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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Manually annotated by BRENDA team
gene DARS2
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-
Manually annotated by BRENDA team
no activity in Sulfolobus tokodaii
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-
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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
-
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
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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 70°C, but its thermostability increases when complexed to tRNAAsn. tRNAAsn is very stable, as its melting temperature is 85°C. the GatCAB is poorly protected against heat inactivation, as its denaturation starts at 40°C, but when complexed in the transamidosome, the GatCAB becomes fully thermostable at 85°C
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
Helicobacter pylori (strain ATCC 700392 / 26695)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12870
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1 * 12870, calculated from amino acid sequence
15500
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gel filtration
49074
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x * 49074, sequence calculation
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 Tris–HCl buffer, pH 7.0, hanging-drop vapour-diffusion method, 26°C, 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, 24°C, 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 42°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ND-AspRS is stable in low and high salt
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 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
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
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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