Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4.

摘要: Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction their prosthetic heme to reactive fragments that irreversibly bind the protein. We have attempted characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver 3A4 as target CuOOH-mediated inactivation, and a battery protein characterization approaches [chemical (CNBr) proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, mass spectrometric analyses]. The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion CuOOH-inactivated pertain two distinct active site domains. One peptides corresponds proximal helix L/Cys-region peptide 429-450 domain others K-region (peptide 359-386 domain). Although precise residue(s) targeted remain be identified, we narrowed down region attack within 17 amino acid (429-445) stretch 55-amino L/Cys domain. Furthermore, although exact structures heme-modifying nature adduction established conclusively, incremental masses approximately 302 314 Da detected by electrospray analyses heme-modified are consistent with dipyrrolic fragment comprised either pyrrole ring A-D or B-C, known soluble product peroxidative degradation, modifying species.