Isoleucine-tRNA ligase (type 2)

 

Isoleucine-tRNA ligase (also known as Isoleucyl-tRNA synthetase) is an alpha monomer that belongs to class Ia tRNA synthetases. It activates not only the cognate substrate L-isoleucine but also the structurally similar L-valine in the first, amino-acylation step. In a second, "editing" step, the synthetase itself rapidly hydrolyses only the valylated products. For this two-step substrate selection, a "double-sieve" mechanism has been proposed to explain the high substrate fidelity and low copy error of the tRNA synthetase.

Escherichia coli isoleucyl-tRNA synthetasehas been shown to contain two enzyme-bound zinc atoms per polypeptide chain. The N-terminal zinc ion may play a structural role. Although the two zinc domains are always conserved, the zinc ligands are not, so that there can be both zinc atoms, no zinc atoms or only one of the two. The C-terminal zinc ion is essential (for function in vivo). It plays an important roles in aminoacylation of tRNA(Ile) [PMID:7488160, PMID:8672449].

 

Reference Protein and Structure

Sequence
P56690 UniProt (6.1.1.5) IPR023586 (Sequence Homologues) (PDB Homologues)
Biological species
Thermus thermophilus HB8 (Bacteria) Uniprot
PDB
1ile - ISOLEUCYL-TRNA SYNTHETASE (2.5 Å) PDBe PDBsum 1ile
Catalytic CATH Domains
3.40.50.620 CATHdb (see all for 1ile)
Cofactors
Magnesium(2+) (1), Zinc(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.1.1.5)

L-isoleucine zwitterion
CHEBI:58045ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
AMP 3'-end(1-) residue
CHEBI:78442ChEBI
→
3'-(L-isoleucyl)adenylyl zwitterionic group
CHEBI:78528ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: Isoleucine translase, Isoleucine-tRNA synthetase, Isoleucine-transfer RNA ligase, Isoleucyl-tRNA synthetase, Isoleucyl-transfer RNA synthetase, Isoleucyl-transfer ribonucleate synthetase,

Enzyme Mechanism

Introduction

The zwitterionic leucine substrate attacks the alpha phosphate of ATP, forming an activated, adenylated, amino acid. The 2' end of the specific tRNA molecule attacks at the activated phospho-ester bond. The anionic, tetrahedral intermediate collapses, generating the tRNA-isoleucine adduct and AMP.

Catalytic Residues Roles

UniProt PDB* (1ile)
Trp518, Trp558 Trp518A, Trp558A The bulky tryptophan residues prevent the binding of larger amino acids within the adenylation site. These residues form the first molecular sieve, introducing rough substrate specificity between large and small amino acids [PMID:9554847, PMID:10966471] van der waals interaction, steric role
Lys591, Lys594 Lys591A, Lys594A Help stabilise the reactive intermediates formed during the course of the reaction. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser
Gln554, Asp85, Pro46 Gln554A, Asp85A, Pro46A Form the second molecular sieve site, which discriminates between wrongly activated amino acids, such as valine and the correct substrate for father catalysis. hydrogen bond acceptor, steric role
*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

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

References

  1. Nureki O et al. (1998), Science, 280, 578-582. Enzyme Structure with Two Catalytic Sites for Double-Sieve Selection of Substrate. DOI:10.1126/science.280.5363.578. PMID:9554847.
  2. Fukai S et al. (2003), RNA, 9, 100-111. Mechanism of molecular interactions for tRNAVal recognition by valyl-tRNA synthetase. DOI:10.1261/rna.2760703. PMID:12554880.
  3. Ibba M et al. (2000), Annu Rev Biochem, 69, 617-650. Aminoacyl-tRNA Synthesis. DOI:10.1146/annurev.biochem.69.1.617. PMID:10966471.
  4. Fersht AR (1977), Biochemistry, 16, 1025-1030. Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase. DOI:10.1021/bi00624a034. PMID:321008.

Step 1. The bulky tryptophan residues prevent the binding of larger amino acids within the adenylation site. The zwitterionic leucine substrate attacks the alpha phosphate of ATP, forming an activated, adenylated, amino acid.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp85A attractive charge-charge interaction, steric role, hydrogen bond acceptor
Trp518A steric role, van der waals interaction
Lys591A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Pro46A steric role, van der waals interaction
Lys594A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Trp558A steric role, van der waals interaction
Gln554A steric role, hydrogen bond acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation

Step 2. Amino acids similar in size to isoleucine which slip through the first 'sieve' and are mistakenly adenylated, such as valine, are channelled to the site of the second, finer molecular sieve. This site uses a precise molecular architecture to discriminate between wrongly activated amino acids, such as valine and the correct substrate for father catalysis. Once bound within this second site, catalytically activated hydrolysis deactivates the amino acid, preventing it from attaching to the isoleucine tRNA. The residue Thr232 is thought to be responsible for amino acid recognition at this site. This two sieve mechanism further reduces the risk of missense mutations in later protein synthesis [PMID:9554847, PMID:12554880, PMID:321008]. In this step, the 2' end of the specific tRNA molecule attacks at the activated phospho-ester bond

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp85A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond acceptor
Trp518A van der waals interaction
Lys591A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Pro46A van der waals interaction
Lys594A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Trp558A van der waals interaction
Gln554A hydrogen bond acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, overall reactant used, rate-determining step

Step 3. The anionic, tetrahedral intermediate collapses, generating the tRNA-isoleucine adduct and AMP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp85A hydrogen bond acceptor
Trp518A van der waals interaction
Lys591A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Pro46A van der waals interaction
Lys594A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Trp558A van der waals interaction
Gln554A hydrogen bond acceptor

Chemical Components

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

Step 1. The bulky tryptophan residues prevent the binding of larger amino acids within the adenylation site. The zwitterionic leucine substrate attacks the alpha phosphate of ATP, forming an activated, adenylated, amino acid.

Step 2. Amino acids similar in size to isoleucine which slip through the first 'sieve' and are mistakenly adenylated, such as valine, are channelled to the site of the second, finer molecular sieve. This site uses a precise molecular architecture to discriminate between wrongly activated amino acids, such as valine and the correct substrate for father catalysis. Once bound within this second site, catalytically activated hydrolysis deactivates the amino acid, preventing it from attaching to the isoleucine tRNA. The residue Thr232 is thought to be responsible for amino acid recognition at this site. This two sieve mechanism further reduces the risk of missense mutations in later protein synthesis [PMID:9554847, PMID:12554880, PMID:321008]. In this step, the 2' end of the specific tRNA molecule attacks at the activated phospho-ester bond

Step 3. The anionic, tetrahedral intermediate collapses, generating the tRNA-isoleucine adduct and AMP.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday