Glutathione disulfide inactivates, destabilizes, and enhances proteolytic susceptibility of fructose-1,6-bisphosphate aldolase.
Disulfides (glutathione disulfide, cystine, cystamine) caused a first-order inactivation of rabbit-muscle fructose-1,6-bisphosphate aldolase at pH values of 7.4 and above. Inactivation by glutathione disulfide was partially reversed by reducing agents, but the enzyme became irreversibly inactivated with time. The disulfide-inactivated aldolase had a lower transition temperature and enthalpy of denaturation than the native enzyme. In addition, the disulfide-inactivated enzyme was extensively degraded by proteinases, whereas the native enzyme was resistant. Mixed disulfides were formed; a maximum ratio of 4-5 mol of glutathione/mol of the aldolase tetramer was found. The number of titratable--SH groups on aldolase decreased by 16 (out of 32 total on the control enzyme) after inactivation by glutathione disulfide, indicating that other oxidation reactions in addition to those resulting in mixed disulfides occurred. The substrate, fructose 1,6-bisphosphate, prevented inactivation of aldolase by glutathione disulfide, the formation of glutathione-enzyme mixed disulfides, thermodynamic destabilization of the enzyme, and a decrease of--SH groups on the enzyme. These data indicate that covalent modification of aldolase by biological disulfides is important in modulating enzyme stability and vulnerability to proteinases as well as enzyme activity and that the substrate protects against modification by disulfides.
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002918:Chymotrypsin / Q000378:metabolism*; D004220:Disulfides / Q000032:analysis; D004226:Dithioerythritol / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019803:Glutathione Disulfide; D005980:Glutathione Reductase / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits
D004220:Disulfides; D010446:Peptide Fragments; D004226:Dithioerythritol; D004228:Dithionitrobenzoic Acid; D005980:Glutathione Reductase; D002918:Chymotrypsin; D005634:Fructose-Bisphosphate Aldolase; D005978:Glutathione; D019803:Glutathione Disulfide