Chemical modification of D-amino acid oxidase. Amino acid sequence of the tryptic peptides containing tyrosine and lysine residues modified by fluorodinitrobenzene.
D-Amino acid oxidase can be inactivated by covalent modification of predominantly tyrosine residue(s) at pH 7.4 by a low molar excess of fluorodinitrobenzene, which appears to act as an active site-directed reagent (Nishino, T., Massey, V., and Williams, C. H., Jr. (1980) J. Biol. Chem. 255, 3610-3616). Peptide mapping by high performance liquid chromatography of tryptic digests of protein modified with radiolabeled reagent revealed two major radioactive fractions with substantially different retention times which were not observed in protein modified in the presence of benzoate, a potent competitive inhibitor. Isolation and sequence analysis of the major radiolabeled peptides, as well as other direct chemical analyses, are presented which unambiguously demonstrate that these fractions represent modification of two different regions of the protein. The majority of the radiolabel was found within a 61-amino acid residue peptide containing an O-DNP-tyrosine residue exclusively at position 17. The substantial sequence surrounding this tyrosine residue indicates that it is different from that shown to react with N-chloro-D-leucine (Ronchi, S., Galliano, M., Minchiotti, L., Curti, B., Rudie, N. G., Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 6044-6046). The second fraction consisted of a 12-residue peptide containing an epsilon-DNP-lysine residue at position 5. Together, these two modified amino acids represented 0.89 mol of DNP incorporated/protein monomer. Both modifications must contribute to inactivation to account for the 90% decrease in enzymatic activity. Evidence is presented which suggests that both groups are within the active center of the enzyme and are modified in a mutually exclusive manner.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D004139:Dinitrofluorobenzene / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008239:Lysine; D009578:Nitrobenzenes / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013552:Swine; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine
D009578:Nitrobenzenes; D010446:Peptide Fragments; D014443:Tyrosine; D004139:Dinitrofluorobenzene; D003605:D-Amino-Acid Oxidase; D014357:Trypsin; D008239:Lysine