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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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 705 Da, 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.

Section snippets

Materials

DLPC, NADPH, BSA, tBMP, deuterium oxide, d6-pyridine, and catalase were purchased from Sigma Chemical (St. Louis, MO). 7-EFC was purchased from Molecular Probes (Eugene, OR). TFA was obtained from Pierce (Rockford, IL).

Enzyme expression and purification

Rabbit P450 2B4 in the pLW01 expression vector (generously provided by Dr. Lucy Waskell, VA Medical Center, Ann Arbor, MI) was expressed in Escherichia coli C41(DE3) cells according to the method of Bridges et al. [23]. P450 2B4 was purified as described previously by Hanna et

Inactivation of P450 2B4 by tBMP

In a reconstituted system containing NADPH, tBMP caused a time- and concentration-dependent loss in P450 2B4-mediated 7-EFC O-deethylation activity (Fig. 1). The loss in 7-EFC O-deethylation activity was dependent on NADPH, indicating that catalytic activity is required for the inactivation of P450 2B4 by tBMP. The kinetic rate constants were determined from the slopes of the lines when the logarithm of the percent activity remaining (7-EFC O-deethylation) was plotted versus time (Fig. 1,

Discussion

A number of acetylenic compounds have been identified as mechanism-based inactivators of various P450 2B enzymes [7], [13], [15], [16], [31], [32], [33]. The mechanism, as well as the rate of inactivation, differs significantly among the P450s and between different inactivators. Small changes in the structure of the inactivators can have significant effects on the inactivation. tBMP and tert-butyl acetylene (tBA), two structurally similar acetylenic compounds, have been identified as

Acknowledgements

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.

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