Guanyl-specific ribonuclease Sa
Three different strains of Streptomyces aureofaciens produce the homologous ribonucleases Sa, Sa2 and Sa3. They are all guanylate endoribonucleases, highly specifically hydrolysing the phosphodiester bonds of RNA at the 3'-side of guanosine nucleotides. These enzymes belong to the prokaryotic subgroup of microbial ribonucleases, and are some of the smallest enzymes known.
Reference Protein and Structure
- Sequence
-
Q53752
(3.1.4.8)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Streptomyces aureofaciens (Bacteria)
- PDB
-
1pyl
- Crystal structure of Ribonuclease Sa2
(1.507 Å)
- Catalytic CATH Domains
-
3.10.450.30
(see all for 1pyl)
Enzyme Reaction (EC:3.1.27.3)
+
+
→
+
Alternative enzyme names: Aspergillus oryzae ribonuclease, RNase F(1), RNase G, RNase N(1), RNase N(2), RNase Sa, RNase T(1), Binase, Guanyl-specific RNase, Guanyloribonuclease, Ribonuclease C2, Ribonuclease Ch, Ribonuclease F(1), Ribonuclease N(1), Ribonuclease N(3), Ribonuclease PP1, Ribonuclease SA, Ribonuclease U(1), Ribonuclease guaninenucleotido-2'-transferase (cyclizing), RNase N1, Ribonuclease T1, RNase N2,
Enzyme Mechanism
Introduction
The cleavage of substrate by ribonucleases is a two-step reaction: the first step is a transesterification of which a nucleophilic displacement at the phosphorus atom of the 5' leaving group by the 2' entering oxygen atom takes place, forming a guanosine 2',3'-cyclophosphate intermediate, which is hydrolysed in the second step to yield 3' guanylic acid. The mechanism proceeds as follows:
- The 2' hydroxyl group of the substrate is made more nucleophilic by deprotonation by Glu 56.
- The 2' hydroxyl attacks the phosphorus attached to the 3' carbon of the same ribose ring, displacing the 5' oxygen of the next nucleotide. The charge on the trigonal bipyramidal transition state is stabilised by Arg 67 and Arg 71.
- The 5' oxygen of the next nucleotide is made a better leaving group by protonation by His 86.
Catalytic Residues Roles
| UniProt | PDB* (1pyl) | ||
| Glu122 | Glu56A | Deprotonates the substrate 2' hydroxyl nucleophile in the first step, and reprotonates the 2' hydroxyl leaving group in the second step of the reaction. | proton shuttle (general acid/base) |
| Arg137, Arg133 | Arg71A, Arg67A | Polarises the bonds between the oxygens of the phosphate group and phosphorus atom, causing electron deficiency on the phosphorus atom and, consequently, enhancing formation of the cyclophosphate intermediate. Also helps to stabilise the negatively charged, pentacovalent phosphate transition state. | activator, transition state stabiliser |
| His152 | His86A | Protonates the 5' leaving group in the first step and deprotonates water in the second step of the reaction. | proton shuttle (general acid/base) |
*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
References
- Yakovlev GI et al. (2003), Protein Sci, 12, 2367-2373. Contribution of active site residues to the activity and thermal stability of ribonuclease Sa. DOI:10.1110/ps.03176803. PMID:14500895.
- Bauerová-Hlinková V et al. (2009), FEBS J, 276, 4156-4168. Structure of RNase Sa2 complexes with mononucleotides - new aspects of catalytic reaction and substrate recognition. DOI:10.1111/j.1742-4658.2009.07125.x. PMID:19558492.
- Sevcík J et al. (2004), Acta Crystallogr D Biol Crystallogr, 60, 1198-1204. Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2. DOI:10.1107/s0907444904009035. PMID:15213380.
- Sevcik J et al. (1993), Eur J Biochem, 216, 305-305. Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate. DOI:10.1111/j.1432-1033.1993.tb18146.x. PMID:8396032.
- Takahashi K et al. (1970), Adv Biophys, 1, 53-98. Ribonuclease T1, Structure and function. PMID:4364371.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Arg67A | activator, transition state stabiliser |
| Arg71A | activator, transition state stabiliser |
| Glu56A | proton shuttle (general acid/base) |
| His86A | proton shuttle (general acid/base) |