Cloned (Comment) | Organism |
---|---|
subcloning in strain DH5alpha, expression of His-tagged wild-type and mutant enzymes in strain BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D251W | site-directed mutagenesis, editing site mutant, the substrate specificity and charging fidelity is retained | Escherichia coli |
M336A | site-directed mutagenesis, editing site mutant, the mutant shows a small increase in leucine editing activity | Escherichia coli |
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 |
R249F | site-directed mutagenesis, editing site mutant, editing activity of Leu-tRNALeu is decreased | Escherichia coli |
R249F/T252A | site-directed mutagenesis, editing site mutant, the T252A mutation uncouples specificity | Escherichia coli |
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 |
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 |
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 |
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 |
V338D | site-directed mutagenesis, editing site mutant, the mutant shows reduced post-transfer editing activity compared to the wild-type enzyme | Escherichia coli |
V338E | site-directed mutagenesis, editing site mutant, the mutant shows reduced post-transfer editing activity compared to the wild-type enzyme | Escherichia coli |
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 |
V338F/T252A | site-directed mutagenesis, editing site mutant, the T252A mutation uncouples specificity | Escherichia coli |
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] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics of recombinant His-tagged wild-type and mutant enzymes | Escherichia coli | |
0.0007 | - |
tRNALeu | wild-type enzyme | Escherichia coli | |
0.0009 | - |
tRNALeu | mutant V338A | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-leucine + tRNALeu | Escherichia coli | - |
AMP + diphosphate + L-leucyl-tRNALeu | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from strain BL21(DE3) by nickel affinity chromatography to homogeneity | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-leucine + tRNALeu | - |
Escherichia coli | AMP + diphosphate + L-leucyl-tRNALeu | - |
? | |
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 | - |
? |
Subunits | Comment | Organism |
---|---|---|
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 |
Synonyms | Comment | Organism |
---|---|---|
Leucyl-tRNA synthetase | - |
Escherichia coli |
LeuRS | - |
Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.12 | - |
tRNALeu | wild-type enzyme | Escherichia coli | |
0.4 | - |
tRNALeu | mutant V338A | Escherichia coli | |
0.4 | - |
tRNALeu | mutant V338D | Escherichia coli | |
0.4 | - |
tRNALeu | mutant V338E | Escherichia coli | |
0.4 | - |
tRNALeu | mutant V338F | Escherichia coli | |
0.4 | - |
tRNALeu | mutant V338L | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |