Identification of methionine-110 as the residue covalently modified in the electrophilic inactivation of D-amino-acid oxidase by O-(2,4-dinitrophenyl) hydroxylamine.

The reaction of O-(2,4-dinitrophenyl)hydroxylamine with D-amino-acid oxidase leads to complete inactivation which can be protected against by the competitive inhibitor benzoate [D'Silva, C., Williams, C. H., Jr., & Massey, V. (1986) Biochemistry 25, 5602-5608]. The residue modified has been identified as methionine-110. Differential high-performance liquid chromatography mapping of tryptic digests of D-amino-acid oxidase modified in the absence and presence of benzoate allows the isolation of a single methionine-containing tryptic peptide corresponding to residues 100-115 and referred to as T6-T7. In unmodified enzyme, the bond involving Arg-108 is readily cleaved and T6 and T7 are isolated. Brief treatment of peptide T6-T7 with carboxypeptidase Y released residues 112-115, and the residual peptide was isolated in good yield. Further treatment of this peptide (residues 100-111) with carboxypeptidase Y released Val and an unknown amino acid that comigrated with synthetically prepared S-aminomethionine sulfonium salt. The unknown compound and S-aminomethionine break down to methionine on treatment with dithiothreitol.