Thioredoxin Catalyzes the Reduction of Insulin Disulfides by Dithiothreitol and Dihydrolipoamide

• When the two interchain disulfides of insulin are split by reduction, the free B chain will aggregate and precipitate from neutral solutions at low concentration. This well known phenomenon (14) was used in this study to develop a rapid and simple spectrophotometric assay for thiol-mediated protein disulfide reduction. The quantitative relationship between disulfide reduction and the onset of a rate of precipitation was calculated from extrapolation of the rate of NADPH oxidation in the presence of NADPH and thioredoxin reductase as the reducing system for thioredoxin. The two interchain disulfides of insulin are split by thioredoxin at similar rates (5). The K,,, value for insulin with thioredoxin is 11 pM (5). The concentration of insulin in the turbidimetric assay (130 pM) is thus sufficiently high to saturate the thioredoxin-(SH)2 reaction. The main result of this study is the demonstration of thioredoxin as a dithiol-disulfide oxidoreductase that catalyzes the reduction of protein disulfides by dithiothreitol or dihydrolipoamide. Chemically reduced ribonuclease has also been found to be a very efficient dithiol substrate for thioredoxin.’ This result is consistent with a general function of thioredoxin in catalyzing oxidoreduction between suitable dithiols and exposed protein disulfides. The enzymatic mechanism of thioredoxin involves the reduction and oxidation of an exposed active site disulfide bridge (1) with the structure: The disulfide in thioredoxin-S2 and the dithiol in thioredoxin-( SH)z both have unusual reactivities in thiol-disulfide interchange reactions. The second order rate constant for the reduction of thioredoxin by dithiothreitol obtained from the stopped flow fluorescence measurements is compared with measurements by Creighton for model disulfides (19) in Table V. Apparently, thioredoxin-Sz reacts between 2 to 3 orders of magnitude faster at physiological pH values than the model disulfides. Since thioredoxin-(SH)t shows an apparent reactivity with insulin that is around lo4 times higher at pH 7.0 than dithiothreitol (5), the combined effect of these two rates explains the catalytic action of thioredoxin in reducing insulin with dithiothreitol. Thioredoxin-Sz and thioredoxin-(SH)2 may thus be regarded as two species of an enzyme working in a ping-pong reaction. The local conformational change in thioredoxin upon oxidoreduction observed by tryptophan fluorescence (11) and nuclear magnetic resonance spectroscopy(20) explains the differences in reactivity of the two species of thioredoxin. Dihydrolipoamide was a very good dithiol substrate for thioredoxin. This provides a mechanism for NADH-dependent reduction of disulfides to sulfhydryl groups through the operation of lipoamide dehydrogenase. Roman0 and Nickerson (21) have described the existence of a specific NADHdependent cystine reductase (EC 1.6.4.1) or NADH-cystine transhydrogenase in yeast. The cystine reductase activity has not been purified and characterized but could be identical to the combined effect of NADH, lipoamide, lipoamide dehydrogenase, and thioredoxin. Eldjarn and Bremer (22) and Skrede (23) have studied disulfide reduction in mitochondria and shown that reduction of cystamine is NADH and lipoic acid-dependent and occurs by the lipoamide dehydrogenase enzyme in the cY-oxoacid complexes. The studies of Tietze (24) also suggests mitochondria as the place of NADH-dependent disulfide reduction since only NADPH was observed to support this process in 100,OOO X g supernatants of rat liver. Is there any physiological basis for the rapid reaction between dihydrolipoamide and thioredoxin-Sz? Some recent results regarding the functional organization of the pyruvate dehydrogenase complex in E. coli suggest a possible mechanism (25, 26). Lipoic acid is bound in an amide linkage to the dihydrolipoyl transacetylase component of the pyruvate and a-ketoglutarate dehydrogenase multienzyme complexes in E. coli and mitochondria. Studies on the site coupling in electron and acetyl group transfer in the E. coli pyruvate dehydrogenase complex has suggested (26) that only half of the a-lipoyl moieties are coupled to lipoamide dehydrogenase and NADH formation. The other half of the dihydrolipoamide groups may have a yet unidentified electron acceptor (26). Experiments with isolated E. coli pyruvate dehydrogenase complex should decide if this can be thioredoxin. Thioredoxin has been found in mitochondrial preparations from calf liver (27). The coupling between pyruvate decarboxylation and the reduction of thioredoxin may of course represent an unknown substrate cycle. This could operate in cells lacking thioredoxin reductase, as erythrocytes seem to do.3 It should also be noted that a novel role for lipoic acid in oxidative phosphorylation has been suggested recently (28). The fast reduction of thioredoxin-Sz by dihydrolipoamide observed in this study represents the only kinetically favorable reaction between any of the three compounds, lipoic acid, glutathione and thioredoxin. The reduction of the disulfides in these ubiquitous compounds is catalyzed by the three similar but specific dehydrogenases called NADH-lipoamide dehydrogenase, NADPH-glutathione reductase and NADPHthioredoxin reductase. The electrode potentials (E’o values), of the Lip&, GSSG, and thioredoxin-S2 couples are similar or -0.29, -0.25, and -0.26 V, respectively (3). The small molecules GSH and lipoic acid show no particularly rapid thiol-disulfide interchange rates (3). Thioredoxin-Sn is only very slowly reduced by the monothiol GSH (29) nor does thioredoxin-(SH)z react rapidly with GSSG (5). Thioredoxin was originally considered to be a substrate cofactor for the two enzymes thioredoxin reductase and ribonucleotide reductase (30). As shown here, it should rather be regarded as a small disulfide reductase enzyme. This applies for the thioredoxins from E. coli, yeast, and mammalian cells which are homologous proteins2 that all give high activity in the insulin turbidimetric assay.