Information on EC 6.1.1.23 - aspartate-tRNAAsn ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.23
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RECOMMENDED NAME
GeneOntology No.
aspartate-tRNAAsn ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-aspartate + tRNAAsx = AMP + diphosphate + L-aspartyl-tRNAAsx
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
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aspartate and asparagine metabolism
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SYSTEMATIC NAME
IUBMB Comments
L-aspartate:tRNAAsx ligase (AMP-forming)
When this enzyme acts on tRNAAsp, it catalyses the same reaction as EC 6.1.1.12, aspartate---tRNA ligase. It has, however, diminished discrimination, so that it can also form aspartyl-tRNAAsn. This relaxation of specificity has been found to result from the absence of a loop in the tRNA that specifically recognizes the third position of the anticodon [1]. This accounts for the ability of this enzyme in, for example, Thermus thermophilus, to recognize both tRNAAsp (GUC anticodon) and tRNAAsn (GUU anticodon). The aspartyl-tRNAAsn is not used in protein synthesis until it is converted by EC 6.3.5.6, asparaginyl-tRNA synthase (glutamine-hydrolysing), into asparaginyl-tRNAAsn.
CAS REGISTRY NUMBER
COMMENTARY hide
9027-32-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
gene aspS, ND-AspRS; strain NRC-1, gene aspS
SwissProt
Manually annotated by BRENDA team
gene DARS2
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-
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum
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UniProt
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum DSM 1053
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UniProt
Manually annotated by BRENDA team
no activity in Sulfolobus tokodaii
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-
-
Manually annotated by BRENDA team
no activity in Sulfolobus tokodaii 7
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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during tRNA-dependent asparagine formation, tRNAAsn promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-tRNA from non-discriminating aspartyl-tRNA synthetase active site to the GatCAB amidotransferase site. A transamidosome particle is formed by two GatCABs, two dimeric nondiscriminating-AspRSs and four tRNAsAsn molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl-tRNAAsn without dissociation of the complex. The transamidosome constitutes a transfer-ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + Asp-tRNAAsn
show the reaction diagram
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only the enzyme AspRS2 aspartylates tRNAAsn
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-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsx
AMP + diphosphate + L-aspartyl-tRNAAsx
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 + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsx
AMP + diphosphate + L-aspartyl-tRNAAsx
show the reaction diagram
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-
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
required at 0.1-3 M, salt dependence profile, overview
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
heptapeptide-nucleotide microcin C
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McC, an antimicrobial nucleotide peptide that targets and strongly inhibits aspartyl-tRNA synthetase. The inhibitor cannot be processed by the nonspecific oligopeptidases PepA, PepB, or PepN, the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC, structure, massspectrometric analysis of McC produced by wild-type Escherichia coli cells, overview
L-aspartol adenylate
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i.e. Asp-ol-AMP, a stable analogue of the natural reaction intermediate L-aspartyl adenylate, biphasic, competitive inhibition, differential inhibition of tRNAAsp and tRNAAsn aspartylation by the enzyme, overview
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.005 - 0.03
aspartate
0.033 - 0.28
ATP
0.00006 - 2.23
tRNAAsn
0.00003 - 0.87
tRNAAsp
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014 - 0.6
tRNAAsn
0.021 - 7.2
tRNAAsp
additional information
additional information
Helicobacter pylori
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the wild-type enzyme has a kcat for tRNAAsp that is 60% higher than that of tRNAAsn
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.041 - 0.215
L-aspartol adenylate
additional information
additional information
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inhibition kinetics
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
activities of wild-type and mutant enzymes with different tRNA substrates
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 85
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transamidosome complex: ND-AspRS is thermostable up to 70C, but its thermostability increases when complexed to tRNAAsn. tRNAAsn is very stable, as its melting temperature is 85C. the GatCAB is poorly protected against heat inactivation, as its denaturation starts at 40C, but when complexed in the transamidosome, the GatCAB becomes fully thermostable at 85C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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the partners involved in tRNA-dependent Asn formation assemble into a ternary complex called the transamidosome, consisting of the ND-AspRS, GatCAD amidotransferase, and tRNAAsn
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15500
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gel filtration
300000
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ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn, theoretical
380000
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ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn, determined by gel filtration
400000
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ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn, determined by static light scattering, SLS
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 49074, sequence calculation
homodimer
structure modelling, overview
monomer
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1 * 12870, calculated from amino acid sequence; 1 * 15500, gel filtration
trimer
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ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
post-translational modifications in the N-terminal extension of AspRS neutralizing the lysine-rich motif contained in this domain
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified AspRS, sitting drop vapour diffusion method, 0.0002 ml of 9 mg/ml protein is mixed with 0.0002 ml of reservoir solution containing 0.2 M lithium sulfate, 0.1 M Bis-Tris, pH 5.5, and 23% w/v PEG 3350, X-ray diffraction structure determination and analysis at 2.8 A resolution
N-terminal anticodon-binding domain of non-discriminating aspartyl-tRNA synthetase, hanging drop vapor diffusion method, using 30% (w/v) PEG 4000, 100 mM ammonium sulfate, 100 mM sodium citrate at pH 5.6
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purified recombinant enzyme, 5-8 mg/ml in 20 mM TrisHCl buffer, pH 7.0, hanging-drop vapour-diffusion method, 26C, from 0.002 ml protein solution is mixed with 0.002 ml of reservoir solution containing 100 mM sodium HEPES buffer, pH 7.5, containing 100 mM NaCl and 1.6 M (NH4)2SO4, equilibration against 0.7 ml reservoir solution, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement
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purified recombinant enzyme, X-ray diffraction structure determination and analysis at 2.3 A resolution
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hanging drop technique, 24C, 5 days in Crystal Screen I or II, solutions containing either polyethylene glycol or ethylene glycol
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a complete dataset of the transamidosome is collected to 3 A resolution
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purified 520 kDa transamidosome complex formed by two dimeric nondiscriminating-AspRSs, two trimeric GatCABs, and four tRNAsAsn molecules, X-ray diffraction structure determination and analysis at 3.0 A resolution, molecular replacement
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 42
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the recombinant purified N-terminal anticodon-binding domain of non-discriminating aspartyl-tRNA synthetase is stable up to 42C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ND-AspRS is stable in low and high salt
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, purified recombinant enzyme, in 50% glycerol, and 33 mM phosphate, pH 7.4, 3 mM Tris-HCl, 1.5 mM 2-mercaptoethanol, and 0.5 mM phenylmethanesulfonyl fluoride, stable for months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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Methanothermobacter thermautotrophicum
Ni-NTA column chromatography, and gel filtration
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recombinant enzyme
recombinant enzyme from Escherichia coli strain BL21(DE3)
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recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography
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recombinant His-tagged AsnRS from Escherichia coli strain DH5alpha by nickel affinity chromatography
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recombinant His-tagged AspRS, cleavage of the N-terminal His-tag
recombinant His-tagged enzyme from strain ADD1976 by nickel affinity chromatography
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recombinant His-tagged wild-type and mutant ND-AspRS in Escherichia coli strain trpA34 by nickel affinity chromatography
the transamidosome complex is isolated by preparative gel filtration on a Superdex G200 column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Escherichia coli trypA34 missense mutant transformed with heterologous ND-aspS gene
expression in Escherichia coli
expression in JM101Tr cells
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expression in YBC-603 cells
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expression of C-terminally His-tagged enzyme in strain ADD1976
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for expression in Escherichia coli cells
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gene aspS, DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and mutant enzymes
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gene aspS, phylogenetic tree, expression of His-tagged wild-type and mutant ND-AspRS in Escherichia coli strain trpA34, carrying a D60N mutation in trpA leading to tryptophan auxotrophy, co-expression of tRNAAsn leading to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNAAsn
gene DARS2, DNA and amino acid analysis of wild-type and mutant enzyme
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overexpression in Escherichia coli
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overexpression in Escherichia coli strain BL21(DE3)
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overexpression of His-tagged AsnRS in Escherichia coli strain DH5alpha, the enzyme is toxic when heterologously overexpressed in Escherichia coli, because of sequestration of tRNAAsn as Asp-tRNAAsn, this toxicity is rescued upon coexpression of the Helicobacter pylori Asp/Glu-Adt, EC 6.3.5.6
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overproduced from the cloned gene in Pseudomonas aeruginosa
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recombinant expression of the N-terminally His-tagged AspRS
the N-terminal anticodon-binding domain of non-discriminating aspartyl-tRNA synthetase is expressed in Escherichia coli BL21(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H28Q
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wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp. Mutation H28Q leads to a reverse tRNA preference
H77K/H28Q
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wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp. Mutation P77K/H28Q leads to a reverse tRNA preference
P77K
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wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp. Mutation P77K leads to a reverse tRNA preference and a 3fold increase in specificity for tRNAASp over tRNAAsn
H26A
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
H26A/P84A
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
H26A/P84K
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
H26Q
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
H26Q/P84A
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
H26Q/P84K
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
P84A
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
P84K
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
H26A
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site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
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H26Q
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site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
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H26Q/P84A
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site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
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P84A
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site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
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P84K
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site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRNA substrate specificity compared to the wild-type enzyme, overview
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L81N
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site-directed mutagenesis, the mutation in the anticodon binding domain doubles the kcat for tRNAAsn as compared to the wild-type enzyme
L81N/L86M
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site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme, the L81N/L86M mutant does not follow Michaelis-Menten kinetics
L86M
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site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme
G83K
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aspartylation reaction of the mutant enzyme is 55% as fast as the wild-type enzyme; site-directed mutagenesis, the mutation increases the specificity of tRNAAsp charging over that of tRNAAsn by 4.2fold
H31L
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aspartylation reaction of the mutant enzyme is 84% as fast as the wild-type enzyme; aspartylation reaction of the mutant enzyme is 92% as fast as the wild-type enzyme; site-directed mutagenesis, the mutation increases the specificity of tRNAAsp charging over that of tRNAAsn by 3.5fold
R485K
-
a catalytic site mutant
additional information
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