Catalysis of thiol/disulfide exchange: single-turnover reduction of protein disulfide-isomerase by glutathione and catalysis of peptide disulfide reduction.
Protein disulfide-isomerase, a protein localized to the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes the posttranslational formation and rearrangement of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl group per mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order in GSH concentration. The enzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of both substrates (Km for GSH much greater than 14 mM, Km for CYIQNC much greater than 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster than the corresponding uncatalyzed reactions.
Gilbert
H F
HF
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
D002384:Catalysis; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione