His···Asp Catalytic Dyad of Ribonuclease A:  Conformational Stability of the Wild-Type, D121N, D121A, and H119A Enzymes

Abstract

Residue His119 acts as an acid/base during the cleavage/hydrolysis reactions catalyzed by bovine pancreatic ribonuclease A (RNase A). In the native enzyme, His119 forms a hydrogen bond with Asp121. This His...Asp dyad is conserved in all homologous pancreatic ribonucleases of known sequence. Yet, replacing Asp121 with an asparagine or alanine residue does not have a substantial effect on either structure or function [Schultz, L. W., Quirk, D. J., and Raines, R. T. (1998) Biochemistry 37, 8886-8898]. Here, the pH dependencies 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 deltadeltaG(o) = 2.0 kcal/mol, from a total of 9.0 kcal/mol. The magnitude of this loss is similar to that to transition-state binding during catalysis. As the pH decreases, the aspartate residue becomes protonated and deltadeltaG(o) decreases. D121N RNase A and D121A RNase A are approximately equivalent in conformational stability. This equivalence arises from compensating changes to enthalpy and entropy. A general analytical method was developed to determine the value of the pKa 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 pKa values of approximately 2.4 and 3.4 in the native and denatured states, respectively, of wild-type RNase A. This change in pKa can account fully for the differential effects of pH on the conformational stabilities of the wild-type and variant proteins. We conclude that the His...Asp catalytic dyad in pancreatic ribonucleases has two significant roles: (1) to position the proper tautomer of His119 for catalysis and (2) to enhance the conformational 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.

KFERQ Sequence in Ribonuclease A-mediated Cytotoxicity

“…Values of T m for RNase A, its variants, and ONC are listed in Table I. The T m values of both wild-type RNase A and G88R RNase A in PBS were determined to be 63°C, respectively; this value is similar to those reported previously (16,43). K7A RNase A, K7A/G88R RNase A, R10A RNase A, and R10A/G88R RNase A were found to have T m values of 63, 62, 60, and 62°C, respectively.…”

Complexity of Protein Energy Landscapes Studied by Solution NMR Relaxation Dispersion Experiments

“…12, 17 In addition, these studies show that the dynamics of cluster 2 are not pH dependent until a value of 4.5 is reached, at which there is a slight elevation in k ex at all 4 temperatures. This may suggest that the conserved residue D121 18, 56 plays a role in these motions, given its pK a ~ 3.0, 21 and possibly its interaction with H119 and the local water network. Changes in local water networks in lysozyme and RNase A were previously found to affect motion 57, 58 .…”

Point Mutations in Protein Globular Domains: Contributions from Function, Stability and Misfolding

“…On the other hand, it has been pointed out that some functional residues can contribute to both function and stability, giving a positive function-stability correlation. 6,26,29,30 Altogether, there is no consensus on the evolutionary relationship between function and stability.…”

“…Some functional sites have been reported to be suboptimal in terms of stability, [16][17][18][19][20][21][22][23][24] leading to the principle of functionstability trade-off. [16][17][18][19][20][21][22][23][24] Some sites contribute to both stability and function, 6,16,[25][26][27][28][29][30][31][32][33] which led to the opposing hypothesis of a coupling between stability and function. 6,26,29,30 Similarly, the evolutionary relationship between stability and misfolding is not clear.…”