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 ensem bles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-di sulfide ensemble to two native-like three-disulfide species, des-[65-72] an d des-[40-95], that convert to the native structure during oxidative format ion of the fourth disulfide bond. Under the same regeneration conditions, w ith oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 mu M protein disulfide isomeras e (PDI) was found to lead to catalysis of each disulfide-formation step, in cluding the rate-limiting rearrangement steps in which the native-like inte rmediates des-[65-72] and des-[40-95] are formed. The changes in the distri bution 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 PDT, which we o bserved earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism. (C) 2000 Academ ic Press.