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Literature summary for 6.1.1.10 extracted from

  • Casina, V.C.; Lobashevsky, A.A.; McKinney, W.E.; Brown, C.L.; Alexander, R.W.
    Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site (2011), Biochemistry, 50, 763-769.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of an N-terminally His6-tagged MetRS monomer in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
D369A site-directed mutagenesis in the MetRS SCF, the mutant shows reduced transfer RNA aminoacylation and 125fold loss in tRNAMet aminoacylation efficiency compared to the wild-type enzyme Escherichia coli
D369K/K295D site-directed mutagenesis in the MetRS SCF, the mutant shows reduced transfer RNA aminoacylation compared to the wild-type enzyme Escherichia coli
D369N site-directed mutagenesis in the MetRS SCF, the mutant shows reduced transfer RNA aminoacylation and 60fold loss in tRNAMet aminoacylation efficiency compared to the wild-type enzyme Escherichia coli
K295A site-directed mutagenesis in the MetRS SCF, the mutant shows reduced transfer RNA aminoacylation compared to the wild-type enzyme Escherichia coli
K295V site-directed mutagenesis in the MetRS SCF, the mutant shows reduced transfer RNA aminoacylation compared to the wild-type enzyme Escherichia coli
additional information replacement of amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase, EC6.1.1.18, motif or with alanine residues. Chimeric variants retain significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to RNA aminoacylation than does amino acid identity. In contrast, chimeras are significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic parameters for tRNAMet aminoacylation and for MetRS-catalyzed diphosphate exchange of wild-type enzyme and mutants, overview Escherichia coli
0.0039
-
tRNAMet pH 7.5, 25°C, wild-type enzyme Escherichia coli
0.006
-
tRNAMet pH 7.5, 25°C, mutant D369K/K295D Escherichia coli
0.0102
-
tRNAMet pH 7.5, 25°C, mutant KA295V Escherichia coli
0.0177
-
tRNAMet pH 7.5, 25°C, mutant K295V Escherichia coli
0.018
-
tRNAMet pH 7.5, 25°C, mutant D369A Escherichia coli
0.026
-
tRNAMet pH 7.5, 25°C, mutant D369N Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-methionine + tRNAMet Escherichia coli
-
AMP + diphosphate + L-methionyl-tRNAMet
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged MetRS monomer from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-methionine + tRNAMet
-
Escherichia coli AMP + diphosphate + L-methionyl-tRNAMet
-
?

Synonyms

Synonyms Comment Organism
MetRS
-
Escherichia coli
More the enzyme is a class I aminoacyl-tRNA synthetase Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.037
-
tRNAMet pH 7.5, 25°C, mutant D369A Escherichia coli
0.042
-
tRNAMet pH 7.5, 25°C, mutant KA295V Escherichia coli
0.05
-
tRNAMet pH 7.5, 25°C, mutant D369K/K295D Escherichia coli
0.11
-
tRNAMet pH 7.5, 25°C, mutant D369N Escherichia coli
0.3
-
tRNAMet pH 7.5, 25°C, mutant K295V Escherichia coli
1
-
tRNAMet pH 7.5, 25°C, wild-type enzyme 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

General Information

General Information Comment Organism
additional information the catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates, structural comparisons of class Ia and Ib enzymes, overview. Structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2
-
tRNAMet pH 7.5, 25°C, mutant D369A Escherichia coli
4
-
tRNAMet pH 7.5, 25°C, mutant D369N Escherichia coli
4
-
tRNAMet pH 7.5, 25°C, mutant KA295V Escherichia coli
8
-
tRNAMet pH 7.5, 25°C, mutant D369K/K295D Escherichia coli
17
-
tRNAMet pH 7.5, 25°C, mutant K295V Escherichia coli
250
-
tRNAMet pH 7.5, 25°C, wild-type enzyme Escherichia coli