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

Abstract

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°C, corresponding to 185 U/mg polarographically at 37°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′‐dithio‐bis(‐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. Copyright © 1993, Wiley Blackwell. All rights reserved