Acid catalysis of the formation of the slow-folding species of RNase A: Evidence that the reaction is proline isomerization

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Abstract
Unfolded [bovine pancreatic] RNase A contains an equilibrium mixture of 2 forms, a slow-folding form (U1) and a fast-folding form (U2). If U1 is produced after unfolding by the slow cis-trans isomerization of proline residues about X-Pro imide bonds, then the formation of U1 should be catalyzed by strong acids. Therefore, the rate of formation of U1 was measured at different HClO4 concentrations. After rapid unfolding of the native protein in concentrated HClO4 at 0.degree., the slow formation of U1 was measured by use of refolding assays. Catalysis of its formation was found at HClO4 concentrations above 5 M. The uncatalyzed reaction follows apparent first-order kinetics but, in the acid-catalyzed range, 2 reactions are found. The faster reaction produces 2/3 of the slow-folding species and shows acid catalysis above 5 M HClO4. Catalysis of the slower reaction begins at 8 M HClO4. The faster reaction shows a 100-fold increase in rate of 10.6 M HClO4 over the rate of the uncatalyzed reaction at 5 M. The activation enthalpy of the uncatalyzed reaction was measured in 2 sets of unfolding conditions: .**GRAPHIC**. is 21.5 kcal/mol (1 kcal = 4.2 .times. 103 J) in 3.3 M HClO4 and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5. Both acid catalysis of the formation of U1 and its high activation enthalpy are consistent with the rate-limiting step being cis-trans isomerization either of X-Pro imide bonds or of peptide bonds. The rate of the uncatalyzed reaction is in the range expected for proline isomerization and is 0.1% of that of peptide bond isomerization; thus, the simplest explanation for the formation of U1 is proline isomerization. Earlier data, showing that the kinetic properties of the U1 .dblarw. U2 reaction in refolding conditions differ from those of proline isomerization, can be explained if there is kinetic coupling between early steps in the folding of U1 and its conversion to U2. The existence of 2 acid-catalyzed reactions that are distinguished by the HClO4 concentration at which catalysis begins suggests that at least 2 essential proline residues produce slow-folding species of RNase A by isomerization after unfolding. Because protonation of imide bonds is responsible for acid catalysis of proline isomerization, the slower reaction probably involves an imide bond with a low pK. It may be the bond connecting Lys-41 and Pro-42, because the positive charge on Lys-41 could make this bond more difficult to protonate.