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 homog eneity by a combination of freeze/thawing, isoelectric precipitation a nd 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 stab ility. 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 analysi s 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 e nzymically active in their native form. The enzyme was inactivated by all sulfhydryl-modifying reagents tested. Inactivation by 5,5'-dithio- bis(-2-nitrobenzoate) was correlated with a modification of up to 2 mo l/mol protein of the six cysteine residues present in the monomer. Ide ntification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl-modifying reagent mono bromobimane. In the presence of a substrate amino acid, under anaerobi c conditions, the protein could be protected from modification and, th us, inactivation by this reagent. Peptide mapping by reverse-phase chr omatography of endoproteinase Glu-C-digested monobromobimane-labeled e nzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid un der anaerobic conditions. Isolation and sequencing of the labeled pept ide led to the identification of Cys298 as the reactive cysteine resid ue.