Thiol-disulfide exchange between glutaredoxin and glutathione.
Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione or an intramolecular disulfide with the C-proximal cysteine. The C-proximal cysteine is not known to be involved in the catalytic mechanism. The stability of the mixed disulfide with glutathione has been investigated in detail using a mutant variant of yeast glutaredoxin 1, in which the C-proximal active site cysteine has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions and the protonation of glutathione thiolate. An algorithm for the analysis of this type of reaction by ITC was developed and showed that the interaction is enthalpy driven with a large entropy penalty. The applicability of the method was verified by a mass spectrometry-based approach, which gave a standard reduction potential of -295 mV for the mixed disulfide. In another set of experiments, the pK(a) value of the active site cysteine was determined. In line with what has been observed for other glutaredoxins, this cysteine was found to have a very low pK(a) value. The glutathionylation of glutaredoxin was shown to have a substantial effect on the thermal stability of the protein as revealed by differential scanning calorimetry.
Iversen
Rasmus
R
Department of Biology, University of Copenhagen, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
Andersen
Peter Anders
PA
Jensen
Kristine Steen
KS
Winther
Jakob R
JR
Sigurskjold
Bent W
BW
D002384:Catalysis; D004220:Disulfides / Q000737:chemistry*; D054477:Glutaredoxins / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D054477:Glutaredoxins; D013438:Sulfhydryl Compounds; D005978:Glutathione