Catalysis of the oxidative folding of bovine pancreatic ribonuclease A by protein disulfide isomerase.
The major oxidative folding pathways of bovine pancreatic ribonuclease A at pH 8.0 and 25 degrees C involve a pre-equilibrium steady state among ensembles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-disulfide ensemble to two native-like three-disulfide species, des-[65-72] and des-[40-95], that convert to the native structure during oxidative formation of the fourth disulfide bond. Under the same regeneration conditions, with oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 microM protein disulfide isomerase (PDI) was found to lead to catalysis of each disulfide-formation step, including the rate-limiting rearrangement steps in which the native-like intermediates des-[65-72] and des-[40-95] are formed. The changes in the distribution of intermediates were also determined in the presence and absence of PDI at three different temperatures (with the DTT redox system) as well as at 25 degrees C (with the glutathione redox system). The results indicate that the acceleration of the formation of native protein by PDI, which we observed earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism.
Shin
H C
HC
Baker Laboratory of Chemistry and Chemical Biology, Ithaca, NY, 14853-1301, USA.
Scheraga
H A
HA
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D012259:Ribonuclease, Pancreatic; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004229:Dithiothreitol