Effect of mutation of proline 93 on redox unfolding/folding of bovine pancreatic ribonuclease A

Both the reductive unfolding and oxidative regeneration of a P93A mutant and wild-type RNase A have been studied at 15 degreesC and pH 8.0. The rate of reduction of the 40-95 disulfide bond is accelerated about 120-fold by the P93A mutation, while the reduction of the 65-72 disulfide bond is not accelerated by this mutation (within the experimental error). Moreover, the reduction of native P93A to des[40-95] is about 10 times faster than the further reduction of the same des[40-95] species. These results demonstrate that the reduction of the mutant proceeds through a local unfolding event and provides strong support for our model in which the reduction of wild-type RNase A to the des species proceeds through two independent local conformational unfolding events. The oxidative regeneration rate of the P93A mutant is comparable to that of wild-type RNase A, suggesting that a cis 92-93 peptide group that is present in native wildtype RNase A and in native des[40-95], is not obligatory for the formation of the third (final) native disulfide bond of des[40-95] by reshuffling from an unstructured 3S precursor. Thus, the trans to cis isomerization of the Tyr92-Pro93 peptide group during the regeneration of wild-type RNase A may occur after the formation of the third native disulfide bond.