-105
Number of pages	11
Journal	Archives of Biochemistry and Biophysics
Volume	425
Issue number	1
DOIs	https://doi.org/10.1016/j.abb.2004.03.014
State	Published - May 1 2004
Externally published	Yes

Bibliographical note

Funding Information:
We thank Jamie Chun and Hsia-lien Lin for preparing purified P450 2B4 and reductase. We also thank Dr. Scott Wohler at the Biochemical Research NMR Core Facility at the University of Michigan for his help with performing and interpreting the NMR experiments. This work was supported in part by NIH Grants CA 16954 and GM 07767.

Keywords

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

Access

10.1016/j.abb.2004.03.014

OpenUrl availability

Full text

Cite this

@article{b6f82743571b4d419085c068b82af9c7,

title = "The mechanism-based inactivation of P450 2B4 by tert-butyl 1-methyl-2-propynyl ether: Structural determination of the adducts to the P450 heme",

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.",

keywords = "Cytochrome P450, Heme adduct, Mechanism-based inactivation, NMR spectroscopy, tert-Butyl 1-methyl-2-propynyl ether",

author = "{Von Weymarn}, {Linda B.} and Blobaum, {Anna L.} and Hollenberg, {Paul F.}",

note = "Funding Information: We thank Jamie Chun and Hsia-lien Lin for preparing purified P450 2B4 and reductase. We also thank Dr. Scott Wohler at the Biochemical Research NMR Core Facility at the University of Michigan for his help with performing and interpreting the NMR experiments. This work was supported in part by NIH Grants CA 16954 and GM 07767.",

year = "2004",

month = may,

day = "1",

doi = "10.1016/j.abb.2004.03.014",

language = "English (US)",

volume = "425",

pages = "95--105",

journal = "Archives of Biochemistry and Biophysics",

issn = "0003-9861",

publisher = "Academic Press Inc.",

number = "1",

}

TY - JOUR

T1 - The mechanism-based inactivation of P450 2B4 by tert-butyl 1-methyl-2-propynyl ether

T2 - Structural determination of the adducts to the P450 heme

AU - Von Weymarn, Linda B.

AU - Blobaum, Anna L.

AU - Hollenberg, Paul F.

N1 - Funding Information: We thank Jamie Chun and Hsia-lien Lin for preparing purified P450 2B4 and reductase. We also thank Dr. Scott Wohler at the Biochemical Research NMR Core Facility at the University of Michigan for his help with performing and interpreting the NMR experiments. This work was supported in part by NIH Grants CA 16954 and GM 07767.

PY - 2004/5/1

Y1 - 2004/5/1

N2 - 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.

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.

KW - Cytochrome P450

KW - Heme adduct

KW - Mechanism-based inactivation

KW - NMR spectroscopy

KW - tert-Butyl 1-methyl-2-propynyl ether

UR - http://www.scopus.com/inward/record.url?scp=1842736562&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842736562&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2004.03.014

DO - 10.1016/j.abb.2004.03.014

M3 - Article

C2 - 15081898

AN - SCOPUS:1842736562

SN - 0003-9861

VL - 425

SP - 95

EP - 105

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

IS - 1

ER -

The mechanism-based inactivation of P450 2B4 by tert-butyl 1-methyl-2-propynyl ether: Structural determination of the adducts to the P450 heme

Abstract

Bibliographical note

Keywords

Access

OpenUrl availability

Other files and links

Fingerprint

Cite this