REGENERATION OF 3-DISULFIDE MUTANTS OF BOVINE PANCREATIC RIBONUCLEASE-A MISSING THE 65-72-DISULFIDE BOND - CHARACTERIZATION OF A MINOR FOLDING PATHWAY OF RIBONUCLEASE-A AND KINETIC ROLES OF CYS65 AND CYS72

The oxidative regeneration pathways of two three-disulfide mutants of bovine pancreatic ribonuclease A (RNase A) missing the 65-72 disulfide bond, [C65S,C72S] and [C65A,C72A], have been studied by using oxidize d dithiothreitol (DTTOX) as an oxidizing agent and 2-aminoethylmethane -thiosulfonate thiosulfonate (AEMTS) as a thiol-blocking agent at 25 d egrees C and pH 8.0. These mutants are analogues of the des-[65-72] in termediate, which is one of the two major three-disulfide intermediate s that follow after the transition states in the regeneration pathways of wild-type RNase A [Rothwarf, D. M., Li, Y.-J., and Scheraga, H. A. (1998) Biochemistry 37, 3760-3766, 3767-3776.]. Both mutants folded t hrough the same pathway but at a rate lower than that of the wild-type protein. The major rate-determining step in the regeneration of these mutants was determined to be the oxidation from the two-disulfide int ermediates (2S) to the post-transition-state three-disulfide intermedi ate (3S), suggesting the existence of a minor oxidation pathway (2S - -> 3S, where 3S* is des-[65-72]) in the regeneration of the wild-type protein, in addition to one of the two major mutants disulfide-rearra ngement pathways (3S --> des-[65-72]). The regeneration intermediates of these mutants (R, 1S, 2S, and 3S) participate in a steady state wit h a kinetic behavior resembling that of the wild-type protein. However , the apparent equilibrium constants (K-eq(av)) in the steady state, a veraged with statistical factors for these mutants, are significantly smaller than those for the wild-type protein, indicating that the inte rmediates in the regeneration of the mutants are relatively less stabl e by 0.32 kcal/mol. This difference is due to the decrease in the aver age rate constants for intramolecular disulfide-bond formation (k(intr a)(av)) for the mutant proteins. Loop entropy calculations indicate th at the increase in the average length of all possible disulfide loops of the mutants due to the replacement of Cys65 and Cys72 is not suffic ient to account for the observed reduction of the values of k(intra)(a v), for the mutants. Therefore, it is the removal of energetic factors (arising from the loss of the 65-72 disulfide loop) that leads to dec eleration of the regeneration of the mutant proteins. The formation of the 65-72 disulfide loop in the regeneration of wild-type RNase A app ears to facilitate the subsequent folding events.