Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase.
At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.
Lyles
M M
MM
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Gilbert
H F
HF
D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction / Q000187:drug effects; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
D012259:Ribonuclease, Pancreatic; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D019803:Glutathione Disulfide