title: An essential lysyl residue (Lys208) in the substrate-binding site of porcine FAD-containing monooxygenase.
abstract: The substrate (amine)-binding site of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5'-phosphate (pyridoxal-P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal-P. Upon reduction of pyridoxal-P-treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal-P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal-P-treated FMOs indicated that the lysyl residues were modified by pyridoxal-P. The about 74% inactivation of the enzymic activity on covalent pyridoxal-P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N-dimethylaniline. The FMO covalently modified with pyridoxal-P in the presence or absence of N,N-dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse-phase high-performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal-P and was detected at 325 nm in the absence of N,N-dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186-208. These results suggest that Lys208 plays an important role in the substrate (amine)-binding site of FMO.
authors.affiliation: Department of Biochemistry, Kagawa Medical School, Japan.
authors.forename: R F
authors.initials: RF
authors.lastname: Wu
authors.forename: Y
authors.initials: Y
authors.lastname: Ichikawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D005260:Female; D008239:Lysine / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
chemical_list: D010446:Peptide Fragments; D009249:NADP; D011732:Pyridoxal Phosphate; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D008239:Lysine