Literature summary for 6.1.1.4 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.

Search for more literature for 6.1.1.4

Cloned(Commentary)

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

Protein Variants

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 [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

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-

Purification (Commentary)

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

Substrates and Products (Substrate)

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

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

Synonyms	Comment	Organism
Leucyl-tRNA synthetase	-	Escherichia coli
LeuRS	-	Escherichia coli

Turnover Number [1/s]

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

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

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Escherichia coli

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Release 2023.1 (January 2023)