On the Reaction Mechanism of Yeast Glutathione Reductase

Abstract

Glutathione reductase has been purified from a number of sources, Escherichiu coli (l), yeast (2, 3), peas (4), rat tissues (5, 6), and erythrocytes (7). In all of the cases, the enzyme appears to be a flavoprotein, containing flavin adenine dinucleotide as prosthetic group (8). In view of the fact that the over-all reaction catalyzed by the enzyme, GSSG + TPNH + H+ & 2 GSH + TPN+, is formally so similar to that catalyzed by lipoyl dehydrogenase, another flavoprotein enzyme, oxidized lipoate (-S-S-) + DPNH + H+ F! reduced lipoate ((-SH),) + DPN+, it is of obvious interest to determine whether the two enzymes have similar reaction mechanisms. In the case of pig heart lipoyl dehydrogenase, it has been found that a protein disulfide linkage (9-11) is concerned in catalysis, in close concert with the FAD prosthetic group. The anaerobic addition of reducing substrate (either DPNH or dihydrolipoyl derivatives) results in the formation of a stable 2-electron reduction intermediate which has been shown to be extremely important in catalysis (12). This intermediate appears to be a complex between the enzyme flavin semiquinone and a sulfur radical formed from the active center disulfide (13). The intermediate has a very characteristic absorption spectrum, quite different from that found with half-reduced forms of other flavoproteins. Our interest in the possible similarity of lipoyl dehydrogenase and glutathione reductase was considerably aroused by the published spectra of yeast glutathione reductase (3) which showed characteristics very similar to those found with lipoyl dehydrogenase. It was suggested therefore that the two enzymes may have very similar reaction mechanisms (14), i.e. that in glutathione reductase a protein disulfide active center might also be operative. This suggestion appeared to have been discounted by later results of Colman and Black (15) who showed by experiments with W-labeled N-ethylmaleimide that only 1 sulfhydryl group was liberated on treatment of the enzyme with TPNH. In this paper, we wish to report on some studies with glutathione reductase which indicate that this enzyme does in fact possess a basically identical reaction mechanism to that of lipoyl dehydrogenase