His - Asp catalytic dyad of ribonuclease A: Conformational stability of the wild-type, D121N, D121A, and H119A enzymes

Residue His119 acts as an acid/base during the cleavage/hydrolysis reaction s catalyzed by bovine pancreatic ribonuclease A (RNase A). In the native en zyme, His119 forms a hydrogen bond with Asp 121, This His...Asp dyad is con served in all homologous pancreatic ribonucleases of known sequence. Yet, r eplacing Asp121 with an asparagine or alanine residue does not have a subst antial effect on either structure or function [Schultz, L. W., Quirk, D, J. , and Raines, R. T. (1998) Biochemistry 37, 8886-8898], Here, the pH depend encies of the conformational stabilities of wild-type RNase A and the D121N , D121A, and H119A variants were determined by monitoring thermal stability over the pH range 1.2-6.0. Replacing Asp121 with an asparagine or alanine residue results in a loss of conformational stability at pH 6.0 of Delta De lta G degrees = 2.0 kcal/mol, from a total of 9.0 kcal/mol. The magnitude o f this loss is similar to that to transition-state binding during catalysis , As the pH decreases, the aspartate residue becomes protonated and Delta D elta G degrees decreases, D121N RNase A and D121A RNase A are approximately equivalent in conformational stability. This equivalence arises from compe nsating changes to enthalpy and entropy. A general analytical method was de veloped to determine the value of the pK(a) of a residue in the native and denatured states of a protein by comparing the pH-stability profile of the wild-type protein with that of a variant in which the ionizable residue is replaced with a nonionizable one. Accordingly, Asp121 was found to have pK( a) values of approximately 2.4 and 3.4 in the native and denatured states, respectively, of wild-type RNase A. This change in pK(a) can account fully fur the differential effects of pH on the conformational stabilities of the wild-type and variant proteins. We conclude that the His...Asp catalytic d yad in pancreatic ribonucleases has two significant roles: (1) to position the proper tautomer of His119 for catalysis and (2) to enhance the conforma tional stability of the native enzyme. Most enzymic residues contribute to catalysis or stability (or neither). Asp121 of RNase A is a rare example of a residue that contributes equally to both.