Thiol/disulfide redox equilibrium between glutathione and glycogen debranching enzyme (amylo-1,6-glucosidase/4-alpha-glucanotransferase) from rabbit muscle.
Rabbit skeletal muscle glycogen debranching enzyme is inactivated in a kinetically biphasic manner by GSSG at pH 8.0. The rapid phase results in the loss of 30% activity, while the slower phase leads to total enzyme inactivation. Both the glucosidase and the transferase activities of the enzyme are inhibited by GSSG. The inactivation by disulfides is fully and rapidly reversed in a biphasic manner by reduction with excess reduced dithiothreitol or GSH. After a fast initial recovery of 70% of the initial activity, the remaining 30% of the activity is recovered more slowly. Equilibration of the enzyme with a redox buffer of GSH and GSSG shows a monophasic equilibration of the activity. The ratio of GSH/GSSG where the enzyme is 50% active (R0.5) is 0.06 +/- 0.03. The R0.5 does not vary significantly with the total concentration of glutathione species suggesting formation of protein-SSG mixed disulfides. The ratios of the observed second-order rate constants for GSSG inactivation and GSH reactivation do not lead to a correct value of the observed thiol/disulfide oxidation equilibrium constant. Although the enzyme has sulfhydryl groups, the oxidation of which leads to activity changes, the kinetic and thermodynamic resistance to oxidation suggests that the enzyme is not likely to be subject to regulation by thiol/disulfide exchange in vivo.
D000818:Animals; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors*; D005978:Glutathione / Q000494:pharmacology*; D006004:Glycogen Debranching Enzyme System / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013816:Thermodynamics
D004220:Disulfides; D006004:Glycogen Debranching Enzyme System; D013438:Sulfhydryl Compounds; D005964:Glucosyltransferases; D005978:Glutathione; D004229:Dithiothreitol