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