Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4.
Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fragments that irreversibly bind to the protein. We have attempted to characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches [chemical (CNBr) and proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, and mass spectrometric analyses]. The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains. One of the peptides isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain). Although the precise residue(s) targeted remain to be identified, we have narrowed down the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain. Furthermore, although the exact structures of the heme-modifying fragments and the nature of the adduction remain to be established conclusively, the incremental masses of approximately 302 and 314 Da detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species.
He
K
K
Department of Cellular and Molecular Pharmacology, Liver Center, University of California, San Francisco 94143, USA.
Bornheim
L M
LM
Falick
A M
AM
Maltby
D
D
Yin
H
H
Correia
M A
MA
D000595:Amino Acid Sequence; D001555:Benzene Derivatives / Q000494:pharmacology*; D001665:Binding Sites; D002250:Carbon Radioisotopes / Q000378:metabolism; D003488:Cyanogen Bromide; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D006868:Hydrolysis; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases
D001555:Benzene Derivatives; D002250:Carbon Radioisotopes; D065607:Cytochrome P-450 Enzyme Inhibitors; D016877:Oxidants; D010455:Peptides; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D003488:Cyanogen Bromide; C007164:cumene hydroperoxide