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 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. Study holds ProTherm entries: 9467, 9468, 9469, 9470, 9471, 9472, 9473, 9474 Extra Details: bovine; hydrogen bond; thermal stability;,conformational stability; catalytic dyad

Submission Details

ID: Gsp8JkLP4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Quirk DJ;Park C;Thompson JE;Raines RT,Biochemistry (1998) His...Asp catalytic dyad of ribonuclease A: conformational stability of the wild-type, D121N, D121A, and H119A enzymes. PMID:9922164
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE