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Information on EC 6.1.1.5 - isoleucine-tRNA ligase and Organism(s) Escherichia coli and UniProt Accession P00956
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6.1.1.5 isoleucine-tRNA ligaseSpecify your search results
Word Map
- 6.1.1.5
- synthetases
- aminoacyl-trna
- aminoacylation
-
isoleucylation
- valyl-trna
-
misactivated
- methionyl-trna
- leurs
-
mischarged
-
pseudomonic
-
post-transfer
-
noncognate
- valrs
-
mupirocin-resistant
-
misacylated
-
anticodons
-
aarss
-
kmsks
- glnrs
-
trna-dependent
-
pretransfer
- lysyl-trna
- molecular biology
- medicine
- drug development
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
isoleucyl-trna synthetase, ilers, iars2, isoleucyl trna synthetase, iles2, ile-trna synthetase, isoleucine-trna synthetase, mitochondrial isoleucyl-trna synthetase, iles1, isoleucyl-transfer rna synthetase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
IleRS
IRS
Isoleucine translase
Isoleucine--tRNA ligase
Isoleucine-transfer RNA ligase
Isoleucine-tRNA synthetase
Isoleucyl-transfer ribonucleate synthetase
Isoleucyl-transfer RNA synthetase
Isoleucyl-tRNA synthetase
Mupirocin resistance protein
Synthetase, isoleucyl-transfer ribonucleate
IleRS
-
-
-
-
IRS
-
-
-
-
Isoleucine translase
-
-
-
-
Isoleucine--tRNA ligase
-
-
-
-
Isoleucine-transfer RNA ligase
-
-
-
-
Isoleucine-tRNA synthetase
-
-
-
-
Isoleucyl-transfer ribonucleate synthetase
-
-
-
-
Isoleucyl-transfer RNA synthetase
-
-
-
-
Isoleucyl-tRNA synthetase
-
-
-
-
Mupirocin resistance protein
-
-
-
-
Synthetase, isoleucyl-transfer ribonucleate
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + L-isoleucine + tRNAIle = AMP + diphosphate + L-isoleucyl-tRNAIle
catalytic and kinetic mechanism analysis, reaction cycle, overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
esterification
aminacylation
deacylation
esterification
-
-
-
-
aminacylation
-
-
-
-
deacylation
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
L-isoleucine:tRNAIle ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY
9030-96-0
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Ile-tRNAIle + 3-mercaptopropionate
S-Isoleucyl-3-mercaptopropionate + tRNAIle
-
-
-
?
Ile-tRNAIle + cysteine
tRNAIle + isoleucylcysteine
-
D- and L-isomer of Lys
D-isoleucylcysteine and L-isoleucylcysteine
?
Ile-tRNAIle + L-cysteine methyl ester
tRNAIle + isoleucyl-L-cysteine methyl ester
-
-
-
?
Ile-tRNAIle + N-acetylcysteine
S-isoleucyl-N-acetylcysteine + tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
r
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
usage of purified recombinant tRNAGAU Ile (with G1-C72 instead of the wild-type A1-U72 sequence) overexpressed in Escherichia coli strain BL21(DE3)
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
the anticodon for methionine and isoleucine tRNAs differ by a single nucleotide, changing this nucleotide in an isoleucine tRNA is sufficient to change aminoacylation specificity to methionine
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
formation of an aminoacyl adenylate reaction intermediate
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
the binding region of the adenine moiety contains a wide hydrophobic pocket large enough to afford three linear aromatic rings
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
physiological function is Thr formation of Ile-tRNA and editing of inadvertently misactivated homocysteine
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
a two-step reaction, the first of which, the amino acid activation step, is reversible, while the second aminoacylation step is not, the amino acid editing site for LeuRS resides within the homologous CP1 domain containing a conserved threonine conferring amino acid substrate recognition, editing mechanism, some positions of the site are idiosyncratic to IleRS, residues Arg249, Asp251, Thr252, Met336, and Val338 are involved,overview
-
-
?
additional information
?
-
enzymatic reactions catalyzed by IleRS include amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme, in the synthetic pathway. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) ortRNA-dependent (3). Misacylated tRNA is deacylated through posttransferediting, overview
-
-
?
additional information
?
-
-
enzymatic reactions catalyzed by IleRS include amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme, in the synthetic pathway. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) ortRNA-dependent (3). Misacylated tRNA is deacylated through posttransferediting, overview
-
-
?
additional information
?
-
wild-type an dmutant enzymes IleRS are tested in reactions with both L-valine and L-isoleucine, neither wild-type nor D342A IleRS significantly deacylates Ile-tRNAIle under steady-state conditions
-
-
?
additional information
?
-
-
wild-type an dmutant enzymes IleRS are tested in reactions with both L-valine and L-isoleucine, neither wild-type nor D342A IleRS significantly deacylates Ile-tRNAIle under steady-state conditions
-
-
?
additional information
?
-
-
discrimination of 20 amino acids in aminoacylation of modified tRNAIle-C-C-A(3'NH2)
-
-
?
additional information
?
-
-
Ile + ATP + enzyme/Ile-AMP-enzyme + diphosphate, isoleucine-dependent ATP-diphosphate exchange
-
-
?
additional information
?
-
-
Ile + ATP + enzyme/Ile-AMP-enzyme + diphosphate, isoleucine-dependent ATP-diphosphate exchange
-
-
?
additional information
?
-
-
Ile + ATP + enzyme/Ile-AMP-enzyme + diphosphate, isoleucine-dependent ATP-diphosphate exchange
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-
?
additional information
?
-
-
Ile + ATP + enzyme/Ile-AMP-enzyme + diphosphate, isoleucine-dependent ATP-diphosphate exchange
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
r
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
-
-
-
?
ATP + L-isoleucine + tRNAIle
AMP + diphosphate + L-isoleucyl-tRNAIle
-
physiological function is Thr formation of Ile-tRNA and editing of inadvertently misactivated homocysteine
-
-
?
additional information
?
-
enzymatic reactions catalyzed by IleRS include amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme, in the synthetic pathway. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) ortRNA-dependent (3). Misacylated tRNA is deacylated through posttransferediting, overview
-
-
?
additional information
?
-
-
enzymatic reactions catalyzed by IleRS include amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme, in the synthetic pathway. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) ortRNA-dependent (3). Misacylated tRNA is deacylated through posttransferediting, overview
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Zn2+
Mg2+
Zinc
additional information
polyamines can replace part of the Mg2+ ions in the aminoacyl-tRNA synthetase reactions, spermidine can replace Mg2+ (KME3) and Mg2+ (KME42), which are involved in the forward and backward transfer reaction, while the competition with Mg2+ (KMR) is much weaker, kinetics, overview
Mg2+
required. In addition to the Mg2+ in MgATP or Mg-diphosphate, only two tRNA-bound Mg2+ are required to explain the magnesium dependence in the best-fit mechanism. The first Mg2+ might be present in all steps before the second activation and is obligatory in the first reorganizing step and transfer step. The second Mg2+ is present only at the transfer step, whereas elsewhere it prevents the reaction, including the activation reaction
Mg2+
-
at least 2 bound Mg2+ or spermidines required for the binding of tRNA to the enzyme
Mg2+
-
optimum at very low concentration (0.5-0.7 mM), slight inhibition at higher concentration
Mg2+
-
enzyme-bound isoleucyl-AMP can be formed in the absence of Mg2+ and spermine
Zinc
-
a single zinc atom which is coordinated to ligands is contained in the catalytic domain, a second, functionally essential zinc is bound to ligands at the C-terminal end of the 939 amino acid polypeptide, the average zinc environment contains predominantly sulfur ligands with a Zn-S distance of 2.33 A
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
chloride
100 mM KCl causes 50% inhibition if the ionic strength is kept constant with potassium acetate. The KappM (tRNA) value is increased from 570 nm to 1370 nM when the KCl concentration is increased from 0 to 200 mM. Potassium acetate inhibits weakly, but K2SO4 inhibits stronger than KCl
K+
potassium acetate inhibits weakly, but K2SO4 inhibits stronger than KCl. KCl and potassium acetate inhibit above 50 mM concentrations when high enough K+ concentration for full activity is reached
2',3'-dialdehyde of tRNAile
-
used to label the binding site for the 3'end of tRNA on the synthetase, incubation of the reagent with IleRS results in a rapid loss of tRNAIle aminoacylation and isoleucine-dependent isotopic ATP-PPi exchange activities
-
2,3-dideoxy-adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
-
IC50: 0.0064 mM
2-deoxy-adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
-
IC50: 0.28 mM
2-iodo-L-isoleucine-NHSO2-AMP
-
highly potent inhibitor, hydrophobic interaction of the 2-substituent of the inhibitor with the adenine binding site of the enzyme
3-deoxy-adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
-
IC50: 0.035 mM
adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
-
IC50: 0.000265 mM
ester analogues of isoleucyl adenylate
-
with or without cyclic substitutents at the adenine moiety
hydroxamate analogues of isoleucyl adenylate
-
with or without cyclic substitutents at the adenine moiety
isoleucyl isovanilloids
-
e.g. the isovanillic hydroxamate and amide analogue
isoleucyl vanilloids
-
e.g. the vanillic hydroxamate with a phenolic hydoxyl at the para-position
pyridoxal 5'-diphospho-5'-adenosine
-
affinity labeling reagent for the ATP-binding site, incubation of the reagent with IleRS results in a rapid loss of tRNAIle aminoacylation and isoleucine-dependent isotopic ATP-PPi exchange activities
spermine
-
catalyzes ATP-diphosphate exchange, no inhibition of specific aminoacylation of tRNAIle
pseudomonic acid
-
bifunctional inhibitor with characteristics of both isoleucine and ATP
additional information
-
the ribose of ATP/AMP can be substituted by its biosteres acyclic amide, hydroxamate, dihydroisooxazole, and dihydrooxazole, binding structure, overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
spermidine
-
at least 2 bound Mg2+ or spermidines required for binding of tRNA to the enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
kinetic analysis, rapid equilibrium determinations, steady-state kinetics. The analysis strongly suggests an additional activation step, formation of a new isoleucyl-AMP before the isoleucyl-tRNA is freed from the enzyme. The removal of Ile-tRNA is possible without the formation of Ile-AMP if both isoleucine and ATP are bound to the E-Ile-tRNA complex, but this route covers only 11% of the total formation of Ile-tRNA. In addition to the Mg2+ in MgATP or Mg-diphosphate, only two tRNA-bound Mg2+ are required to explain the magnesium dependence in the best-fit mechanism. The first Mg2+ might be present in all steps before the second activation and is obligatory in the first reorganizing step and transfer step. The second Mg2+ is present only at the transfer step, whereas elsewhere it prevents the reaction, including the activation reaction
-
additional information
additional information
single-turnover kinetic analysis
-
additional information
additional information
-
single-turnover kinetic analysis
-
additional information
additional information
steady-state kinetics of activation of cognate L-Ile and noncognate amino acid L-Val by Y59 IleRS mutant variants, and transfer kinetics, as well as tRNA-dependent hydrolysis of cognate isoleucyl-AMP, overview
-
additional information
additional information
-
steady-state kinetics of activation of cognate L-Ile and noncognate amino acid L-Val by Y59 IleRS mutant variants, and transfer kinetics, as well as tRNA-dependent hydrolysis of cognate isoleucyl-AMP, overview
-
additional information
additional information
-
Km value of mutant enzynes with altered metal-binding sites
-
additional information
additional information
-
steady-state parameters for tRNA-independent pre-transfer editing by IleRS and its mutants determined by varying concentrations of noncognate valine, overview. Kinetic method to distinguish among three models for pre-transfer editing by IleRS, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0064
2,3-dideoxy-adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
Escherichia coli
-
IC50: 0.0064 mM
0.28
2-deoxy-adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
Escherichia coli
-
IC50: 0.28 mM
0.035
3-deoxy-adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
Escherichia coli
-
IC50: 0.035 mM
0.000265
adenosine-5-[(2S,3S)-2-amino-3-methylpentanoyl]-sulfamate
Escherichia coli
-
IC50: 0.000265 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
under error-prone conditions Streptomyces griseus IleRS is able to rescue the growth of an Escherichia coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability
physiological function
physiological function
-
hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes, e.g. IleRS, occurs in a spatially separate domain inserted into the catalytic Rossmann fold. tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. Balance between pretransfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate, overview. In IleRS both pre- and post-transfer editing are important
additional information
evolution
enzyme IleRS is a class I aaRS enzyme built around the conserved N-terminal Rossmann fold catalytic domain, which encloses the synthetic site. Phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. The accuracy of Ile-tRNAIle synthesis may be entirely ensured by the powerful post-transfer editing domain, which is absolutely conserved through evolution. The origin of discrimination against valine in the synthetic reaction is evolutionarily conserved in IleRS, overview
evolution
the enzyme belongs to the class I amino acyl-tRNA synthetases (aaRS)
physiological function
isoleucyl-tRNA synthetase (IleRS) is responsible for decoding of isoleucine codons in all three domains of life. Besides isoleucine, IleRS also activates non-cognate valine with a discrimination factor as low as 200 and thus it requires editing to enhance accuracy of isoleucyltRNAIle (Ile-tRNAIle) synthesis. Enzyme IleRS is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity as an optional property. Some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability. Specificity of the editing pathways, overview
physiological function
the accuracy of protein synthesis relies on the capacity of aminoacyl-tRNA synthetases (aaRS) to couple cognate amino acids and tRNAs in a two-step reaction that defines the genetic code. In the first step, the amino acid is activated by condensation with ATP to form an enzyme-bound aminoacyl-adenylate (aa-AMP) intermediate with release of pyrophosphate. The second step comprises attack by the terminal 2'- or 3'-OH group of tRNA on the carbonyl carbon atom of aa-AMP, followed by transfer of the aminoacyl moiety to tRNA and release of AMP. The amino acid activation and transfer steps occur within the synthetic active site located in the catalytic domain. Enzyme IleRS possesses an inactivated post-transfer editing domain still capable of robust tRNA-dependent editing. The pretransfer editing activity resides within the synthetic site. Specific recognition of tRNAs by cognate aaRSs is ensured by a network of interactions, based on direct and indirect recognition elements that are embedded in all levels of tRNA structure. Noncognate amino acids that structurally and chemically resemble the cognate substrates are often not well-distinguished in the synthetic reactions alone, so that discrimination is based in part on inherent aaRS-based hydrolytic editing
additional information
a Rossmann fold peptide is loacted directly N-terminal to the strictly conserved HIGH motif. The class I IleRS Rossmann fold accommodates both synthetic and tRNA-dependent pretransfer hydrolysis pathways within the synthetic site. Residue Y59 acts as a gatekeeper of the IleRS synthetic site
additional information
-
a Rossmann fold peptide is loacted directly N-terminal to the strictly conserved HIGH motif. The class I IleRS Rossmann fold accommodates both synthetic and tRNA-dependent pretransfer hydrolysis pathways within the synthetic site. Residue Y59 acts as a gatekeeper of the IleRS synthetic site
additional information
the simultaneous presence of Ile-tRNA and Ile-AMP can cause additional possibilities to proofreading mechanisms of the enzyme, existence of an additional activation step, formation of a new isoleucyl-AMP before the isoleucyl-tRNA is freed from the enzyme. The removal of Ile-tRNA is possible without the formation of Ile-AMP if both isoleucine and ATP are bound to the E-Ile-tRNA complex, but this route covers only 11% of the total formation of Ile-tRNA
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
the amino acid editing site for IleRS resides within the homologous CP1 domain: threonine-rich peptide and a second conserved GTG region that are separated by about 100 amino acids comprise parts of the hydrolytic editing site, comparison to LeuRS, some positions of the site are idiosyncratic to IleRS, tertiary and primary structure analysis of the amino acid editing site, overview
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D342A
site-directed mutagenesis, the mutant exhibits slightly reduced aminoacylation activity compared to the wild-type enzyme, the post-transfer editing deficient D342A IleRS accumulates AMP by pretransfer editing and by tRNA misacylation when the noncognate aa-AMP evades this hydrolytic reaction, neither wild-type nor D342A IleRS significantly deacylates Ile-tRNAIle under steady-state conditions
Y59F
site-directed mutagenesis, mutation of a conserved residue located within the active site of bacterial IleRS, directly adjacent to the binding site for the 3'-terminal hydroxyl group of tRNA, aminacylation activity is about 35fold reduced compared to the wild-type enzyme
Y59F/D342A
site-directed mutagenesis, the mutant activity is similar to the wild-type
Y59T
site-directed mutagenesis, mutation of a conserved residue located within the active site of bacterial IleRS, directly adjacent to the binding site for the 3'-terminal hydroxyl group of tRNA, Km and kcat values measured for Y59T are increased by 10fold and decreased by 5fold, respectively, for both isoleucine and valine substrates compared to the wild-type enzyme, aminacylation activity is about 12fold reduced
Y59T/D342A
site-directed mutagenesis, kinetic analysis of Y59F/D342A IleRS does not provide reliable results because of the very slow aminoacylation/misacylation
AIleRS
-
mutant enzymes IleRS(C922S) and AIleRS with replacement of Cys922 through Ala939 with a 33 amino acid peptide unable to bind zinc. Mutant enzymes have altered zinc binding and aminoacylation activity
D342A
-
site-directed mutagenesis, the IleRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations
IleRS(C922S)
-
mutant enzymes IleRS(C922S) and AIleRS with replacement of Cys922 through Ala939 with a 33 amino acid peptide unable to bind zinc. Mutant enzymes have altered zinc binding and aminoacylation activity
T243R
-
site-directed mutagenesis, the mutant retains tRNA-independent editing at a level identical to the WT enzyme and shows increased ATP hydrolysis compared to the wild-type enzyme
T243R/D342A
-
site-directed mutagenesis, the IleRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations
additional information
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant His-tagged IleRS from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene ileS, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Yanagisawa, T.; Lee, J.T.; Wu, H.C.; Kawakami, M.
Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli
J. Biol. Chem.
269
24304-24309
1994
Escherichia coli
Glasfeld, E.; Landro, J.A.; Schimmel, P.
C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase
Biochemistry
35
4139-4145
1996
Escherichia coli
Jakubowski, H.
Proofreading in trans by an aminoacyl-tRNA synthetase. A model for single site editing by isoleucyl-tRNA synthetase
Nucleic Acids Res.
24
2505-2510
1996
Escherichia coli
Zhou, L.; Rosevear, P.R.
Mutation of the carboxy terminal zinc finger of E. coli isoleucyl-tRNA synthetase alters zinc binding and aminoacylation activity
Biochem. Biophys. Res. Commun.
216
648-654
1995
Escherichia coli
Auld, D.S.; Schimmel., P.
Switching recognition of two tRNA synthetases with an amino acid swap in a designed peptide
Science
267
1994-1996
1995
Escherichia coli
Landro, J.A.; Schmidt, E.; Schimmel, P.
Thiol ligation of two zinc atoms to a class I tRNA synthetase. Evidence for unshared thiols and role in amino acid binding and utilization
Biochemistry
33
14213-14220
1994
Escherichia coli
Xu, B.; Trawick, B.; Krudy, G.A.; Phillips, R.M.; Zhou.L.; Rosevear, P.R.
Probing the metal binding sites of E. coli isoleucyl-tRNA synthetase
Biochemistry
33
398-402
1994
Escherichia coli
Airas, R.K.
Analysis of the isoleucyl-tRNA synthetase reaction by total rate equation. Magnesium and spermidine in the tRNA kinetics
Eur. J. Biochem.
210
451-454
1992
Escherichia coli, Escherichia coli B / ATCC 11303
Schimmel, P.; Landro, J.A.; Schidt, E.
Evidence for distinct locations for metal binding sites in two closely related class I tRNA synthetases
J. Biomol. Struct. Dyn.
11
571-581
1993
Escherichia coli
Glasfeld, E.; Schimmel, P.
Zinc-dependent tRNA binding by a peptide element within a tRNA synthetase
Biochemistry
36
6739-6744
1997
Escherichia coli
Grosjean, H.; Charlier, J.
Comparative studies on the isoleucyl-tRNA synthetases from Bacillus stearothermophilus and Escherichia coli. II. The effect of magnesium ions in the transacylation reaction
FEBS Lett.
18
342-346
1971
Geobacillus stearothermophilus, Escherichia coli
Steinmetz Kayne , M.; Cohn, M.
Cation requirements of isoleucyl-tRNA synthetase from Escherichia coli
Biochem. Biophys. Res. Commun.
46
1285-1291
1972
Escherichia coli
Holler, E.
Isoleucyl transfer ribonucleic acid synthetase of Escherichia coli B. Effects of magnesium and spermine on the amino acid activation reaction
Biochemistry
12
1142-1149
1973
Escherichia coli, Escherichia coli B / ATCC 11303
Durekovic, A.; Flossdorf, J.; Kula, M.R.
Isolation and properties of isoleucyl-tRNA synthetase from Escherichia coli MRE 600
Eur. J. Biochem.
36
528-533
1973
Escherichia coli, Escherichia coli MRE 600
Carr, A.C.; Igloi, G.L.; Penzer, G.R.; Plumbridge, J.A.
The effect of spermine anf Mg2+ on the catalytic mechanism of isoleucine:tRNA ligase
Eur. J. Biochem.
54
169-173
1975
Escherichia coli, Escherichia coli B / ATCC 11303
Hammer-Raber, B.; Rainey, P.M.; Holler, E.
L-Isoleucyl-tRNA synthetase aus E. coli. pH-Abhngigkeit der Enzym-Substrat Komplexbildung
Hoppe-Seyler's Z. Physiol. Chem.
357
295
1976
Escherichia coli
-
Moe, J.G.; Piszkiewicz, D.
Purification of isoleucyl transfer ribonucleic acid synthetase by affinity chromatography on blue dextran-Sepharose
FEBS Lett.
72(1)
147-150
1976
Escherichia coli, Escherichia coli B / ATCC 11303
Hughes, J.; Mellows, G.
Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid
Biochem. J.
176
305-318
1978
Escherichia coli
Freist, W.; Sternbach, H.; Cramer, F.
Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNAIle-C-C-A(3'NH2)
Eur. J. Biochem.
169
33-39
1987
Escherichia coli, Escherichia coli MRE 600, Saccharomyces cerevisiae
Lee, J.; Kang, S.U.; Kim, S.Y.; Kim, S.E.; Kang, M.K.; Jo, Y.J.; Kim, S.
Ester and hydroxamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases
Bioorg. Med. Chem. Lett.
11
961-964
2001
Escherichia coli
Lee, J.; Kang, S.U.; Kim, S.Y.; Kim, S.E.; Job, Y.J.; Kim, S.
Vanilloid and isovanilloid analogues as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases
Bioorg. Med. Chem. Lett.
11
965-968
2001
Escherichia coli
Lee, J.; Kim, S.E.; Lee, J.Y.; Kim, S.Y.; Kang, S.U.; Seo, S.H.; Chun, M.W.; Kang, T.; Choi, S.Y.; Kim, H.O.
N-Alkoxysulfamide, N-hydroxysulfamide, and sulfamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases
Bioorg. Med. Chem. Lett.
13
1087-1092
2003
Escherichia coli
Kim, S.E.; Kim, S.Y.; KI, S.; Kang, T.; Lee, J.
Deoxyribosyl analogues of methionyl and isoleucyl sulfamate adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetase
Bioorg. Med. Chem. Lett.
15
3389-3393
2005
Escherichia coli
Zhai, Y.; Nawaz, M.H.; Lee, K.W.; Kirkbride, E.; Briggs, J.M.; Martinis, S.A.
Modulation of substrate specificity within the amino acid editing site of leucyl-tRNA synthetase
Biochemistry
46
3331-3337
2007
Escherichia coli
Baouz, S.; Schmitter, J.M.; Chenoune, L.; Beauvallet, C.; Blanquet, S.; Woisard, A.; Hountondji, C.
Primary structure revision and active site mapping of E. coli isoleucyl-tRNA synthetase by means of maldi mass spectrometry
Open Biochem. J.
3
26-38
2009
Escherichia coli, Escherichia coli EM20031
Dulic, M.; Cvetesic, N.; Perona, J.J.; Gruic-Sovulj, I.
Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases
J. Biol. Chem.
285
23799-23809
2010
Escherichia coli
Dulic, M.; Perona, J.J.; Gruic-Sovulj, I.
Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase
Biochemistry
53
6189-6198
2014
Escherichia coli (P00956), Escherichia coli
Airas, R.K.
Kinetic analysis of the isoleucyl-tRNA synthetase mechanism the next reaction cycle can start before the previous one ends
FEBS open bio
8
244-255
2018
Escherichia coli (P00956)
Cvetesic, N.; Dulic, M.; Bilus, M.; Sostaric, N.; Lenhard, B.; Gruic-Sovulj, I.
Naturally occurring isoleucyl-tRNA synthetase without tRNA-dependent pre-transfer editing
J. Biol. Chem.
291
8618-8631
2016
Streptomyces griseus, Escherichia coli (P00956), Escherichia coli, Saccharomyces cerevisiae (P09436), Saccharomyces cerevisiae, Saccharomyces cerevisiae ATCC 204508 / S288c (P09436)
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