Characterization of the active site cysteine residues of the thioredoxin-like domains of protein disulfide isomerase.
The dithiol/disulfide active sites of each of the two isolated thioredoxin-like domains of protein disulfide isomerase (PDI) expressed in Escherichia coli have been characterized in order to understand their catalytic mechanisms and their functions in PDI. In each of the folded domains, as in other proteins of the thioredoxin family, only one of the cysteine residues of the active site sequence -Cys-Gly-His-Cys- is accessible, and its thiol group is highly reactive and has a low pKa value. The kinetics and equilibria have been measured of the reactions between the active site cysteine residues and glutathione, the predominant thiol/disulfide reagent of the endoplasmic reticulum. A disulfide bond can be formed very rapidly between the pair of cysteine residues of each domain, but each disulfide bond is very unstable and reacts rapidly with reduced glutathione. The very low stabilities of these disulfide bonds, which destabilize the protein structures, account for the efficiency with which PDI and each of the isolated domains can introduce disulfide bonds into proteins. These kinetics and equilibrium data go far in helping to understand the catalytic mechanism of PDI and its individual domains.
European Molecular Biology Laboratory, Heidelberg, Germany.
N J
NJ
Darby
T E
TE
Creighton
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003538:Cystamine / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008623:Mercaptoethanol / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry
D004220:Disulfides; D011994:Recombinant Proteins; D013879:Thioredoxins; D008623:Mercaptoethanol; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D003538:Cystamine