Interaction between Tetraethylthiuram Disulfide and the Sulfhydryl Groups of d-Amino Acid Oxidase and of Hemoglobin

From the ¹ From the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Inhibition of the crystallized hog kidney flavoenzyme, d-amino acid oxidase, by tetraethylthiuram disulfide (disulfiram, TETD) has been found to involve the sulfhydryldisulfide exchange reaction; primary products of the interaction between oxidase and reagent are diethyldithiocarbamate ion and a derived protein carrying mixed disulfide linkages, each of which consists of an enzyme thiol sulfur atom linked to a diethyldithiocarbamyl residue. Evidence for this conclusion includes the following observations. One mole (taken as 10⁵ g) of d-amino acid oxidase contains 6 to 8 "immediately reactive" sulfhydryl groups of a total of 12 such residues. The extent of inhibition of the oxidase is a linear function of the amount of TETD added; complete inhibition is attained with 6 to 8 mole eq of TETD. Studies with ³⁵S-TETD reveal, in association with the inhibition, the liberation of 6 to 8 mole eq of diethyldithiocarbamate ion and the fixation to oxidase protein of 6 diethyldithiocarbamyl residues. If the native enzyme is denatured with detergent, 12 mole eq of diethyldithiocarbamate ion are produced directly.

The disulfide character of TETD is requisite for its inhibitory action on d-amino acid oxidase. Related compounds without a disulfide bond, such as tetramethylthiuram monosulfide, do not inhibit the oxidase, whereas a variety of other disulfide reagents, including tetramethylthiuram disulfide, tetrathionate, oxidized glutathione, formamidine disulfide, and 5,5'-dithiobis-(2-nitrobenzoic acid) are inhibitors.

The stoichiometry of reaction between ³⁵S-TETD and human oxyhemoglobin, in both the native and the denatured state, was found to compare favorably with predictions based on the sulfhydryl character of the protein and a comparable sulfhydryl-disulfide exchange reaction.

The inhibition of d-amino acid oxidase by TETD, acting as a sulfhydryl-characterizing reagent, is time-dependent and irreversible; there is an associated conformational change of the oxidase protein. Flavin adenine dinucleotide retards the inhibitory process, whereas GSH prevents, but does not reverse, the inhibition. The over-all inhibitory process conforms to the criteria set forth earlier for the "selective inhibition" of polysulfhydryl enzymes by sulfhydryl-characterizing reagents.

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