Discrimination between native and non-native disulfides by protein-disulfide isomerase.
The folding assistant and chaperone protein-disulfide isomerase (PDI) catalyzes disulfide formation, reduction, and isomerization of misfolded proteins. PDI substrates are not restricted to misfolded proteins; PDI catalyzes the dithiothreitol (DTT)-dependent reduction of native ribonuclease A, microbial ribonuclease, and pancreatic trypsin inhibitor, suggesting that an ongoing surveillance by PDI can test even native disulfides for their ability to rearrange. The mechanism of reduction is consistent with an equilibrium unfolding of the substrate, attack by the nucleophilic cysteine of PDI followed by direct attack of DTT on a covalent intermediate between PDI and the substrate. For native proteins, the rate constants for PDI-catalyzed reduction correlate very well with the rate constants for uncatalyzed reduction by DTT. However, the rate is weakly correlated with disulfide stability, surface exposure, or local disorder in the crystal. Compared with native proteins, scrambled ribonuclease is a much better substrate for PDI than predicted from its reactivity with DTT; however, partially reduced bovine pancreatic trypsin inhibitor (des(14-38)) is not. An extensively unfolded polypeptide may be required by PDI to distinguish native from non-native disulfides.
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
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Gilbert
D000818:Animals; D007611:Aprotinin / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D004220:Disulfides; D007611:Aprotinin; D012259:Ribonuclease, Pancreatic; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D004229:Dithiothreitol