Contribution of active site residues to the activity and thermal stability of ribonuclease Sa.


Abstract

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability. Study holds ProTherm entries: 16727, 16728, 16729, 16730, 16731, 16732, 16733, 16734, 16735, 16736, 16737, 16738, 16739, 16740, 16741, 16742, 16743, 16744, 16745, 16746, 16747, 16748, 16749, 16750, 16751, 16752 Extra Details: Ribonuclease Sa; active-site mutants; catalytic activity; specificity; thermal stability

Submission Details

ID: 9GEDLzqe3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Yakovlev GI;Mitkevich VA;Shaw KL;Trevino S;Newsom S;Pace CN;Makarov AA,Protein Sci. (2003) Contribution of active site residues to the activity and thermal stability of ribonuclease Sa. PMID:14500895
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 PDB Entries

Structure ID Release Date Resolution Structure Title
1C54 2001-11-28 SOLUTION STRUCTURE OF RIBONUCLEASE SA
1T2H 2004-12-21 1.0 Y81W mutant of RNase Sa from Streptomyces aureofaciens
1LNI 2002-07-31 1.0 CRYSTAL STRUCTURE ANALYSIS OF A RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS AT ATOMIC RESOLUTION (1.0 A)
4GHO 2013-08-14 1.1 Crystal Structure Analysis of Streptomyces aureofaciens Ribonuclease S24A mutant
1T2I 2004-12-21 1.1 T76W mutant of RNase Sa from Streptomyces aureofaciens
1ZGX 2006-08-08 1.13 Crystal structure of ribonuclease mutant
1RGE 1996-10-14 1.15 HYDROLASE, GUANYLORIBONUCLEASE
1RGH 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
1RGF 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
1YNV 2005-07-19 1.2 Asp79 makes a large, unfavorable contribution to the stability of RNase Sa
1RGG 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
4J5K 2014-05-28 1.23 Crystal structure analysis of Streptomyces aureofaciens ribonuclease Sa Y51F mutant
1I8V 2001-09-19 1.25 CRYSTAL STRUCTURE OF RNASE SA Y80F MUTANT
4J5G 2014-05-28 1.31 Crystal structure analysis of Streptomyces aureofaciens ribonuclease Sa T95A mutant
1BOX 1999-12-29 1.6 N39S MUTANT OF RNASE SA FROM STREPTOMYCES AUREOFACIENS
3A5E 2010-08-04 1.6 Crystal structure of 5K RNase Sa
1GMP 1993-10-31 1.7 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1AY7 1999-03-02 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
1I70 2001-09-19 1.7 CRYSTAL STRUCTURE OF RNASE SA Y86F MUTANT
1GMR 1993-10-31 1.77 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1UCK 2003-09-09 1.8 Mutants of RNase Sa
1SAR 1992-04-15 1.8 DETERMINATION AND RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURES OF RIBONUCLEASE SA AND ITS COMPLEX WITH 3'-GUANYLIC ACID AT 1.8 ANGSTROMS RESOLUTION
1GMQ 1993-10-31 1.8 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
2SAR 1992-04-15 1.8 DETERMINATION AND RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURES OF RIBONUCLEASE SA AND ITS COMPLEX WITH 3'-GUANYLIC ACID AT 1.8 ANGSTROMS RESOLUTION
1UCI 2003-09-09 1.8 Mutants of RNase Sa
1UCJ 2003-09-09 1.81 Mutants of RNase Sa
1UCL 2003-09-09 1.82 Mutants of RNase Sa
1RSN 1995-12-07 2.0 RIBONUCLEASE (RNASE SA) (E.C.3.1.4.8) COMPLEXED WITH EXO-2',3'-CYCLOPHOSPHOROTHIOATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Guanyl-specific ribonuclease Sa P05798 RNSA_KITAU