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Literature summary extracted from

  • Dulic, M.; Cvetesic, N.; Perona, J.J.; Gruic-Sovulj, I.
    Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases (2010), J. Biol. Chem., 285, 23799-23809.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.1.5 overexpression of His-tagged IleRS in Escherichia coli strain BL21 (DE3) Escherichia coli
6.1.1.9 overexpression of His-tagged ValRS in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
6.1.1.5 D342A site-directed mutagenesis, the IleRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations Escherichia coli
6.1.1.5 T243R site-directed mutagenesis, the mutant retains tRNA-independent editing at a level identical to the WT enzyme and shows increased ATP hydrolysis compared to the wild-type enzyme Escherichia coli
6.1.1.5 T243R/D342A site-directed mutagenesis, the IleRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations Escherichia coli
6.1.1.9 D286A site-directed mutagenesis, the ValRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations Escherichia coli
6.1.1.9 K277P site-directed mutagenesis, the ValRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations Escherichia coli
6.1.1.9 K277P/D286A site-directed mutagenesis, the ValRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.1.1.5 additional information
-
additional information steady-state parameters for tRNA-independent pre-transfer editing by IleRS and its mutants determined by varying concentrations of noncognate valine, overview. Kinetic method to distinguish among three models for pre-transfer editing by IleRS, overview Escherichia coli
6.1.1.5 0.6
-
ATP pH 7.5, 37°C, mutant T243R/D342A, in presence of tRNA Escherichia coli
6.1.1.5 0.7
-
ATP pH 7.5, 37°C, mutant T234R, in presence of tRNA Escherichia coli
6.1.1.5 2.4
-
ATP pH 7.5, 37°C, mutant D342A, in presence of tRNA Escherichia coli
6.1.1.5 4.4
-
ATP pH 7.5, 37°C, wild-type IleRS, in presence of tRNA Escherichia coli
6.1.1.9 additional information
-
additional information steady-state parameters for tRNA-independent pre-transfer editing by ValRS and its mutants determined by varying concentrations of noncognate threonine, overview Escherichia coli
6.1.1.9 5.8
-
ATP pH 7.5, 37°C, mutant D286A, in presence of tRNA Escherichia coli
6.1.1.9 9.4
-
ATP pH 7.5, 37°C, wild-type ValRS, in presence of tRNA Escherichia coli
6.1.1.9 10.7
-
ATP pH 7.5, 37°C, mutant K277P/D286A, in presence of tRNA Escherichia coli
6.1.1.9 13.4
-
ATP pH 7.5, 37°C, mutant K277P, in presence of tRNA Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.5 Mg2+ assay at Escherichia coli
6.1.1.9 Mg2+ assay at Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.1.1.5 ATP + L-isoleucine + tRNAIle Escherichia coli
-
AMP + diphosphate + L-isoleucyl-tRNAIle
-
?

Organism

EC Number Organism UniProt Comment Textmining
6.1.1.5 Escherichia coli
-
-
-
6.1.1.9 Escherichia coli
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.1.5 recombinant His-tagged IleRS from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography Escherichia coli
6.1.1.9 recombinant His-tagged ValRS from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography Escherichia coli

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
6.1.1.5 additional information
-
rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS Escherichia coli
6.1.1.9 additional information
-
in ValRS transfer to tRNA is 200fold faster than hydrolysis Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.1.5 ATP + L-isoleucine + tRNAIle
-
Escherichia coli AMP + diphosphate + L-isoleucyl-tRNAIle
-
?
6.1.1.9 ATP + L-valine + tRNAVal
-
Escherichia coli AMP + diphosphate + L-valyl-tRNAVal
-
?

Synonyms

EC Number Synonyms Comment Organism
6.1.1.5 IleRS
-
Escherichia coli
6.1.1.5 Isoleucyl-tRNA synthetase
-
Escherichia coli
6.1.1.5 More the enzyme is a class I aminoacyl-tRNA synthetase Escherichia coli
6.1.1.9 More the enzyme is a class I aminoacyl-tRNA synthetase Escherichia coli
6.1.1.9 ValRS
-
Escherichia coli
6.1.1.9 Valyl-tRNA synthetase
-
Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.1.1.5 37
-
assay at, aminoacylation and deacylation reactions Escherichia coli
6.1.1.9 37
-
assay at, aminoacylation and deacylation reactions Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.1.1.5 0.29
-
ATP pH 7.5, 37°C, mutant T243R/D342A, in presence of tRNA Escherichia coli
6.1.1.5 0.48
-
ATP pH 7.5, 37°C, mutant D342A, in presence of tRNA Escherichia coli
6.1.1.5 1.04
-
ATP pH 7.5, 37°C, mutant T234R, in presence of tRNA Escherichia coli
6.1.1.5 1.56
-
ATP pH 7.5, 37°C, wild-type IleRS, in presence of tRNA Escherichia coli
6.1.1.9 0.28
-
ATP pH 7.5, 37°C, mutant D286A, in presence of tRNA Escherichia coli
6.1.1.9 0.34
-
ATP pH 7.5, 37°C, mutant K277P, in presence of tRNA Escherichia coli
6.1.1.9 0.48
-
ATP pH 7.5, 37°C, mutant K277P/D286A, in presence of tRNA Escherichia coli
6.1.1.9 12.9
-
ATP pH 7.5, 37°C, wild-type ValRS, in presence of tRNA Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.1.5 7.5
-
assay at, aminoacylation and deacylation reactions Escherichia coli
6.1.1.9 7.5
-
assay at, aminoacylation and deacylation reactions Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.1.5 ATP
-
Escherichia coli
6.1.1.9 ATP
-
Escherichia coli

General Information

EC Number General Information Comment Organism
6.1.1.5 physiological function hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes, e.g. IleRS, occurs in a spatially separate domain inserted into the catalytic Rossmann fold. tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. Balance between pretransfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate, overview. In IleRS both pre- and post-transfer editing are important Escherichia coli
6.1.1.9 physiological function hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes, e.g. ValRS, occurs in a spatially separate domain inserted into the catalytic Rossmann fold, location and mechanisms of pre-transfer hydrolysis of misactivated amino acids, overview. The rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates. Editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain Escherichia coli

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.1.1.5 0.2
-
ATP pH 7.5, 37°C, mutant D342A, in presence of tRNA Escherichia coli
6.1.1.5 0.35
-
ATP pH 7.5, 37°C, wild-type IleRS, in presence of tRNA Escherichia coli
6.1.1.5 0.48
-
ATP pH 7.5, 37°C, mutant T243R/D342A, in presence of tRNA Escherichia coli
6.1.1.5 1.49
-
ATP pH 7.5, 37°C, mutant T234R, in presence of tRNA Escherichia coli
6.1.1.9 0.03
-
ATP pH 7.5, 37°C, mutant K277P, in presence of tRNA Escherichia coli
6.1.1.9 0.05
-
ATP pH 7.5, 37°C, mutant D286A, in presence of tRNA Escherichia coli
6.1.1.9 0.05
-
ATP pH 7.5, 37°C, mutant K277P/D286A, in presence of tRNA Escherichia coli
6.1.1.9 1.37
-
ATP pH 7.5, 37°C, wild-type ValRS, in presence of tRNA Escherichia coli