Abstract
The holoenzyme form ofRhodotorula gracilisd-amino acid oxidase, an 80-kDa homodimer, reacted only to a limited extent with general thiol reagents (2,2′-dithiodipyridine, 5,5′-dithiobis(2-nitrobenzoic acid), andN-[7-(dimethylamino)-4-methylcoumarinyl]maleimide) (60% residual activity), whereas the monomeric apoprotein was completely inactivated and denatured by these reagents. To investigate the presence of thiol residue(s) in the active site of the enzyme, the apoprotein was reconstituted with the 8-(methylsulfonyl)-FAD chemical-affinity probe. Competitive inhibition between this analogue and FAD for apoprotein binding was observed. The covalent attachment of the flavin analogue to the apoprotein was complete after ≈20 h of incubation and the flavinylated enzyme, containing 8-(cysteinyl)-FAD, was monomeric and inactive. After HPLC isolation of the flavin-labeled tryptic peptides, Cys208 was identified as the only cysteine to react with the FAD analogue. These results show that a single cysteine ofR. gracilisd-amino acid oxidase reacts with the flavin analogue and that this is located near or at the FAD-binding domain.
Published Version
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