Identification of active-site peptides from 3H-labeled 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 using amino acid sequencing and mass spectrometry.
2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of rat cytochrome P450 (P450) 2B1 with 1.3 mol of adduct bound per mole of P450 inactivated [Roberts, E.S., Hopkins, N.E., Alworth, W.L., & Hollenberg, P.F. (1993) Chem. Res. Toxicol. 6, 470-479]. Further studies have shown that 2EN is also an efficient mechanism-based inactivator of the 7-ethoxycoumarin O-deethylase activity of rabbit P450 2B4 with 0.83 mol of adduct bound per mole of P450. Cleavage of [3H]2EN-inactivated 2B1 with cyanogen bromide, separation of the peptides by HPLC, and further purification of the radiolabeled fraction by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) led to the identification by autoradiography of a radiolabeled peptide (M(r) approximately 3000). Amino acid sequence analysis of the first 12 N-terminal residues revealed the sequence ISLLSLFFAGTE corresponding to positions 290-301 in the protein. When the radiolabeled fraction from the HPLC separation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), peaks at m/z 2722.5 and 2890.6 were detected. The lower mass peak corresponds to the molecular ion (average mass) of the cyanogen bromide peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the same peptide with a bound 2-naphthylacetyl group (theoretical 2890.4). When [3H]2EN-inactivated 2B4 was treated with cyanogen bromide, the peptides were separated by HPLC, and the fractions were analyzed by Tricine-SDS-PAGE, two radiolabeled peptides (M(r) = 5000 and 8000) were identified by autoradiography. Amino acid sequence analysis of the first 11 residues revealed identical N-termini with the sequence EKDKSDPSSEF corresponding to positions 273-283.(ABSTRACT TRUNCATED AT 250 WORDS)
Roberts
E S
ES
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Hopkins
N E
NE
Zaluzec
E J
EJ
Gage
D A
DA
Alworth
W L
WL
Hollenberg
P F
PF
D015660:7-Alkoxycoumarin O-Dealkylase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017421:Sequence Analysis; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D014316:Tritium
D065607:Cytochrome P-450 Enzyme Inhibitors; D009281:Naphthalenes; D010446:Peptide Fragments; D014316:Tritium; C065012:2-ethynylnaphthalene; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D015660:7-Alkoxycoumarin O-Dealkylase; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D003488:Cyanogen Bromide