Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Yun
C H
CH
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Hammons
G J
GJ
Jones
G
G
Martin
M V
MV
Hopkins
N E
NE
Alworth
W L
WL
Guengerich
F P
FP
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D001713:Biphenyl Compounds; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009281:Naphthalenes; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D010777:Photochemistry; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment
D000345:Affinity Labels; D001386:Azides; D001713:Biphenyl Compounds; D065607:Cytochrome P-450 Enzyme Inhibitors; D009281:Naphthalenes; C065012:2-ethynylnaphthalene; C077839:4-azido-1,1'-biphenyl; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2