Covalent Modification of Thr302 in Cytochrome P450 2B1 by the Mechanism-Based Inactivator 4-tert-Butylphenylacetylene

Abstract

The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence 296 FFAGTSSTTLR 308 in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 Å, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s.

Identification of the Residue in Human CYP3A4 That Is Covalently Modified by Bergamottin and the Reactive Intermediate That Contributes to the Grapefruit Juice Effect

“…For example, the Thr302 residue modified in CYP2B1, CYP2B4, and CYP2B6 by tert-butylphenylacetylene is located in the I-helix and is only ϳ6 Å away from the heme iron Lin et al, 2010Lin et al, , 2011, whereas the residues of 3A4 modified by BG and raloxifene are all relatively far away from the heme iron. For mechanism-based inactivation of CYP3A4 by raloxifene, Tyr75 (ϳ25 Å away from heme iron) and Cys239 (ϳ21 Å away from heme iron) were identified as the two sites that are covalently modified by the diquinone methide intermediate of raloxifene (Pearson et al, 2007;Yukinaga et al, 2007).…”

“…The control and BG-inactivated CYP3A4 (500 pmol) were digested with 2 g of trypsin in 100 l of 50 mM ammonium bicarbonate buffer, pH 8.0, at 37°C for 18 h, as described previously (Lin et al, 2010). The digested peptides were centrifuged at 16,000g, and the clear supernatant was subjected to analysis using a C18 reversed-phase column (Jupiter, 5 m, 2.0 Ј 100 mm; Phenomenex).…”

“…The mass shift of 388 Da was used to identify the covalently modified peptide in the tryptic digest by using the SEQUEST database search (Lin et al, 2010). The modified peptide was then analyzed by LC-MS/MS to determine the identity of the DHBG-modified residue.…”

Thr302 Is the Site for the Covalent Modification of Human Cytochrome P450 2B6 Leading to Mechanism-Based Inactivation by tert-Butylphenylacetylene

“…BPA-inactivated CYP2B6 (500 pmol) was digested with 2 g of trypsin in 100 l of 50 mM ammonium bicarbonate buffer (pH 8.0) at 22°C for 18 h as described previously (Lin et al, 2010). The digested peptides were centrifuged at 16,000g, and the clear supernatant was subjected to analysis by mass spectrometry using a C18 reverse-phase column (Jupiter, 5 m, 2.0 ϫ 100 mm; Phenomenex).…”

“…In recent studies, a potent mechanism-based inactivator, 4-tert-butylphenylacetylene (BPA), for the inactivation of CYP2B1 and CYP2B4 has been characterized. We have had success in analyzing the BPA-inactivated apoprotein by LC-MS analysis (Lin et al, , 2010Zhang et al, 2009). Here, we used BPA as a probe to test whether it can also act a mechanism-based inactivator of CYP2B6 and, most importantly, whether we can identify the adducted apoprotein and the residue that is covalently modified by BPA using LC-MS/MS analysis.…”