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Literature summary extracted from

  • Zhai, Y.; Nawaz, M.H.; Lee, K.W.; Kirkbride, E.; Briggs, J.M.; Martinis, S.A.
    Modulation of substrate specificity within the amino acid editing site of leucyl-tRNA synthetase (2007), Biochemistry, 46, 3331-3337.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.1.4 subcloning in strain DH5alpha, expression of His-tagged wild-type and mutant enzymes in strain BL21(DE3) Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
6.1.1.4 D251W site-directed mutagenesis, editing site mutant, the substrate specificity and charging fidelity is retained Escherichia coli
6.1.1.4 M336A site-directed mutagenesis, editing site mutant, the mutant shows a small increase in leucine editing activity Escherichia coli
6.1.1.4 M336F/T252A site-directed mutagenesis, editing site mutant, the T252A mutation uncouples specificity, M336F/T252A double LeuRS mutant exhibited only slightly increased leucylation activity relative to the T252A single mutation Escherichia coli
6.1.1.4 R249F site-directed mutagenesis, editing site mutant, editing activity of Leu-tRNALeu is decreased Escherichia coli
6.1.1.4 R249F/T252A site-directed mutagenesis, editing site mutant, the T252A mutation uncouples specificity Escherichia coli
6.1.1.4 R249T site-directed mutagenesis, editing site mutant, the mutant shows increased activity with tRNALeu, but even higher activity with tRNAIle compared to the wild-type enzyme Escherichia coli
6.1.1.4 R249T/D251W site-directed mutagenesis, editing site mutant, the mutant shows decreased hydrolysis of mischarged Ile-tRNALeu compared to the wild type enzyme Escherichia coli
6.1.1.4 T252A site-directed mutagenesis, the mutation uncouples specificity and shows a 24-fold increase in hydrolytic activity compared to the wild-type enzyme, introduction of the large aromatic residue at Arg249 or Val338 rescued leucylation activity of the T252A mutation Escherichia coli
6.1.1.4 V338A site-directed mutagenesis, editing site mutant, it shows increased post-transfer editing activity of Leu-tRNALeu compared to the wild-type enzyme Escherichia coli
6.1.1.4 V338D site-directed mutagenesis, editing site mutant, the mutant shows reduced post-transfer editing activity compared to the wild-type enzyme Escherichia coli
6.1.1.4 V338E site-directed mutagenesis, editing site mutant, the mutant shows reduced post-transfer editing activity compared to the wild-type enzyme Escherichia coli
6.1.1.4 V338F site-directed mutagenesis, editing site mutant, single introduction of the bulky phenylalanine residue nearly abolished post-transfer editing activity and facilitated mischarging of both isoleucine and valine to tRNALeu, 3000fold reduced activity Escherichia coli
6.1.1.4 V338F/T252A site-directed mutagenesis, editing site mutant, the T252A mutation uncouples specificity Escherichia coli
6.1.1.4 V338L site-directed mutagenesis, editing site mutant, the mutant shows reduced post-transfer editing activity compared to the wild-type enzyme Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.1.1.4 additional information
-
 additional information kinetics of recombinant His-tagged wild-type and mutant enzymes Escherichia coli
6.1.1.4 0.0007
-
 tRNALeu wild-type enzyme Escherichia coli
6.1.1.4 0.0009
-
 tRNALeu mutant V338A Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.4 Mg2+
-
 Escherichia coli
6.1.1.5 Mg2+
-
 Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.1.4 ATP + L-leucine + tRNALeu
-
 Escherichia coli AMP + diphosphate + L-leucyl-tRNALeu
-
 ?
6.1.1.4 ATP + L-leucine + tRNALeu a two-step reaction, the first of which, the amino acid activation step, is reversible, while the second aminoacylation step is not, the amino acid editing site for LeuRS resides within the homologous CP1 domain, some positions are idiosyncratic to LeuRS including a conserved arginine conferring amino acid substrate recognition, it complements other sites in the amino acid binding pocket of the editing active site of Escherichia coli LeuRS, including Thr252 and Val338, the latter is second to the first, which collectively fine-tune amino acid specificity to confer fidelity, editing mechanism, residues Arg249, Asp251, Thr252, Met336, and Val338 are involved, overview Escherichia coli AMP + diphosphate + L-leucyl-tRNALeu
-
 ?
6.1.1.5 ATP + L-isoleucine + tRNAIle
-
 Escherichia coli AMP + diphosphate + L-isoleucyl-tRNAIle
-
 ?
6.1.1.5 ATP + L-isoleucine + tRNAIle a two-step reaction, the first of which, the amino acid activation step, is reversible, while the second aminoacylation step is not, the amino acid editing site for LeuRS resides within the homologous CP1 domain containing a conserved threonine conferring amino acid substrate recognition, editing mechanism, some positions of the site are idiosyncratic to IleRS, residues Arg249, Asp251, Thr252, Met336, and Val338 are involved,overview Escherichia coli AMP + diphosphate + L-isoleucyl-tRNAIle
-
 ?

Subunits

EC Number Subunits Comment Organism
6.1.1.4 More the amino acid editing site for LeuRS resides within the homologous CP1 domain: threonine-rich peptide and a second conserved GTG region that are separated by about 100 amino acids comprise parts of the hydrolytic editing site, comparison to IleRS, tertiary and primary structure analysis of the amino acid editing site, overview Escherichia coli
6.1.1.5 More the amino acid editing site for IleRS resides within the homologous CP1 domain: threonine-rich peptide and a second conserved GTG region that are separated by about 100 amino acids comprise parts of the hydrolytic editing site, comparison to LeuRS, some positions of the site are idiosyncratic to IleRS, tertiary and primary structure analysis of the amino acid editing site, overview Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
6.1.1.4 Leucyl-tRNA synthetase
-
 Escherichia coli
6.1.1.4 LeuRS
-
 Escherichia coli
6.1.1.5 IleRS
-
 Escherichia coli
6.1.1.5 Isoleucyl-tRNA synthetase
-
 Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.1.1.4 0.12
-
 tRNALeu wild-type enzyme Escherichia coli
6.1.1.4 0.4
-
 tRNALeu mutant V338A Escherichia coli
6.1.1.4 0.4
-
 tRNALeu mutant V338D Escherichia coli
6.1.1.4 0.4
-
 tRNALeu mutant V338E Escherichia coli
6.1.1.4 0.4
-
 tRNALeu mutant V338F Escherichia coli
6.1.1.4 0.4
-
 tRNALeu mutant V338L Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.1.4 7.5
-
 assay at Escherichia coli
6.1.1.5 7.5
-
 assay at Escherichia coli

Cofactor

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