PDBsum entry: 1c8w

Hydrolase

PDB id: 1c8w

Contents

Protein chain

124 a.a. *

Ligands

ACT

Metals

_CL ×3

Waters ×115

* Residue conservation analysis

PDB id:

1c8w

Name:

Hydrolase

Title:

Thr45gly variant of ribonuclease a

Structure:

Protein (ribonuclease a). Chain: a. Fragment: rnase a. Engineered: yes. Mutation: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Organ: pancreas. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.176

Authors:

B.R.Kelemen,R.T.Sweeney,L.W.Schultz,R.T.Raines

Key ref:

B.R.Kelemen et al. (2000). Excavating an active site: the nucleobase specificity of ribonuclease A. Biochemistry, 39, 14487-14494. PubMed id: 11087402 DOI: 10.1021/bi001862f

Date:

30-Jul-99 Release date: 01-May-02

Protein chain

P61823  (RNAS1_BOVIN) -  Ribonuclease pancreatic

Seq: 150 a.a.

Struc: 124 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.27.5 - Pancreatic ribonuclease.
• Reaction: Endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotides ending in C-P or U-P with 2',3'-cyclic phosphate intermediates.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biochemical function: nucleic acid binding (6 terms)

DOI no: 10.1021/bi001862f

Biochemistry 39:14487-14494 (2000)

PubMed id: 11087402

Excavating an active site: the nucleobase specificity of ribonuclease A.

B.R.Kelemen, L.W.Schultz, R.Y.Sweeney, R.T.Raines.

ABSTRACT

Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine nucleotides. When bound in the active site, the base of a pyrimidine nucleotide forms hydrogen bonds with the side chain of Thr45. Here, the role of Thr45 was probed by using the wild-type enzyme, its T45G variant, X-ray diffraction analysis, and synthetic oligonucleotides as ligands and substrates. Catalytic specificity was determined with the fluorogenic substrate: 6-carboxyfluorescein approximately dArXdAdA approximately 6-carboxytetramethylrhodamine (6-FAM approximately dArXdAdA approximately 6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 10(6)-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM approximately d(CAA) is 50-fold greater than for 6-FAM approximately d(AAA). T45G RNase A cleaves 6-FAM approximately dArAdAdA approximately 6-TAMRA 10(2)-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-A resolution by X-ray diffraction analysis, does not reveal new potential interactions with a nucleobase. Indeed, the two enzymes have a similar affinity for 6-FAM approximately d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM approximately d(AU(F)AA), where U(F) is 2'-deoxy-2'-fluorouridine. The conformation of the pentofuranosyl ring in dU(F) is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM approximately d(AU(F)AA) is 50-fold lower than for 6-FAM approximately d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple roles-interacting favorably with pyrimidine nucleobases but unfavorably with purine nucleobases. Moreover, a ribose-like ring disfavors the interaction of Thr45 with a pyrimidine nucleobase, suggesting that Thr45 enhances catalysis by ground-state destabilization.