The homologous rat cytochrome P450s 2B1 and 2B2 differ by 13 amino acids. A chimeric construct of P450 2B1/2B2 was used in conjunction with several site-directed mutants to identify key residues involved in the inactivation of P450 2B1 by two acetylenic compounds, 17α-ethynylestradiol (17EE) and tertbutyl 1-methyl-2-propynyl ether (tBMP). 17EE is a mechanism-based inactivator of P450 2B1 but not of P450 2B2. We show here that tBMP is also a mechanism-based inactivator of P450 2B1 and not P450 2B2. Minimal loss in 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) activity was observed when P450 2B1 G478A was incubated with either inactivator, suggesting that this residue plays a role in the inactivation. However, P450 2B2 A478G behaved like wild-type P450 2B2, indicating that this residue alone is not sufficient for inactivation. A chimeric construct of P450 2B1/2B2 that is essentially P450 2B1 with five residues of P450 2B2 (including residue 478), was not inactivated by either tBMP or 17EE, suggesting that these five residues are important for inactivation. Sequential mutagenesis of the chimeric construct to quadruple (S407T-N417D-A419T-G478A) and triple (S407T-N417D-A419T) mutants of P450 2B1 did not result in inactivation by either inactivator. However, the triple mutant with mutations only in non-substrate recognition site (SRS) regions still exhibits wild-type P450 2B1 7-EFC O-deethylation activity with a Km value of 25 μM and Vmax of 8 nmol/min/nmol P450. These results demonstrate that substitution of three non-SRS residues in P450 2B1 leads to protection against inactivation of 2B enzymes by these two acetylenic compounds.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Oct 2004|