Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl

• Published in: Biochemistry (Easton) 1992-11, Vol.31 (43), p.10556-10563
• Main Authors: Yun, Chul Ho, Hammons, George J, Jones, Gina, Martin, Martha V, Hopkins, Nancy Eddy, Alworth, William L, Guengerich, F. Peter
• Format: Article
• Language: English
• Subjects: 2-ethynylnaphthalene; 4-azidobiphenyl; Affinity Labels; Amino Acid Sequence; Animals; Azides; Biphenyl Compounds; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; endoplasmic reticulum; Humans; man; Microsomes, Liver - enzymology; Molecular Sequence Data; Naphthalenes; Oxidoreductases - antagonists & inhibitors; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Peptide Mapping; photoaffinity labelling; Photochemistry; Rabbits; Rats; Sequence Alignment; utilization
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00158a019

2-Ethynylnaphthalene (2EN) had previously been demonstrated to be a mechanism-based inactivator of rat cytochrome P450 (P450) 1A2 [Hammons, G.J., Alworth, W.L., Hopkins, N.E., Guengerich, F. P., & Kadlubar, F. F. (1989) Chem. Res. Toxicol. 2, 367-374]. In this work 2EN was also demonstrated to be a useful inactivator of rabbit P450 1A2 (k(inactivation) 0.094 min-1, K(i) 11 microM) but it did not inactivate human P450 1A2, although the sequences of the three proteins are approximately 80% identical. Rat and rabbit P450 1A2 were modified by incubation with NADPH-P450 reductase, NADPH, and [3H]2EN to levels of 0.35 and 0.47 nmol of adduct (nmol of P450)-1, respectively. In each case only a single tryptic peptide was labeled; recovery of labeled peptides was low under the acidic HPLC conditions. The rabbit P450 1A2 peptide FQELMAAVGR (positions 175-184) and the rat P450 1A2 peptide L(S)QQYGDVLQIR (positions 67-78) were identified. 4-Azidobiphenyl (4-N3BP) was developed as a photoaffinity label for P-450 1A2 proteins because of its similarity to 4-aminobiphenyl, a known substrate for the enzymes. 4-N3BP was shown to be photolyzed with 350-nm light and radioactive label could be incorporated into rat P450 1A2. Labeling of the protein was found to be saturable with increasing concentrations of 4-N3BP and up to 0.59 nmol of label could be incorporated (nmol P450 1A2)-1. The substrate 4-aminobiphenyl and the competitive inhibitor 7,8-benzoflavone blocked photolabeling of P450 1A2 with 4-N3BP, and 4-N3BP inhibited N-hydroxylation of 4-aminobiphenyl by P450 1A2 in the usual enzyme assay.