Cloned (Comment) | Organism |
---|---|
gene ileS, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D342A | site-directed mutagenesis, the mutant exhibits slightly reduced aminoacylation activity compared to the wild-type enzyme, the post-transfer editing deficient D342A IleRS accumulates AMP by pretransfer editing and by tRNA misacylation when the noncognate aa-AMP evades this hydrolytic reaction, neither wild-type nor D342A IleRS significantly deacylates Ile-tRNAIle under steady-state conditions | Escherichia coli |
Y59F | site-directed mutagenesis, mutation of a conserved residue located within the active site of bacterial IleRS, directly adjacent to the binding site for the 3'-terminal hydroxyl group of tRNA, aminacylation activity is about 35fold reduced compared to the wild-type enzyme | Escherichia coli |
Y59F/D342A | site-directed mutagenesis, the mutant activity is similar to the wild-type | Escherichia coli |
Y59T | site-directed mutagenesis, mutation of a conserved residue located within the active site of bacterial IleRS, directly adjacent to the binding site for the 3'-terminal hydroxyl group of tRNA, Km and kcat values measured for Y59T are increased by 10fold and decreased by 5fold, respectively, for both isoleucine and valine substrates compared to the wild-type enzyme, aminacylation activity is about 12fold reduced | Escherichia coli |
Y59T/D342A | site-directed mutagenesis, kinetic analysis of Y59F/D342A IleRS does not provide reliable results because of the very slow aminoacylation/misacylation | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics of activation of cognate L-Ile and noncognate amino acid L-Val by Y59 IleRS mutant variants, and transfer kinetics, as well as tRNA-dependent hydrolysis of cognate isoleucyl-AMP, overview | Escherichia coli | |
0.0019 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59F/D342A | Escherichia coli | |
0.0026 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59F | Escherichia coli | |
0.0046 | - |
L-isoleucine | pH 7.5, 37°C, recombinant wild-type enzyme | Escherichia coli | |
0.0052 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant D342A | Escherichia coli | |
0.0383 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59T/D342A | Escherichia coli | |
0.0415 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59T | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli | |
Zn2+ | required | 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 | - |
? | |
additional information | Escherichia coli | enzymatic reactions catalyzed by IleRS include amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme, in the synthetic pathway. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) ortRNA-dependent (3). Misacylated tRNA is deacylated through posttransferediting, overview | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P00956 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-isoleucine + tRNAIle | - |
Escherichia coli | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
? | |
ATP + L-isoleucine + tRNAIle | usage of purified recombinant tRNAGAU Ile (with G1-C72 instead of the wild-type A1-U72 sequence) overexpressed in Escherichia coli strain BL21(DE3) | Escherichia coli | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
? | |
additional information | enzymatic reactions catalyzed by IleRS include amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme, in the synthetic pathway. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) ortRNA-dependent (3). Misacylated tRNA is deacylated through posttransferediting, overview | Escherichia coli | ? | - |
? | |
additional information | wild-type an dmutant enzymes IleRS are tested in reactions with both L-valine and L-isoleucine, neither wild-type nor D342A IleRS significantly deacylates Ile-tRNAIle under steady-state conditions | Escherichia coli | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
EcIleRS | - |
Escherichia coli |
IleRS | - |
Escherichia coli |
Isoleucyl-tRNA synthetase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
10.7 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59T/D342A | Escherichia coli | |
11.4 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59T | Escherichia coli | |
21 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59F | Escherichia coli | |
23 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59F/D342A | Escherichia coli | |
47 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant D342A | Escherichia coli | |
55 | - |
L-isoleucine | pH 7.5, 37°C, recombinant wild-type enzyme | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the class I amino acyl-tRNA synthetases (aaRS) | Escherichia coli |
additional information | a Rossmann fold peptide is loacted directly N-terminal to the strictly conserved HIGH motif. The class I IleRS Rossmann fold accommodates both synthetic and tRNA-dependent pretransfer hydrolysis pathways within the synthetic site. Residue Y59 acts as a gatekeeper of the IleRS synthetic site | Escherichia coli |
physiological function | the accuracy of protein synthesis relies on the capacity of aminoacyl-tRNA synthetases (aaRS) to couple cognate amino acids and tRNAs in a two-step reaction that defines the genetic code. In the first step, the amino acid is activated by condensation with ATP to form an enzyme-bound aminoacyl-adenylate (aa-AMP) intermediate with release of pyrophosphate. The second step comprises attack by the terminal 2'- or 3'-OH group of tRNA on the carbonyl carbon atom of aa-AMP, followed by transfer of the aminoacyl moiety to tRNA and release of AMP. The amino acid activation and transfer steps occur within the synthetic active site located in the catalytic domain. Enzyme IleRS possesses an inactivated post-transfer editing domain still capable of robust tRNA-dependent editing. The pretransfer editing activity resides within the synthetic site. Specific recognition of tRNAs by cognate aaRSs is ensured by a network of interactions, based on direct and indirect recognition elements that are embedded in all levels of tRNA structure. Noncognate amino acids that structurally and chemically resemble the cognate substrates are often not well-distinguished in the synthetic reactions alone, so that discrimination is based in part on inherent aaRS-based hydrolytic editing | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
270 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59T | Escherichia coli | |
280 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59T/D342A | Escherichia coli | |
8100 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59F | Escherichia coli | |
9040 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant D342A | Escherichia coli | |
12000 | - |
L-isoleucine | pH 7.5, 37°C, recombinant wild-type enzyme | Escherichia coli | |
12100 | - |
L-isoleucine | pH 7.5, 37°C, recombinant mutant Y59F/D342A | Escherichia coli |