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Literature summary extracted from

  • Dulic, M.; Perona, J.J.; Gruic-Sovulj, I.
    Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase (2014), Biochemistry, 53, 6189-6198 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.1.5 gene ileS, recombinant expression of His-tagged wild-type and mutant enzymes 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 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
6.1.1.5 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
6.1.1.5 Y59F/D342A site-directed mutagenesis, the mutant activity is similar to the wild-type Escherichia coli
6.1.1.5 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
6.1.1.5 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.1.1.5 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
6.1.1.5 0.0019
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59F/D342A Escherichia coli
6.1.1.5 0.0026
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59F Escherichia coli
6.1.1.5 0.0046
-
 L-isoleucine pH 7.5, 37°C, recombinant wild-type enzyme Escherichia coli
6.1.1.5 0.0052
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant D342A Escherichia coli
6.1.1.5 0.0383
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59T/D342A Escherichia coli
6.1.1.5 0.0415
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59T Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.5 Mg2+ required Escherichia coli
6.1.1.5 Zn2+ required 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
-
 ?
6.1.1.5 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

EC Number Organism UniProt Comment Textmining
6.1.1.5 Escherichia coli P00956
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.1.5 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography 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
-
 ?
6.1.1.5 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
-
 ?
6.1.1.5 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 ?
-
 ?
6.1.1.5 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

EC Number Synonyms Comment Organism
6.1.1.5 EcIleRS
-
 Escherichia coli
6.1.1.5 IleRS
-
 Escherichia coli
6.1.1.5 Isoleucyl-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 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 10.7
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59T/D342A Escherichia coli
6.1.1.5 11.4
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59T Escherichia coli
6.1.1.5 21
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59F Escherichia coli
6.1.1.5 23
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59F/D342A Escherichia coli
6.1.1.5 47
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant D342A Escherichia coli
6.1.1.5 55
-
 L-isoleucine pH 7.5, 37°C, recombinant wild-type enzyme Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.1.5 7.5
-
 assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.1.5 ATP
-
 Escherichia coli

General Information

EC Number General Information Comment Organism
6.1.1.5 evolution the enzyme belongs to the class I amino acyl-tRNA synthetases (aaRS) Escherichia coli
6.1.1.5 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
6.1.1.5 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 [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.1.1.5 270
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59T Escherichia coli
6.1.1.5 280
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59T/D342A Escherichia coli
6.1.1.5 8100
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59F Escherichia coli
6.1.1.5 9040
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant D342A Escherichia coli
6.1.1.5 12000
-
 L-isoleucine pH 7.5, 37°C, recombinant wild-type enzyme Escherichia coli
6.1.1.5 12100
-
 L-isoleucine pH 7.5, 37°C, recombinant mutant Y59F/D342A Escherichia coli