Ribonuclease 4

 

The RNase 4 family is unique among RNase enzymes, displaying the highest level of sequence similarity and encompassing the shortest polypeptide chain. It is the only one showing high specificity due to unique substrate interactions.

 

Reference Protein and Structure

Sequence
P34096 UniProt (3.1.27.-) IPR001427 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
2rnf - X-RAY CRYSTAL STRUCTURE OF HUMAN RIBONUCLEASE 4 IN COMPLEX WITH D(UP) (2.4 Å) PDBe PDBsum 2rnf
Catalytic CATH Domains
3.10.130.10 CATHdb (see all for 2rnf)
Click To Show Structure

Enzyme Reaction (EC:4.6.1.18)

messenger RNA
CHEBI:33699ChEBI
+
water
CHEBI:15377ChEBI
→
3'-end ribonucleotide(1-) residue
CHEBI:74896ChEBI
+
5'-end ribonucleotide(1-) residue
CHEBI:137923ChEBI
Alternative enzyme names: Ceratitis capitata alkaline ribonuclease, RNase, RNase A, RNase I, S-genotype-asssocd. glycoproteins, SLSG glycoproteins, Alkaline ribonuclease, Endoribonuclease I, Gene S glycoproteins, Gene S locus-specific glycoproteins, Pancreatic RNase, Ribonuclease, Ribonuclease I, Ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, Ribonucleic phosphatase,

Enzyme Mechanism

Introduction

Rnase 4 uses an extended binding site to increase its specificity for hydrolysis at the phosphate on the 3' side of a uridine nucleotide. Initially His12 deprotonates the uridine 2'OH group which then attacks the 3'phosphate group to form a penta-coordinated intermediate, stabilised by Lys40. Collapse of this high energy intermediate occurs at the same time as the 5'OH group of the following nucleotide accepts a proton from His116, resulting in lysis of the phosphodiester bond. This half of the chain then leaves the active site. The five membered ring formed by the previous steps is opened by activated water to reform the 3' phosphate group. His116 acts as the general base deprotonating water which then attacks the phosphate group to form the high energy penta-coordinate intermediate. His12 then acts as a general acid, donating a proton to the 2' position group as the intermediate collapses forming the 2'OH group and 3' phosphate group. The enzyme is thus returned to its initial protonation state and the cleaved RNA chain leaves the active site.

Catalytic Residues Roles

UniProt PDB* (2rnf)
Lys68 Lys40(41)A Electrostatic stabilisation of the penta-coordinate intermediate. electrostatic stabiliser
His40 His12(13)A Deprotonation of the 2'OH group causing nucleophilic attack. Protonation of the 2'OH group forming a good leaving group. proton acceptor, proton donor
His144 His116(117)A Protonation of the 5'OH group forming a good leaving group. Deprotonation of water causing nucleophilic attack. proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, intramolecular nucleophilic addition, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, bimolecular nucleophilic addition, native state of enzyme regenerated

References

  1. Terzyan SS et al. (1999), J Mol Biol, 285, 205-214. The three-dimensional structure of human RNase 4, unliganded and complexed with d(up), reveals the basis for its uridine selectivity. DOI:10.1006/jmbi.1998.2288. PMID:9878400.
  2. Liang S et al. (2016), FEBS J, 283, 912-928. Structural basis of substrate specificity in porcine RNase 4. DOI:10.1111/febs.13646. PMID:26748441.
  3. Hsu C et al. (2003), J Mol Biol, 326, 1189-1201. Solution Structure of the Cytotoxic RNase 4 from Oocytes of Bullfrog Rana catesbeiana. DOI:10.1016/S0022-2836(02)01472-9.
  4. Esposito L et al. (2000), J Mol Biol, 297, 713-732. The ultrahigh resolution crystal structure of ribonuclease A containing an isoaspartyl residue: hydration and sterochemical analysis. DOI:10.1006/jmbi.2000.3597. PMID:10731423.
  5. Shapiro R et al. (1989), Biochemistry, 28, 7401-7408. Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin. Alanine derivatives inhibit angiogenin-induced angiogenesis. DOI:10.1021/bi00444a038. PMID:2479414.

Step 1. His12 deprotonates the 2'OH which activates it to nucleophilically attack the 3' phosphate to form a penta-coordinated intermediate, stabilised by Lys40.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys40(41)A electrostatic stabiliser
His12(13)A proton acceptor

Chemical Components

proton transfer, ingold: intramolecular nucleophilic addition, intermediate formation, overall reactant used

Step 2. The simultaneous protonation of the 5' OH by His116 and the collapse of the oxyanion intermediate results in the cleavage of the phosphodiester bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys40(41)A electrostatic stabiliser
His116(117)A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

Step 3. His116 abstracts a proton from a water which activates it to attack the 3' phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys40(41)A electrostatic stabiliser
His116(117)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 4. His12 then acts as a general acid, donating a proton to the 2' position group as the intermediate collapses forming the 2'OH group and 3' phosphate group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys40(41)A electrostatic stabiliser
His12(13)A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. His12 deprotonates the 2'OH which activates it to nucleophilically attack the 3' phosphate to form a penta-coordinated intermediate, stabilised by Lys40.

Step 2. The simultaneous protonation of the 5' OH by His116 and the collapse of the oxyanion intermediate results in the cleavage of the phosphodiester bond.

Step 3. His116 abstracts a proton from a water which activates it to attack the 3' phosphate.

Step 4. His12 then acts as a general acid, donating a proton to the 2' position group as the intermediate collapses forming the 2'OH group and 3' phosphate group.

Products.

Contributors

Gemma L. Holliday, Charity Hornby