T. Schrader et Jr. Andreesen, EVIDENCE FOR THE FUNCTIONAL IMPORTANCE OF CYS298 IN D-AMINO-ACID OXIDASE FROM TRIGONOPSIS-VARIABILIS, European journal of biochemistry, 218(2), 1993, pp. 735-744
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.