title: Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide.
abstract: Thioredoxin from Escherichia coli was shown to catalyze the reduction of insulin disulfides by dithiothreitol. A quantitative assay was developed which measures the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the free insulin B chain. Thioredoxin, at 5 microM concentration, accelerated the reaction between 0.130 mM insulin and 1.0 mM dithiothreitol at pH 7 around 20-fold. The pH optimum of the reaction was 7.5. Thioredoxins from E. coli and calf liver showed similar specific activities. Stopped flow fluorescence measurements of the rate of reduction of thioredoxin-S2 by dithiothreitol showed a second order rate constant of 1647 M-1 s-1 at pH 7.2. This is between 10(2) to 10(3) times larger than the reaction between insulin or linear model disulfides and dithiothreitol. It is consistent with a ping-pong mechanism of thioredoxin catalysis since reduced thioredoxin is known to react very fast with insulin. Thioredoxin also catalyzed lipoamide-dependent reduction of the insulin disulfides in a coupled system with NADH, lipoamide, and lipoamide dehydrogenase. The fast spontaneous reaction between dihydrolipoamide and thioredoxin-S2 provides a mechanism for NADH or pyruvate-dependent disulfide reduction. The implication of the dithiol-disulfide oxidoreductase activity of thioredoxin for the regulation of enzyme activities by thiol oxidation-reduction control is discussed.
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol; D004926:Escherichia coli / Q000378:metabolism; D007328:Insulin; D007700:Kinetics; D010084:Oxidation-Reduction; D008063:Thioctic Acid / Q000031:analogs & derivatives*; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D007328:Insulin; D013879:Thioredoxins; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase; D004229:Dithiothreitol