title: Identification of methionine-110 as the residue covalently modified in the electrophilic inactivation of D-amino-acid oxidase by O-(2,4-dinitrophenyl) hydroxylamine.
abstract: 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.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors*; D006898:Hydroxylamines / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008715:Methionine; D010446:Peptide Fragments / Q000032:analysis; D013552:Swine
chemical_list: D000596:Amino Acids; D006898:Hydroxylamines; D010446:Peptide Fragments; D008715:Methionine; D003605:D-Amino-Acid Oxidase; C050904:O-(2,4-dinitrophenyl)hydroxylamine