Kinetic analysis of the mechanism of insulin degradation by glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase).
Kinetic studies have been made with glutathione-insulin transhydrogenase, an enzyme which degrades insulin by promoting cleavage of its disulfide bonds via sulfhydryl-disulfide interchange. The degradation of 125I-labeled insulin by enzyme purified from beef pancreas was studied with various thiol-containing compounds as cosubstrates. The apparent Km for insulin was found to be a function of the type and concentration of thiol; values obtained were in the range from 1 to 40 muM. Lineweaver-Burk plots for insulin as varied substrate were linear, whereas those for the thiol substrates were nonlinears: the plots for low molecular weight monothiols (GSH and mercaptoethanol) were parabolic; those for low molecular weight dithiols (dithiothreitol, dihydrolipoic acid, and 2,3-dimercaptopropanol) were apparently linear modified by substrate inhibition; and the plots for protein polythiols (reduced insulin A and B chains and reduced ribonuclease) were parabolic with superposed substrate inhibition. The nonparallel nature of the reciprocal plots for all substrates shows that the enzyme does not follow a ping-pong mechanism. Product inhibition studies were performed with GSH as thiol substrate. Oxidized glutathione was found to be a linear competitive inhibitor vs. both GSH and insulin. The S-sulfonated derivative of insulin A chain was also linearly competitive vs. both substrates. Inhibition by S-sulfonated B chain was competitive vs. insulin; the data eliminated the possibility that this derivative was uncompetitive vs. GSH. Experiments with the cysteic acid derivatives of insulin A and B chains similarly excluded the possibility that these were uncompetitive vs. either substrate. These inhibition studies indicate that the enzyme probably follows a randdom mechanism.
D000818:Animals; D002417:Cattle; D007328:Insulin; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents
D007328:Insulin; D013439:Sulfhydryl Reagents; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione)