Information on EC 4.6.1.16 - tRNA-intron lyase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.6.1.16
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RECOMMENDED NAME
GeneOntology No.
tRNA-intron lyase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
pretRNA = a 3'-half-tRNA molecule with a 5'-OH end + a 5'-half-tRNA molecule with a 2',3'-cyclic phosphate end + an intron with a 2',3'-cyclic phosphate and a 5'-hydroxyl terminus
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
endoribonuclease reaction
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tRNA splicing I
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tRNA splicing II
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SYSTEMATIC NAME
IUBMB Comments
pretRNA lyase (intron-removing; cyclic-2',3'-phosphate-forming)
The enzyme catalyses the final stage in the maturation of tRNA molecules.
CAS REGISTRY NUMBER
COMMENTARY hide
117444-13-0
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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
strain 20B-12-1
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-
Manually annotated by BRENDA team
strain M304
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3'-phospho-double stranded-RNA
?
show the reaction diagram
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-
-
-
?
5'-AAGCGACCGACC*AUA*GCUGCA-3' + H2O
?
show the reaction diagram
-
-
-
-
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5'-AAUGCAGCGGUC*AAA*GGUCGC-3' + H2O
?
show the reaction diagram
-
-
-
-
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pre-t-RNAarcheuka + H2O
?
show the reaction diagram
pre-tRNA
?
show the reaction diagram
pre-tRNA + H2O
?
show the reaction diagram
-
-
-
?
pre-tRNA + H2O
tRNA containing 5'hydroxy and 2',3'-cyclic phosphate termini
show the reaction diagram
-
-
-
?
pre-tRNAArg
?
show the reaction diagram
pre-tRNALeu
?
show the reaction diagram
pre-tRNATrp
?
show the reaction diagram
pretRNA
a 3'-half-tRNA molecule with a 5'-OH end + a 5'-half-tRNA molecule with a 2',3'-cyclic phosphate end + an intron with a 2',3'-cyclic phosphate and a 5'-hydroxyl terminus
show the reaction diagram
siRNA + H2O
?
show the reaction diagram
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RNA kinase responsible for the phosphorylation at the 5' position of tRNA 3' exons
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-
?
tRNA + H2O
?
show the reaction diagram
tRNA + H2O
tRNA containing 5'hydroxy and 2',3'-cyclic phosphate termini
show the reaction diagram
-
The enzyme is responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs.
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-
?
tRNA precursor
a 3'-half-tRNA molecule with a 5'-OH end + a 5'-half-tRNA molecule with a 2',3'-cyclic phosphate end + an intron with a 2',3'-cyclic phosphate and a 5'-hydroxyl terminus
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
pre-tRNA
?
show the reaction diagram
pre-tRNA + H2O
?
show the reaction diagram
O29362
-
-
-
?
pre-tRNA + H2O
tRNA containing 5'hydroxy and 2',3'-cyclic phosphate termini
show the reaction diagram
Q9HIY5
-
-
-
?
pretRNA
a 3'-half-tRNA molecule with a 5'-OH end + a 5'-half-tRNA molecule with a 2',3'-cyclic phosphate end + an intron with a 2',3'-cyclic phosphate and a 5'-hydroxyl terminus
show the reaction diagram
siRNA + H2O
?
show the reaction diagram
-
RNA kinase responsible for the phosphorylation at the 5' position of tRNA 3' exons
-
-
?
tRNA + H2O
?
show the reaction diagram
tRNA + H2O
tRNA containing 5'hydroxy and 2',3'-cyclic phosphate termini
show the reaction diagram
-
The enzyme is responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs.
-
-
?
tRNA precursor
a 3'-half-tRNA molecule with a 5'-OH end + a 5'-half-tRNA molecule with a 2',3'-cyclic phosphate end + an intron with a 2',3'-cyclic phosphate and a 5'-hydroxyl terminus
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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Mg2+ or Ca2+ required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Monovalent cations
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p-hydroxymercuribenzoate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Nonionic detergents
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stimulate activity of the endonuclease in the membrane fraction
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spermidine
spermine
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or spermidine required, enhances extent and accuracy of cleavage, degree of stimulation varies with the pre-tRNA substrate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00025
additional information
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wild-type protein, substrate: 5'-AAGCGACCGACC*AUA*GCUGCA-3' or 5'-AAUGCAGCGGUC*AAA*GGUCGC-3'
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8
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active in the range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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soluble and membrane bound
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aeropyrum pernix (strain ATCC 700893 / DSM 11879 / JCM 9820 / NBRC 100138 / K1)
Aeropyrum pernix (strain ATCC 700893 / DSM 11879 / JCM 9820 / NBRC 100138 / K1)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Nanoarchaeum equitans (strain Kin4-M)
Pyrobaculum aerophilum (strain ATCC 51768 / IM2 / DSM 7523 / JCM 9630 / NBRC 100827)
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15000
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alphabetagammadelta, 1 * 54000, 1 * 44000, 1* 34000, 1 * 15000, SDS-PAGE
21700
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SDS-PAGE
29500
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x * 100000, polypeptide A, + y * 52500, polypeptide B, + z * 40700, polypeptide C, + v * 29500, polypeptide D, + 4 minor polypeptides of MW 140000, 120000, 90000 and 70000 are detected in SDS-PAGE of most highly purified fraction, probably polypeptides B, C, and D exist as a trimeric complex and represent the tRNA splicing endonuclease
37000
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2 * 37000, SDS-PAGE
40700
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x * 100000, polypeptide A, + y * 52500, polypeptide B, + z * 40700, polypeptide C, + v * 29500, polypeptide D, + 4 minor polypeptides of MW 140000, 120000, 90000 and 70000 are detected in SDS-PAGE of most highly purified fraction, probably polypeptides B, C, and D exist as a trimeric complex and represent the tRNA splicing endonuclease
44000
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alphabetagammadelta, 1 * 54000, 1 * 44000, 1* 34000, 1 * 15000, SDS-PAGE
52500
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x * 100000, polypeptide A, + y * 52500, polypeptide B, + z * 40700, polypeptide C, + v * 29500, polypeptide D, + 4 minor polypeptides of MW 140000, 120000, 90000 and 70000 are detected in SDS-PAGE of most highly purified fraction, probably polypeptides B, C, and D exist as a trimeric complex and represent the tRNA splicing endonuclease
54000
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alphabetagammadelta, 1 * 54000, 1 * 44000, 1* 34000, 1 * 15000, SDS-PAGE
100000
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x * 100000, polypeptide A, + y * 52500, polypeptide B, + z * 40700, polypeptide C, + v * 29500, polypeptide D, + 4 minor polypeptides of MW 140000, 120000, 90000 and 70000 are detected in SDS-PAGE of most highly purified fraction, probably polypeptides B, C, and D exist as a trimeric complex and represent the tRNA splicing endonuclease
146000
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deduced from gene sequences of subunits
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
heterotetramer
homodimer
multimer
oligomer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant wild-type and mutant APE-EndAs, hanging-drop vapor diffusion method, 10 mg/ml protein solution is mixed in equal volumes with reservoir solution containing 0.25 M ammonium sulfate, 0.1 M sodium citrate, pH 5.6, 0.9 M lithium sulfate and 1 mM MgCl2, 1 day, equilibration over 0.5 ml reservoir solution, 22°C, X-ray diffraction structure determination and analysis at 2.8 A resolution, molecular replacement
purified recombinant wild-type and mutant EndAs, method screening, sitting drop vapor diffusion technique, mixing of 0.001 ml protein solution with 0.001 ml reservoir solution containing 0.2 M NaCl, 0.1 M phosphate-citrate, pH 4.2, and 10% w/v PEG 3000, equilibration over 0.1 ml reservoir solution, 22°C, X-ray diffraction structure determmination and analysis at 1.7-2.3 A resolution, molecular replacement
purified recombinant AFU-EndA, hanging-drop vapor diffusion method, 10 mg/ml protein solution is mixed in equal volumes with reservoir solution containing 2.2 M ammonium sulfate, 0.2 M potassium sodium tartrate tetrahydrate and 0.1 M sodium citrate, pH 5.6, several days, equilibration over 0.5 ml reservoir solution, 22°C, X-ray diffraction structure determination and analysis at 2.8 A resolution, molecular replacement
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truncated active dimeric enzyme form lacking the N-terminal domain, hanging drop vapour diffusion method, 60 mg/ml protein is mixed with reservoir solution conatining 0.1 M sodium cacodylate, pH 6.5-6.8, 20 mM ammonium sulfate, and 0.3-0.4 M sodium acetate, approximately 30 days, X-ray diffraction structure determination and analysis at 2.0 A resolution
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hanging-drop vapor diffusion method
catalytic subunit, X-ray diffraction structure determination and analysis at 3.1 A resolution
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Crystallization was performed at 44°C by the sitting-drop method using a Hydra II Plus One crystallization robot (Matrix Technology) with approximately 1500 conditions and a ratio of 200 nl precipitants to 200 nl protein solution.
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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rapid inactivation above
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, stable for months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chromatographic methods
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copurification of recombinant His-tagged SULSO alpha-subunit and untagged beta-subunit from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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partial, strain M304
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recombinant EndA from Escherichia coli strain Rosetta 2(DE3) by heat treatment at 70°C for 30 min, followed by heparin affinity chromatography and gel filtration
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant His-tagged subunits by nickel affinity chromatography
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recombinant truncated fully active dimeric enzyme form lacking the N-terminal domain from Escherichia coli strain BLR(DE3) to homogeneity
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recombinant wild-type and mutant EndAs from Escherichia coli strain Rosetta 2(DE3) by heat treatment at 70°C for 30 min, followed by heparin affinity chromatography and gel filtration
recombinant wild-type and mutant EndAs from Escherichia coli strain Rosetta 2(DE3) by heat treatment at 70°C for 30 min, followed by metal affinity chromatography and gel filtration
TALON metal affinity resin column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
epsilon2 endonuclease, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of C-terminally His6-tagged wild-type and mutant enzyme
expressed in Escherichia coli Rosetta-gami (DE3) cells
expression in Eschericha coli
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expression in Escherichia coli
Q975R3 and F9VMU0
expression of genes NEQ205 and NEQ261 in Escherichia coli strain BL21(DE3), phylogenetic analysis
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expression of His6-tagged wild-type and mutant EndAs in Escherichi coli strain Rosetta 2(DE3)
gene AFN, expression of truncated fully active dimeric enzyme form lacking the N-terminal domain in Escherichia coli strain BLR(DE3)
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gene ARCFU, DNA and amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene METJA, DNA and amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene SULSO, DNA and amino acid sequence determination and analysis, coexpression of His-tagged SULSO alpha-subunit and untagged beta-subunit in Escherichia coli strain BL21(DE3)
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gene XENLA, DNA and amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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genes endo1 and endo2, expression as His-tagged subunits forming the heterocomplex
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genes of all four subunits, each of them being essential
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overexpression in Escherichia coli
overexpression of EndA in Escherichia coli strain Rosetta 2(DE3)
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overexpression of wild-type and mutant EndAs in Escherichia coli strain Rosetta 2(DE3)
two distinct genes endA, ORFs ST0358 and STS047, DNA and amino acid sequence determination and analysis of two ORFs encoding the enzyme, one ORF lacks the putative catalytic amino acid residue, individual expression in Escherichia coli reveals that both are components of an enzyme complex
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two genes coding for different subunits
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D49A
site-directed mutagenesis, the mutation does not affect the enzyme activity
E43A
site-directed mutagenesis, the mutation does not affect the enzyme activity
E51A
site-directed mutagenesis, the mutation does not affect the enzyme activity
F50A
site-directed mutagenesis, the mutant shows reduced enzyme activity compared to the wild-type enzyme
P45A
site-directed mutagenesis, the mutant shows reduced enzyme activity compared to the wild-type enzyme
H257A
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residue of the catalytic triad, 28-fold reduction in activity
H257C
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residue of the catalytic triad
H257D
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residue of the catalytic triad
H257N
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residue of the catalytic triad
K179A
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site-directed mutagenesis, the mutant removes the BHB intron from the anticodon loop and the D-loop. However, AFU-CSL K179A can not remove the BHL intron correctly
K287E
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residue of the catalytic triad, no activity in absence of divalent metal ions, activity observed in the presence of specific divalent metal ions
K287Q
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residue of the catalytic triad
K287R
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residue of the catalytic triad
Y246F
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residue of the catalytic triad, 7-fold reduction in activity
Y246F/H257A
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residues of the catalytic triad, no activity
Y246H
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residue of the catalytic triad
D357A
site-directed mutagenesis
K224E
site-directed mutagenesis
C122A
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inactive
H297A
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catalytically inactive
H136A
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site-directed mutagenesis of the catalytically essential residue results in an inactive mutant
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
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