Evidence for the functional importance of Cys298 in D-amino acid oxidase from Trigonopsis variabilis.

D-Amino acid oxidase from Trigonopsis variabilis was purified to homogeneity by a combination of freeze/thawing, isoelectric precipitation and chromatography on Mono Q. This purification procedure required very little working effort. The homogeneous enzyme exhibited a ratio A280/A450 of about 6.5 and was obtained in high yield (63%) and a good stability. Using D-methionine as a substrate, a specific activity of 120 U/mg was determined colorimetrically at 26 degrees C, corresponding to 185 U/mg polarographically at 37 degrees C. Polyclonal antibodies were raised against the homogeneous protein and Western immunoblot analysis showed that the 39-kDa subunit can undergo defined cleavages at the carboxy terminus of amino acid positions 104, 106 and 108, leading to 27-kDa and 12-kDa fragments as revealed by SDS/PAGE, which are still enzymically active in their native form. The enzyme was inactivated by all sulfhydryl-modifying reagents tested. Inactivation by 5,5'-dithiobis(-2-nitrobenzoate) was correlated with a modification of up to 2 mol/mol protein of the six cysteine residues present in the monomer. Identification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl-modifying reagent monobromobimane. In the presence of a substrate amino acid, under anaerobic conditions, the protein could be protected from modification and, thus, inactivation by this reagent. Peptide mapping by reverse-phase chromatography of endoproteinase Glu-C-digested monobromobimane-labeled enzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid under anaerobic conditions. Isolation and sequencing of the labeled peptide led to the identification of Cys298 as the reactive cysteine residue.