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.

Porcine FAD-containing monooxygenase metabolizes lidocaine, bupivacaine and propranolol in vitro

“…The enzymatic activity was determined optically and aerobically by measuring the decrease in absorbance at 340 nm due to the oxidation of NADPH in the presence of a substrate (Wu and Ichikawa, 1995). The reaction mixture comprised FMO (10 Ag protein), 0.1 M K-phosphate buffer, pH 8.0 at 25 jC, and appropriate amounts of NADPH and 0.1 mM to 1.4 mM substrate, in a final volume of 1 ml.…”