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Literature summary extracted from

  • Betha, A.K.; Williams, A.M.; Martinis, S.A.
    Isolated CP1 domain of Escherichia coli leucyl-tRNA synthetase is dependent on flanking hinge motifs for amino acid editing activity (2007), Biochemistry, 46, 6258-6267.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

Protein Variants

EC Number Protein Variants Comment Organism
6.1.1.4 A293D site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme Escherichia coli
6.1.1.4 A293E site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme Escherichia coli
6.1.1.4 A293K site-directed mutagenesis, the post-transfer editing activity of the isolated CP1-domain is enhanced compared to the wild-type enzyme's domain Escherichia coli
6.1.1.4 A293R site-directed mutagenesis, the post-transfer editing activity of the isolated CP1-domain is enhanced compared to the wild-type enzyme's domain Escherichia coli
6.1.1.4 D345A site-directed mutagenesis, the mutation in the isolated CP1-domain abolishes hydrolytic post-transfer editing activity Escherichia coli
6.1.1.4 additional information isolated LeuRS CP1 domain requires idiosyncratic adaptations to confer editing activity independent of the full-length enzyme, the beta-strands, which link the CP1 domain to the aminoacylation core of LeuRS, are required for editing of mischarged tRNALeu, hydrolytic activity is also enhanced by inclusion of short flexible peptides, called hinges, at the end of both LeuRS beta-strands, overview Escherichia coli
6.1.1.4 T247V site-directed mutagenesis, hydrolysis of Ile-tRNALeu is completely abolished Escherichia coli
6.1.1.4 T247V/T248V site-directed mutagenesis, the double mutation abolishes post-transfer editing activity Escherichia coli
6.1.1.4 T248V site-directed mutagenesis, hydrolysis of Ile-tRNALeu is completely abolished Escherichia coli
6.1.1.4 T252A site-directed mutagenesis, the mutation in the full-length LeuRS uncouples specificity and hydrolyzes correctly charged LeutRNALeu Escherichia coli
6.1.1.4 T252Y site-directed mutagenesis, the mutation occupies the amino acid binding pocket and blocks the binding of substrate to abolish editing activity Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.4 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
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.1.1.4 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 affiniry chromatography Escherichia coli

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
6.1.1.4 additional information
-
 hydrolytic post-transfer editing activity of wild-type and D345A mutant LeuRS CP1 domains, activities of truncation mutants, overview 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 the editing domain called CP1 is required for hydrolyzing the incorrectly misaminoacylated noncognate amino acids Ile and Val, the beta-strands, which link the CP1 domain to the aminoacylation core of LeuRS, are required for editing of mischarged tRNALeu, hydrolytic activity is also enhanced by inclusion of short flexible peptides, called hinges, at the end of both LeuRS beta-strands, overview Escherichia coli AMP + diphosphate + L-leucyl-tRNALeu
-
 ?
6.1.1.4 additional information isolated LeuRS CP1 domain requires idiosyncratic adaptations to confer editing activity independent of the full-length enzyme, 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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.1.1.4 37
-
 assay at Escherichia coli

pH Optimum

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

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.1.4 ATP
-
 Escherichia coli