Characterization of fumarate reductase from baker's yeast: essential sulfhydryl group for binding of FAD.
Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr. The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M. The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation. These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule. The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity. Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.
D001051:Apoenzymes / Q000378:metabolism; D003545:Cysteine / Q000037:antagonists & inhibitors / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000378:metabolism*
D001051:Apoenzymes; D005182:Flavin-Adenine Dinucleotide; D004228:Dithionitrobenzoic Acid; D013385:Succinate Dehydrogenase; D003545:Cysteine