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

AUTOR(ES)

Schmid, Franz X.

RESUMO

Unfolded RNase A is known to contain an equilibrium mixture of two 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 has been measured at different HClO4 concentrations. After rapid unfolding of the native protein in concentrated HClO4 at 0°, 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, two reactions are found. The faster reaction produces two-thirds 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 at 10.6 M HClO4 over the rate of the uncatalyzed reaction of 5 M. The activation enthalpy of the uncatalyzed reaction has been measured in two sets of unfolding conditions: ΔH‡ is 21.5 kcal/mol (1 kcal = 4.2 × 103 J) in 3.3 M HClO4 and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5.

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