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.
Schräder
T
T
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Andreesen
J R
JR
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D007525:Isoelectric Focusing; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D005456:Fluorescent Dyes; D013439:Sulfhydryl Reagents; D003605:D-Amino-Acid Oxidase; D003545:Cysteine