Determination of the reduction-oxidation potential of the thioredoxin-like domains of protein disulfide-isomerase from the equilibrium with glutathione and thioredoxin.
Protein disulfide-isomerase (PDI) contains two thioredoxin-like domains with the active-site sequence: Cys-Gly-His-Cys. Reduction of the two active-site disulfides in PDI by NADPH and bovine thioredoxin reductase was not reversible by addition of excess NADP+, consistent with a redox potential (E0') above -200 mV. Redox states of PDI and a mutated Escherichia coli thioredoxin, P34H Trx, were determined by quantitative analysis of cysteine residues by alkylation in equilibrium mixtures of oxidized and reduced forms of the two proteins. From the known E0' of P34H Trx (-235 mV), an E0' value of -190 +/- 10 mV was calculated for PDI. Similarly, with defined redox buffers of glutathione, the redox-active dithiols in PDI were shown to have an equilibrium constant of 3 mM (E0' = -175 +/- 15 mV). The results showed that PDI has a high redox potential and therefore is a good oxidant of nascent protein thiols. Direct transfer of reducing equivalents from PDI to NADP+ via thioredoxin reductase during protein disulfide formation seems unlikely due to the unfavorable equilibrium. The thioredoxin domains in PDI have a widely different redox potential compared with that of thioredoxin. A Pro to His exchange in the active site contributes to half of the change; the other half remains to be identified in the structure of PDI.
Lundström
J
J
Department of Biochemistry, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Holmgren
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D019803:Glutathione Disulfide; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D019803:Glutathione Disulfide