Cloned (Comment) | Organism |
---|---|
overexpression of His-tagged IleRS in Escherichia coli strain BL21 (DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D342A | site-directed mutagenesis, the IleRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations | Escherichia coli |
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 |
T243R/D342A | site-directed mutagenesis, the IleRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | |
0.6 | - |
ATP | pH 7.5, 37°C, mutant T243R/D342A, in presence of tRNA | Escherichia coli | |
0.7 | - |
ATP | pH 7.5, 37°C, mutant T234R, in presence of tRNA | Escherichia coli | |
2.4 | - |
ATP | pH 7.5, 37°C, mutant D342A, in presence of tRNA | Escherichia coli | |
4.4 | - |
ATP | pH 7.5, 37°C, wild-type IleRS, in presence of tRNA | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | assay at | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-isoleucine + tRNAIle | Escherichia coli | - |
AMP + diphosphate + L-isoleucyl-tRNAIle | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged IleRS from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-isoleucine + tRNAIle | - |
Escherichia coli | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
? |
Synonyms | Comment | Organism |
---|---|---|
IleRS | - |
Escherichia coli |
Isoleucyl-tRNA synthetase | - |
Escherichia coli |
More | the enzyme is a class I aminoacyl-tRNA synthetase | Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at, aminoacylation and deacylation reactions | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.29 | - |
ATP | pH 7.5, 37°C, mutant T243R/D342A, in presence of tRNA | Escherichia coli | |
0.48 | - |
ATP | pH 7.5, 37°C, mutant D342A, in presence of tRNA | Escherichia coli | |
1.04 | - |
ATP | pH 7.5, 37°C, mutant T234R, in presence of tRNA | Escherichia coli | |
1.56 | - |
ATP | pH 7.5, 37°C, wild-type IleRS, in presence of tRNA | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at, aminoacylation and deacylation reactions | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
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 |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.2 | - |
ATP | pH 7.5, 37°C, mutant D342A, in presence of tRNA | Escherichia coli | |
0.35 | - |
ATP | pH 7.5, 37°C, wild-type IleRS, in presence of tRNA | Escherichia coli | |
0.48 | - |
ATP | pH 7.5, 37°C, mutant T243R/D342A, in presence of tRNA | Escherichia coli | |
1.49 | - |
ATP | pH 7.5, 37°C, mutant T234R, in presence of tRNA | Escherichia coli |