Information on EC 6.1.1.14 - glycine-tRNA ligase

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The expected taxonomic range for this enzyme is: Archaea, Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
6.1.1.14
-
RECOMMENDED NAME
GeneOntology No.
glycine-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + glycine + tRNAGly = AMP + diphosphate + glycyl-tRNAGly
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
-
tRNA charging
-
-
SYSTEMATIC NAME
IUBMB Comments
glycine:tRNAGly ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
9037-62-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
alpha subunit
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
overproducing strain
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
2 dual-targeted isozymes
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
mitochondrial isoform GlyRS1
UniProt
Manually annotated by BRENDA team
strain H
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
-
glycyl-tRNA synthetase specifically binds to the poliovirus internal ribosome entry site to activate translation initiation
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
AMP + ADP
P1,P3-bis(5'-adenosyl) triphosphate
show the reaction diagram
AMP + ATP
P1,P4-bis(5'-adenosyl) tetraphosphate
show the reaction diagram
AMP + phosphate
ADP
show the reaction diagram
ATP + glycine + tRNAGly
?
show the reaction diagram
ATP + glycine + tRNAGly
AMP + diphosphate + glycyl-tRNAGly
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + glycine + tRNAGly
?
show the reaction diagram
ATP + glycine + tRNAGly
AMP + diphosphate + glycyl-tRNAGly
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Li+
-
slightly stimulates in presence of Mg2+
PO43-
-
phosphorylation and dephosphorylation seem to be a means of regulation
additional information
-
the enzyme is twice as active in Tris-HCl or Hepes-NaOH as it is in potassium phosphate buffer
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-adenylylimidodiphosphate
-
i.e. AMPPNP, a nonsubstrate ATP analogue, competitive inhibition
5,5'-dithiobis(2-nitrobenzoate)
-
-
adenylylmethylenediphosphate
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i.e. AMPPCP, a nonsubstrate ATP analogue, competitive inhibition
alpha,beta-methyleneadenosine 5'-diphosphate
-
i.e. AMPCPP, a nonsubstrate ATP analogue, competitive inhibition
Aminoethyl-AMP
-
-
glycyl sulfamoyl adenosine
-
-
Inorganic sulfide
-
activity is restored by addition of glutathione, cysteine or cysteamine
-
KCl
-
inhibits acylation of yeast tRNA, little effect on acylation of homologous tRNAGly
Nalidixic acid
-
-
novobiocin
-
-
Oxolinic acid
-
-
p-chloromercuribenzoate
p-Chloromercuriphenylsulfonic acid
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activity is not restored by treatment with 2-mercaptoethanol
phosphate
spermine
-
-
additional information
-
reversible denaturation by urea or guanidine-HCl, wild-type enzyme and N-terminally truncated mutant both dissociate to monomers and exhibit a cooperative transition at higher denaturant concentration, wild-type unfolds by a multistate process and 2 parallel state unfoldings, which are absent in the truncated mutant
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013 - 0.37
ATP
0.00035 - 0.67
Gly
0.13 - 0.145
glycine
0.0091
tRNA(C2*71-G2*C71)
-
acceptor stem mutant substrate
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0.0026
tRNA1Gly
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aminoacylation
0.000088 - 0.0033
tRNAGly
0.0018
tRNAGly(C2*G71-C2*A71)
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acceptor stem mutant substrate
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0.0045
tRNAGly(G1*C72-A1*U72)
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acceptor stem mutant substrate
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0.00096
tRNAGly(G1*C72-G1*U72)
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acceptor stem mutant substrate
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0.0031
tRNAGly(U73-A73)
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mutant substrate
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0.0046
tRNAGly(U73-C73)
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mutant substrate
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0.0059
tRNAGly(U73-G73)
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mutant substrate
-
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15.9
tRNA1Gly
Bombyx mori
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aminoacylation
8.1
tRNA2Gly
Bombyx mori
-
aminoacylation
0.19 - 0.73
tRNAGly
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
140 - 1230
tRNAGly
3369
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02
5'-adenylylimidodiphosphate
-
-
0.0008
adenylylmethylenediphosphate
-
-
0.06
alpha,beta-methyleneadenosine 5'-diphosphate
-
-
0.08
AMP
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0074
-
tetrameric enzyme form
0.124
-
purified recombinant enzyme
0.55
-
dimeric enzyme form
1.65
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.6
-
enzyme form B, ATP-diphosphate exchange and glycine-charging activity
8 - 8.5
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8
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enzyme form A, glycine-charging activity
8.5
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enzyme form A, ATP-diphosphate exchange
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 40
-
aminoacylation of tRNAGly
additional information
-
wild-type enzyme is active at 42C, temperature-sensitive mutant enzyme not
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Campylobacter jejuni subsp. jejuni serotype O:23/36 (strain 81-176)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
2-3 * 27000, about, partially processed isozyme GlyRS1, SDS-PAGE
33000
-
2 * 33000 (alpha) + 2 * 80000 (beta), SDS-PAGE
40000
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x * 40000 + x * 78000, alpha-alpha interactions contibute to the stability of the native enzyme while beta-beta interactions do not, SDS-PAGE
51000
-
x * 51000, SDS-PAGE
57500
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2 * 67600 (alpha) + 2 * 57500 (beta), enzyme form isolated in presence of minimal concentrations of dithioerythritol. A dimeric enzyme form is isolated in presence of high concentrations of protease inhibitors and dithioerythritol, SDS-PAGE
66000
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gel filtration, enzyme form E2
67600
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2 * 67600 (alpha) + 2 * 57500 (beta), enzyme form isolated in presence of minimal concentrations of dithioerythritol. A dimeric enzyme form is isolated in presence of high concentrations of protease inhibitors and dithioerythritol, SDS-PAGE
72000
-
x * 72000, wild-type enzyme, SDS-PAGE
76919
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x * 76919, wild-type enzyme, calculation from nucleotide sequence
77530
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x * 77530, calculation from nucleotide sequence
78000
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x * 40000 + x * 78000, alpha-alpha interactions contibute to the stability of the native enzyme while beta-beta interactions do not, SDS-PAGE
81000
-
2 * 30000 (alpha) + 2 * 81000 (beta), enzyme form E1, SDS-PAGE, 2 * 30000, SDS-PAGE, enzyme form E2, E2 is a component of E1, SDS-PAGE
90000
-
2 * 90000, SDS-PAGE
117000
135000 - 142000
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sucrose density gradient centrifugation, dimeric enzyme form
155000
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gel filtration, calculation from sedimentation constant
158000 - 160000
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PAGE under nondenaturing conditions, gel filtration, dimeric enzyme form
160000
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gel filtration, sedimentation velocity
189000
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gel filtration, mitochondrial enzyme
202000
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gel filtration, cytoplasmic enzyme
205000
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high speed sedimentation equilibrium measurement
210000
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sucrose density gradient centrifugation, tetrameric enzyme form
226000
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gel filtration, enzyme form E1
227000
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sedimentation equilibrium ultracentrifugation
230000
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gel filtration
240000
-
PAGE
250000
-
PAGE under nondenaturing conditions, gel filtration, tetrameric enzyme form
270000
-
polyacrylamide 4/30% gradient PAGE
additional information
-
primary structure of both subunits
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer or trimer
2-3 * 27000, about, partially processed isozyme GlyRS1, SDS-PAGE
heterotetramer
homodimer
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
cytoplasmic and mitochondrial isoforms have the same polypeptide sequence except for a 23 amino acid mitochondrial leader peptide
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
alpha-subunit the enzyme and its complexes with ATP, and ATP and glycine, sitting drop vapor diffusion method, using 0.1 M Bis-Tris propane:NaOH pH 7.0 and 0.7 M magnesium formate (complex with glycine), or 0.1 M Tris-HCl pH 8.5 and 0.3 M magnesium chloride (complex with ATP), or 0.1 M Na2HPO4-citrate acid pH 4.2, 0.2 M lithium sulfate and 20% (w/v) PEG 1000 (complex with ATP and glycine)
purified His-tagged wild-type and S581L mutant enzymes, sitting drop vapour diffusion nanocrystallization method, mutant S581L GlyRS from 20% PEG 3350, 0.2 M Na2SO4, 0.1 M Bis-Tris propane, pH 6.5, wild-type GlyRS from 20% PEG 3350, 0.2M sodium bromide, 0.1 M bis-Tris propane pH 8.5, X-ray diffraction structure determination and anaylsis at 2.8A and 3.1 A resolution, respectively
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purified recombinant soluble enzyme, hanging drop vapour diffusion method, 0.001 ml of 8 mg/ml protein in 10 mM HEPES, pH 7.0, with 20 mM NaCl is mixed with 0.001 ml of reservoir solution containing 10% PEG 6000, 0.1 HEPES pH 6.5, 0.01 Tris-HCl, pH 8.5, 0.5 NaCl and 0.1 magnesium acetate at room temperature, 2 days, X-ray diffraction structure determination and analysis at 3.0 A resolution
at 2.75 A resolution
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28
-
half-life of temperature-sensitive mutant: 30 min
50
-
80% loss of activity after 1 min in absence of substrates, 60% loss of activity after 10 min in presence of aminoethyl-AMP and Mg2+
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
aminoethyl-AMP protects from heat inactivation
-
ATP and nonsubstrate ATP analogues render the enzyme more resistant to digestion by several proteases, e.g. thrombin, Arg-C, and chymotrypsin
-
DTT or 25% glycerol stabilizes
-
glycerol required for stabilization
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glycyl-adenylate stabilizes the enzyme
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in absence of protease inhibitors and/or dithioerythritol, the enzyme rapidly loses its activity
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protectors of SH groups at concentrations of 20 mM are required to obtain an optimal aminoacylation rate
-
sensitive to repeated freezing and thawing
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unstable after dilution to less than 0.02 mg/ml in absence of nonspecific carrier proteins
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, enzyme concentration 0.5 mg/ml, 40% glycerol, 30% loss of activity after 8 months
-
-80C, retains approximately 90% of its activity after 3 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 enzyme forms: E1 and E2
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glutathione-Sepharose column chromatography, and gel filtration
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Ni-affinity column chromatography
Ni-NTA column chromatography; Ni-NTA column chromatography
recombinant C-terminally His6-tagged cytosolic enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant enzyme from Escherichia coli, 10fold
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recombinant His-atgged wild-type enzyme and P552F mutant from Escherichia coli
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recombinant His-tagged isozymes GlyRS1 and 2 from Escherichia coli
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant isozyme GlyRS1 from Escherichia coli
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 genes: grs1 encodes mitochondrial and cytoplasmic isozyme, grs2 encodes a mitochondrial dipensable isozyme, expression in Escherichia coli as HIs-tagged proteins, both cytoplasmic and mitochondrial phenotypes of a knockout allele of grs1 are complemented by the expression of the single gene of Schizosaccharomyces pombe, expression of gene grs2 in a grs1-deficient yeast mutant cannot complement the lethal phenotype
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as a fusion protein with 13 kDa biotinylated tag with an apparent MW of 90 kDa; expression in Escherichia coli
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DNA sequence determination and analysis of full-length edd1 wild-type gene, encoding the enzyme, gene contains a chloroplast targeting sequence, determination of the promoter, functional complementation of enzyme-deficient Arabidopsis plants thereby generation of transgenic plants
DNA sequence determination and analysis, chromosomal localization in region 7p15.1-p15.3, the gene contains only 1 transcriptional start point in 3 different cell lines, but 5 potential initiation codons, expression may be translationally regulated, N-terminal mitochondrial targeting sequence
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli Rosetta (DE3) cells
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expressed in Saccharomyces cerevisiae strain InvSc1; expressed in Saccharomyces cerevisiae strain InvSc1
expression in Escherichia coli
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expression of CMT-causing mutant variants and wild-type enzymes in neuroblastoma cells that sprout primitive neurites
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expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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expression of His-tagged wild-type and p552F mutant enzyme in Escherichia coli
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expression of wild-type and N-terminally truncated enzyme, the latter lacking 55 N-terminal amino acid residues, in Escherichia coli BL21(DE3)
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gene GARS, allele-specific expression analysis, expression of wild-type enzyme in Escherichia coli strain BL21(DE3), expression of EGFP-tagged enzyme in COS-7 cells, wild-type GARS-EGFP associates with granules in both the cell body and neurite projections of 42% of EGFP-positive transfected MN-1 cells
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gene GARS, DNA and amino acid sequence determination and analysis, genetic mapping
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gene GARS, lacking the mitochondrial-specific coding region, expression as C-terminally His6-tagged cytosolic enzyme in Escherichia coli strain BL21(DE3)
gene glyS, DNA and amino acid sequence determination and analysis, overexpression in Escherichia coli BL21(DE3)
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gene glyS, GlyRS phylogenetic analysis, deduction of ancestral sequence of GlyRS, and introduction of individual or pairs of ancestral residues into Thermus thermophilus GlyRS resulting in ancestral mutants, which are expressed in Escherichia coli strain BL21(DE3), overview
gene grs1 is essential
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overexpression of isozyme GlyRS1 in Escherichia coli strain TG2
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
P61L
-
mutant enzymes with an altered amino acid binding site. Pro61Leu substitution in the alpha chain confers an elevation of the Km value for Gly, 25fold, and for ATP, 45fold, in the aminoacylation reaction, but only a minor pertubation of the Km for tRNA
A57V
-
found in a screen of 33 patients
P234KY
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naturally occuring mutation in the catalytic domain, the mutation lies in a conserved region and causes Charcot-Marie-Tooth peripheral neuropathies
P244L
-
disease phenotype CMT2D
GarsC201R/+
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mouse model, decreased grip strength, poor skilled motor function, increased total GARS protein at p15, reduction in large diameter axon in sciatic nerve, normal lifespan
GarsC201R/C201R
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mouse model, reduced weight and viability, impaired limb movement, life expectancy 17 days
GarsC201R/XM256
-
mouse model, embryonic lethal
GarsNmf249/+
-
mouse model, sensory and motor deficits, abnormal neuromuscular junction morphology, impaired nerve impule transmission, reduced nerve conduction velocities, loss of large diameter peripheral axons, life expectancy 6-8 weeks
GarsNmf249/Nmf249
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mouse model, embryonic lethal
GarsNmf249/XM256
-
mouse model, embryonic lethal
GarsP278KY/+
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mouse model, sensory and motor deficits, abnormal neuromuscular junction morphology, impaired nerve impule transmission, reduced nerve conduction velocities, loss of large diameter peripheral axons, life expectancy 6-8 weeks
GarsXM256/+
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mouse model, reduced GARS RNA levels, normal neuromuscular junction morphology, normal nerve conduction velocities, normal lifespan
GarsXM256/XM256
-
mouse model, embryonic lethal
P552F
-
temperature-sensitive mutant grs1-1 shows altered substrate specificities, the mutant strain can be complemented by expression of the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reversible denaturation by urea or guanidine-HCl, wild-type enzyme and N-terminally truncated mutant both dissociate to monomers and exhibit a cooperative transition at higher denaturant concentration, wild-type unfolds by a multistate process and 2 parallel state unfoldings, which are absent in the truncated mutant
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
molecular biology
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translation of mRNA for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions -3 to -1 relative to the initiation codon. A/A/R (R: A or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence -3 to -1 from AAA to CGC reduce translation initiation from the UUG codon up to 32fold and resulted in loss of mitochondrial respiration
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