title: Glutaredoxin accelerates glutathione-dependent folding of reduced ribonuclease A together with protein disulfide-isomerase.
abstract: Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 microM reduced and denatured ribonuclease RNase-(SH)8 in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S2)4, a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 microM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'0 of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO3(2-)), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.
authors.affiliation: Medical Nobel Institute for Biochemistry, Karolinska Institute, Stockholm, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Lundström-Ljung
authors.forename: A
authors.initials: A
authors.lastname: Holmgren
mesh_terms: D002384:Catalysis; D003538:Cystamine / Q000378:metabolism; D003543:Cysteamine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D011506:Proteins / Q000494:pharmacology*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D018038:Sodium Selenite / Q000378:metabolism
chemical_list: D054477:Glutaredoxins; D011506:Proteins; D003543:Cysteamine; D010088:Oxidoreductases; D012259:Ribonuclease, Pancreatic; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D018038:Sodium Selenite; D003538:Cystamine