Information on EC 6.1.1.18 - glutamine-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.18
-
RECOMMENDED NAME
GeneOntology No.
glutamine-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-glutamine + tRNAGln = AMP + diphosphate + L-glutaminyl-tRNAGln
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
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Metabolic pathways
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tRNA charging
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SYSTEMATIC NAME
IUBMB Comments
L-glutamine:tRNAGln ligase (AMP-forming)
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CAS REGISTRY NUMBER
COMMENTARY hide
9075-59-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
strain R1
Uniprot
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
strain PAO1
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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the architecture of the GlnRS RNP has differentiated over evolutionary time to maintain glutamine-binding affinity at a weak level, and provides strong evidence for long-distance communication
physiological function
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negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code
additional information
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generation of a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNAGln complex, interaction analysis, detailed overview. Distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Signaling from binding of the tRNA inner elbow, overview. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 A
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + L-glutamate + tRNAGln
AMP + diphosphate + glutamyl-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
primary binding pocket structure, overview
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-
?
ATP + L-glutamine + tRNAGln
AMP + diphosphate + L-glutaminyl-tRNAGln
show the reaction diagram
ATP + L-glutamine + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
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-
-
-
?
ATP + L-glutamine + tRNAGln(CUG)
AMP + diphosphate + L-glutaminyl-tRNAGln(CUG)
show the reaction diagram
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-
-
?
ATP + L-glutamine + tRNAGln(UUG)
AMP + diphosphate + L-glutaminyl-tRNAGln(UUG)
show the reaction diagram
-
-
-
?
additional information
?
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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 + L-glutamate + tRNAGln
AMP + diphosphate + glutamyl-tRNAGln
show the reaction diagram
P00962
-
-
-
?
ATP + L-glutamine + tRNAGln
AMP + diphosphate + L-glutaminyl-tRNAGln
show the reaction diagram
ATP + L-glutamine + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
-
-
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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can partially replace Mg2+ in activation of ATP-diphosphate exchange
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-Methyleneglutamine
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5'-O-[N-(L-glutaminyl)sulfamoyl] adenosine
5,5'-dithiobis(2-nitrobenzoate)
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Br-
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0.1 M, 50% inhibition
Cl-
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0.2 M, 50% inhibition
Glu-AMS
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Glutamic acid 4-hydroxamate
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glutaminol adenylate
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; competitive to glutamine
glutaminol adenylate methyl phosphate ester
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; competitive to glutamine
glutaminyl-beta-ketophosphonate-adenosine
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i.e. Gln-KPA, selective, competitive inhibition of GlnRS, contrast, Gln-KPA inhibits GlnRS by binding competitively but weakly at two distinct sites on the enzyme, the glutamine and the AMP modules of Gln-KPA, connected by the beta-ketophosphonate linker, cannot bind GlnRS simultaneously, and that one Gln-KPA molecule binds the AMP-binding site of GlnRS through its AMP module, whereas another Gln-KPA molecule binds the glutamine-binding site through its glutamine module, mechanism, overview
glutamyl-beta-ketophosphonate-adenosine
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i.e. Glu-KPA, competitive inhibition, non-cognate, binds weakly at one site on the monomeric enzyme
I-
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0.06 M, 50% inhibition
L-Glutamic acid
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competitive inhibition of the wild-type and mutant enzymes
p-hydroxymercuribenzoate
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S-Carbamoylcysteine
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S-Carbamoylserine
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tRNA
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above 0.6 mg/ml
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
putrescine
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can partially replace Mg2+ in activation, with 32% efficiency
spermidine
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can partially replace Mg2+ in activation, with 12% efficiency
spermine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.038 - 660
ATP
0.028 - 17.8
Gln
240
L-glutamate
mutant C229R GlnRS, with tRNAGln
0.05 - 46.3
L-glutamine
0.000019 - 0.31
tRNAGln
0.0001
tRNAGln in unfractionated tRNA
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-
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016 - 6.27
ATP
0.00041 - 0.046
L-glutamate
0.0025 - 6.08
L-glutamine
3.3
tRNAGln
Escherichia coli
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-
additional information
ATP
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0013
5'-O-[N-(L-glutaminyl)sulfamoyl] adenosine
0.00028
glutaminol adenylate
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versus glutamine
0.01
glutaminol adenylate methyl phosphate ester
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versus glutamine
0.65
glutaminyl-beta-ketophosphonate-adenosine
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pH 7.2, 37C, versus L-glutamine
2.8
glutamyl-beta-ketophosphonate-adenosine
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pH 7.2, 37C, versus L-glutamine
17 - 96
L-Glutamic acid
additional information
additional information
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inhibition kinetics
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.1074
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additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2
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ATP-diphosphate exchange
7
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assay at
8.5
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aminoacylation
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature; assay at room temperature
25
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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of seedlings
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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localization of cytoplasmic GlnRS in mitochondrial Gln-tRNA synthesis
Manually annotated by BRENDA team
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enzyme DNA sequence contains a lysine-rich nuclear targeting sequence motif
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65400 - 71200
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sucrose density gradient sedimentation
68500
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electrophoresis of the native enzyme in polyacrylamide gels of various concentrations
138000
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gel filtration
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant full-length GlnRS grown in microbatch in the presence of PEG 3350, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement
vapour-diffusion method. Orthorombic crystals are obtained that belong to space group P2(1)2(1)2(1) and diffract to 2.3 A resolution
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2.5 A resolution; structure of the enzyme with its cognate glutaminyl-tRNA and ATP; the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules
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2.8 A resolution; structure of the enzyme with its cognate glutaminyl-tRNA and ATP
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analysis of the crystal structure of GlnRS-tRNAGln complex bound to the glutaminyl adenylate analogue 5'-O-[N-(L-Gln)sulfamoyl] adenosine
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cocrystallization of the purified enzyme with tRNAGln and inhibitor QSI, X-ray diffraction structure determination at 2.4 A resolution and analysis
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crystal structure of three misacylating mutants of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNAGln and ATP
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crystals of the GlnRS-tRNA(2'H)Gln complex bound to the ATP analog AMPCPP and glutamine are grown by microseeding with crystals of the GlnRS-tRNAGln-ATP ternary complex. Crystals grew in 1-2 weeks by vapor diffusion over a reservoir containing 2 M ammonium sulfate, 10 mM Pipes, pH 7.5, 10 mM MgCl2 and 2 mM DTT
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detailed structural comparison between Met-tRNA synthetase and Gln-tRNA synthetase
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GlnRS-tRNAGln complex, 6.6 mg/ml protein in 10 mM PIPES, pH 7.5, 10 mM MgCl2, and 1.8-5.4 mM tRNA. The tRNA/analog solution is then mixed with equal volumes of a 6.3 mg/ml solution of GlnRS, containing 5mM PIPES, pH 7.0, and 5 mM 2-mercaptoethanol, X-ray diffraction structure determination and analysis at 2.6 A resolution; purified recombinant GlnRS C229R-tRNAGln complex, a protein solution containing 6.3mg/ml GlnRS prepared in 5 mM PIPES, pH 7.0, 5 mM 2-mercaptoethanol, is mixed with the tRNAGln solution, X-ray diffraction structure determination and analysis at 2.6 A resolution
microbatch-under-oil method, using 50 mM NH4Br, 50 mM KC2H3O2, 100 mM HEPES (pH 7.5), and 20% (w/v) polyethylene glycol 20000
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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strain KL301 enzyme is stable, temperature-sensitive mutant enzyme loses about 70% of its activity
additional information
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several noncognate tRNAs protect the enzyme against thermal inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 20 mM potassium phosphate, pH 7.5, 50 mM KCl, 1 mM DTT, 50% glycerol, stable for 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
IgG Sepharose column chromatography and Superdex HiLoad 16/60 gel filtration
Ni-NTA agarose column chromatography
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recombinant His-tagged chimeric mutant enzyme from strain BL21(DE3) or the temperature sensitive strain JP1449(DE3) by nickel affinity chromatography
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recombinant His-tagged GlnRS mutants from strain BL21-DE3 by nickel affinity chromatography
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA sequence determination and analysis, enzyme contains a N-terminal lysine-rich sequence KPKKKKEK, that may function as a nuclear targetng signal as well as in regulation of gene expression, since the 8 amino acid peptide derived from the motif interacts with DNA and changes the DNA molecule form from B to Z form
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Saccharomyces cerevisiae strain BCY123
expression of full length and C-terminal truncated GlnRS, lacking the the Yqey domain, and of the isolated Yqey protein, in Escherichia coli strain ER2566
expression of His-tagged GlnRS mutants in strain BL21-DE3
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expression of the His-tagged chimeric mutant enzyme in Escherichia coli strain BL21(DE3) or the temperature sensitive strain JP1449(DE3)
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gene glnS, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21
overexpression in Escherichia coli
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overexpression of various Myc-tagged enzyme mutants in human embryonic kidney 293 cells
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the vector pET28a is used, Escherichia coli JP1449DE3 cells are used
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A29X
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site-directed mutagenesis
C229R
site-directed mutagenesis, transplanting the conserved arginine residue from glutamyl-tRNA synthetase, EC 6.1.1.17, to glutaminyltRNA synthetase improves the KM of GlnRS for noncognate glutamate
C229R/Q255I
site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Gln, but weakly with L-Glu
C229R/Q255I/S227A/F233Y
site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Gln, but activity with L-Glu
cGluGlnRS
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a chimeric protein, consisting of the catalytic domain of GluRS and the anticodon-binding domain of GlnRS, is constructed
D235A
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saturation mutagenesis, only little complementation of glnS-deficient strain
D486R/L488Q
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the double mutant causes a relaxed tRNA anticodon specificity
D66E
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saturation mutagenesis, 18fold increased Km for glutamine, decreased turnover
D66F
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saturation mutagenesis, highly increased Km for glutamine, 1200fold decrease in activity
D66G
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saturation mutagenesis, only little complementation of glnS-deficient strain
D66H
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saturation mutagenesis, only little complementation of glnS-deficient strain
D66R
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saturation mutagenesis, only little complementation of glnS-deficient strain
E222K
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site-directed mutagenesis, mutational structure-function study, the residue is part of the invariant Hub, the mutation leads to mischarging and affected cognate tRNAGln recognition
E323A
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site-directed mutagenesis, the mutation produces small but consistent 2 to 3fold improvements in glutamine-binding affinity compared to the wild-type enzyme
E34A
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site-directed mutagenesis, the mutant shows highly increased Km and reduced kcat and activity compared to the wild-type enzyme
E34D
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site-directed mutagenesis, the mutant shows highly increased Km and reduced kcat and activity compared to the wild-type enzyme
E34Q
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site-directed mutagenesis, the mutant shows highly increased Km and reduced kcat and activity compared to the wild-type enzyme
E73A
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site-directed mutagenesis, the mutant shows highly increased Km and reduced kcat and activity compared to the wild-type enzyme
E73Q
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site-directed mutagenesis, the mutant shows highly increased Km and reduced kcat and activity compared to the wild-type enzyme, product release remains the rate-limiting step in E73Q
F233D
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saturation mutagenesis, highly increased Km for glutamine, 3700fold decrease in activity
F233L
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saturation mutagenesis, 19fold increased Km for glutamine, decreased turnover
F233Y
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saturation mutagenesis, increased Km for glutamine, increased turnover
K194A
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site-directed mutagenesis, the mutation perturbs the dissociation constant in ATP binding
K401A
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site-directed mutagenesis, the mutant shows reduced kcat compared to the wild-type enzyme
L136A
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site-directed mutagenesis, the mutation perturbs the dissociation constant in ATP binding
N320A
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site-directed mutagenesis, the mutation produces small but consistent 2 to 3fold improvements in glutamine-binding affinity compared to the wild-type enzyme
N336A
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site-directed mutagenesis, the mutation removes contact with the ribose at U38, but does not significantly influence glutamine affinity
N370A
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site-directed mutagenesis, the mutation removes contact with the base of U38, but does not significantly influence glutamine affinity
Q255I
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site-directed mutagenesis, mutational structure-function study, the residue is part of the invariant Hub, the mutation leads to reduced specificity for cognate Gln recognition and increased Glu recognition
Q318A
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site-directed mutagenesis, the mutation produces small but consistent 2 to 3fold improvements in glutamine-binding affinity compared to the wild-type enzyme
Q517A
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site-directed mutagenesis, the mutant shows reduced kcat compared to the wild-type enzyme
R30A
site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Glu
R30K
site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows weak activity with L-Glu
R410A
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site-directed mutagenesis, the mutation removes contact with the base of C34, but does not significantly influence glutamine affinity
R520A
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site-directed mutagenesis, the mutant shows reduced kcat compared to the wild-type enzyme
R545A
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site-directed mutagenesis, the mutant shows reduced kcat compared to the wild-type enzyme
T316A
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site-directed mutagenesis, the mutation produces small but consistent 2 to 3fold improvements in glutamine-binding affinity compared to the wild-type enzyme
T547A
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site-directed mutagenesis, the mutant shows reduced kcat compared to the wild-type enzyme
Y211F
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saturation mutagenesis, 60fold increased Km for glutamine, decreased turnover
Y211F/F233Y
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saturation mutagenesis, increased Km for glutamine, about 6fold decreased activity
Y211G
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saturation mutagenesis, only little complementation of glnS-deficient strain
Y211H
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site-directed mutagenesis, mutational structure-function study, the residue is part of the connection in the quaternary cognate-complex, the mutants shows slow solvation dynamics in the active site
Y211L
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saturation mutagenesis, unaffected Km for glutamine, decreased turnover
Y211S
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saturation mutagenesis, 1700fold decrease in activity
Y240E
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site-directed mutagenesis, active site mutant, 5fold improved glutamic acid recognition in vitro, partial complementation of an enzyme-deficient strain
Y240E/G
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site-directed mutagenesis, mutational structure-function study, the residue is part of the Hub common to ligand-free and quaternary cognate-complex, the mutant shows increased Glu recognition in vitro and in vivo
Y240G
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site-directed mutagenesis, active site mutant, 3fold improved glutamic acid recognition in vitro, partial complementation of an enzyme-deficient strain
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
urea induces equilibrium denaturation of glutaminyl-tRNA synthetase, existence of a stable intermediate state at around 2 M urea, existence of an induced molten globule state in a large multidomain protein which is separated from the native and the denatured protein by high activation energy barriers
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