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

Abstract

The mechanism-based inactivation of human CYP2B6 by tert-butylphenylacetylene (BPA) in the reconstituted system was investigated. The inactivation of CYP2B6 by BPA is time-, concentration-, and NADPH-dependent and exhibits a K(I) of 2.8 μM, a k(inact) of 0.7 min(-1), and a t(1/2) of 1 min. The partition ratio is ∼5. Unlike CYP2B1 and CYP2B4, in addition to the formation of an apoprotein adduct and a glutathione conjugate, a small heme adduct was observed when CYP2B6 was incubated with BPA. The mass increase of the adducted apoprotein and GSH conjugate is 174 Da, equivalent to the mass of one molecule of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B6 was digested with trypsin, and the digest was then analyzed by liquid chromatography-tandem mass spectrometry. A mass shift of 174 Da was used for the SEQUEST database search, and the identity of the modified residue was confirmed by MS/MS fragmentation of the modified peptide. Two residues, Lys274 and Thr302, were identified as having been modified. Further mutagenesis studies have demonstrated that the residue that is modified to result in inactivation is Thr302, not Lys274. Docking studies show that in the enzyme-substrate complex, Thr302 is in close contact with the triple bond of BPA with a distance of 3.8 Å between the terminal carbon of BPA and the oxygen in the hydroxyl group of Thr302. In conclusion, Thr302 of CYP2B6 is covalently modified by a reactive metabolite of BPA, and this modification is responsible for the mechanism-based inactivation.