Information on EC 3.1.13.4 - poly(A)-specific ribonuclease and Organism(s) Homo sapiens

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


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EC NUMBER
COMMENTARY hide
3.1.13.4
-
RECOMMENDED NAME
GeneOntology No.
poly(A)-specific ribonuclease
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
exonucleolytic cleavage of poly(A) to 5'-AMP
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
CAS REGISTRY NUMBER
COMMENTARY hide
110541-21-4
-
215797-47-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
(A)6 + H2O
5'-AMP + ?
show the reaction diagram
-
-
-
-
?
poly(A) + H2O
5'-AMP + ?
show the reaction diagram
-
average substrate length about 200 A
-
-
?
poly(A) + H2O
AMP
show the reaction diagram
-
-
-
-
ir
poly(A) RNA + H2O
5'-AMP + ?
show the reaction diagram
poly(A)-mRNA + H2O
5'-AMP
show the reaction diagram
polyadenylated RNA containing AU-rich elements + H2O
RNA containing AU-rich elements + AMP
show the reaction diagram
-
-
-
?
additional information
?
-
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
poly(A) RNA + H2O
5'-AMP + ?
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
-
can substitute for Mg2+, Mn2+, optimal at 0.01 mM, two binding sites
Mn2+
-
or Mg2+, required
Na+
-
may substitute for K+, but less effective
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl) cytosine
-
-
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N4-benzoyl cytosine
-
-
9-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N6-benzoyl adenine
-
-
ADP
-
purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
ATP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
Ca2+
-
3 mM gradually decreases activity about 15fold
cap analogue 7-methylguanosine(5')ppp(5')G
-
allosteric inhibitor of PARN in the presence of a physiological K+ concentration
CH3COOK
-
-
Co2+
-
3 mM gradually decreases activity about 78fold
deoxyATP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
deoxyGTP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
eIF4E
-
competition between PARN and eIF4E for the 5'-cap
-
GDP
-
purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
GMP
-
purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
GTP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
guanidine hydrochloride
-
full-length enzyme retains most of its activity at guanidine hydrochloride concentrations below 0.5 M, whereas an abrupt decrease of the activity of p54 and p46 is found when guanidine hydrochloride concentration is increased
K3PO4
-
-
Mg2+
-
behaves as a destabilizer of the overall structural stability of PARN, promotes thermal unfolding and aggregation at high temperatures
Mn2+
-
3 mM gradually decreases activity about 5fold
Poly(A)
; strong inhibition of both spermidine- and polyadenylate-binding protein-activated hPAN complex
poly(A)-binding protein PAB 1
-
inhibits; phased poly(A) shortening
-
poly(C)
; strong inhibition of spermidine-, slight inhibition of polyadenylate-binding protein-activated hPAN complex
poly[2'-O-(2,4-dinitrophenyl)]poly-(A)
-
competitive
-
purine nucleotides
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors while purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxy-ribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
-
synthetic fluoro-pyranosyl nucleosides
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synthetic nucleoside analogues bearing a fluoroglucopyranosyl sugar moiety and benzoyl-modified cytosine or adenine as a base can effectively inhibit human PARN
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
K+
-
allosteric activator of PARN and its truncated forms, optimal concentration is around 100 mM, with the increase of the K+ concentration, the enzyme reaches its Vmax at a much lower substrate concentration
polyadenylate-binding protein
or spermidine, required for activity of hPAN complex, cannot stimulate hPAN2 alone
-
spermidine
or polyadenylate-binding protein, required for activity of hPAN2 and of hPAN complex
tristetraprolin
-
required for degradation of polyadenylated RNA containing AU-rich elements
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000236 - 0.00793
poly(A) RNA
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.65 - 7.6
Poly(A)
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.645
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl) cytosine
-
at pH 7.0, 30C
0.21
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N4-benzoyl cytosine
-
at pH 7.0, 30C
0.51
9-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N6-benzoyl adenine
-
at pH 7.0, 30C
0.5
ADP
-
at pH 7.0, 30C
1.6
AMP
-
at pH 7.0, 30C
3.2
ATP
-
at pH 7.0, 30C
0.7
CMP
-
at pH 7.0, 30C
2.8
deoxyATP
-
at pH 7.0, 30C
2
deoxyGTP
-
at pH 7.0, 30C
2.8
GDP
-
at pH 7.0, 30C
4.5
GMP
-
at pH 7.0, 30C
3.3
GTP
-
at pH 7.0, 30C
0.00007
Poly(A)
37C, pH 7.5, spermidine-activated hPAN
0.002
poly(C)
37C, pH 7.5, spermidine-activated hPAN
0.098
poly[2'-O-(2,4-dinitrophenyl)]poly-(A)
-
pH not specified in the publication, temperature not specified in the publication
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
37
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
Hep G2 cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17000
2 * 17000, SDS-PAGE, dimerization of enzyme fragment PARN(443-560)
37000
dimerization of enzyme fragment PARN(443-560), gel filtration
45000
-
x * 45000, recombinant CNOT6L nuclease domain, SDS-PAGE
46000
-
truncated form completely missing the RRM
62000
-
PARN (p62), residues 1-540
73500
-
amino acid analysis
75886
x * 135007, catalytic subunit hPAN2, + x * 75886, regulatory subunit hPAN3, calculated from amino acid sequence
76200
x * 76200, His-tagged full-length enzyme, SDS-PAGE
135007
x * 135007, catalytic subunit hPAN2, + x * 75886, regulatory subunit hPAN3, calculated from amino acid sequence
290000
-
gel filtration, full length enzyme
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer
monomer
-
gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer
tetramer
-
4 * 74000, calculated. Poly(A)-specific ribonuclease PARN can self-associate into tetramer and high-order oligomers both in vitro and in living cells. PARN oligomerization is triggered by the R3H domain,which leads to the solvent-exposed Trp219 fluorophore to become buried in a solvent-inaccessible microenvironment. The RRM and C-terminal domains are involved in modulating the dissociation rate of the tetrameric PARN. Tetramerization does not affect the catalytic behavior of the full-length PARN and truncated enzymes containing the RRM domain. Tetramerization significantly enhances the catalytic activity and processivity of the truncated form with the removal of the RRM and C-terminal domains
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the PARN-RNA-recognition motif domain (residues 445-540) with a bound 7-methylguanosine triphosphate nucleotide shows a remarkable conformational flexibility of the RNA-recognition motif domain, crystal structure is refined at a resolution of 2.1 A, protein folds into a three-stranded antiparallel beta-sheet that is flanked by one alpha-helix connecting beta-strands beta1 and beta2
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free and RNA-bound forms, 20C, hanging-drop method
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in silico model for catalytic mechanism and development of a 3D pharmacophore model. Residues Arg99 and Gln109 are involved in the regulation of catalysis. The natural preference of the enzyme for poly(A) is based on favorable biophysical electrostatic and hydrophobic interactions
-
purified recombinant CNOT6L nuclease domain in complex with AMP and poly(A) DNA, hanging drop vapour diffusion method, 0.001 ml of protein solution, containing 15 mg/ml protein and 1 mM DTT, is mixed with 0.001 ml of reservoir solution and equilibrated over 300 ml reservoir solution, containing 0.1 M HEPES, pH 7.5, 1.1 M ammonium tartrate, and 0.2 M NDSB-201, at 16C, X-ray diffraction structure determination and analysis at 1.94-2.44 A resolution
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 55
-
thermal stability and aggregation kinetic parameters, the aggregation is dominated by a first-order kinetics, increase in t0 and the decrease in k implies that Mg2+ can inhibit the rate of enzyme aggregation, overview. Cofactor Mg2+ is also important to PARN stability against inactivation induced by heat treatment, but promotes thermal aggregation at high temperatures. Mg2+ significantly decreases the rate but increases the aggregate size of the 54 kDa wild-type enzyme in a concentration-dependent manner. Effect of mutations on the Mg2+-dependent enzyme thermal aggregation, overview
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, stable for several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by metal affinity resin
-
by Ni2+ affinity chromatography and gel filtration, purity above 98%
-
Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM.
-
His-tag affinity chromatography HiTrap Q HP and 7-Me-GTP-Sepharose affinity chromatography
-
of the recombinant protein
-
on Ni-NTA resin and by gel filtration, purity above 98%
-
overview on conditions
-
PARNn purified by glutathione-Sepharose 4B, MonoQ and Superdex 200 gel filtration columns. truncated PARN including the putative cap-binding domain purified by TALON affinity resin. full length PARN purified by Ni-NTA column
-
purification of full-length PARN and truncated forms p62 and p54
-
purity above 99%
-
recombinant enzymes
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
-
recombinant His-tagged full-length enzyme and His-tagged enzyme fragment from Escherichia coli strain BL21(DE3)
recombinant wild-type and mutant E240A, D489A, and H529A CNOT6L nuclease domains from Escherichia coli
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
C-terminal-truncated PARN, expression in Escherichia coli
-
cloning and expression of full-length PARN and truncated forms p62 and p54
-
DNA fragment corresponding to amino acids 443-560 in the human PARN sequence cloned into pCR 2.1TOPO vector and subsequently subcloned into the pET19b vector, recombinant PARN or PARN mutants expressed in Escherichia coli strain BL21(DE3)
-
expressed as N-terminal His6-tagged polypeptide in Escherichia coli BL21(DE3)
-
expression in Escherichia coli
-
expression in HEK-293T cells
-
expression of wild-type and mutants of the truncated catalytic domain of human CNOT6L, residues 158-555, in Escherichia coli
-
gene PARN, expression of His-tagged enzyme in Escherichia coli strain Bl21(DE3)
-
His tagged, expression in Escherichia coli BL21 (DE3)
-
His-tagged full-length enzyme, comprising residues 1-639, and His-tagged enzyme fragment, PARN(443-560), containing the RNA recognition motif with a 50 amino acids long C-terminal tail, comprising residues 443-560, are expressed in Escherichia coli strain BL21(DE3)
into vector pET28a, expressed in Escherichia coli BL21(DE3)pLys(S) cells
-
plasmid pET33 containing the human parn gene expressed in Escherichia coli BL21(DE3)
-
R3H domain deletion mutants expressed in Escherichia coli
-
The PARN fragment comprising amino acids 445-540 is amplified from a human cDNA library and cloned into the expression vector, the glutathione S-transferase-PARN-RNA-recognition motif fusion protein is expressed in Escherichia coli BL21(DE3). PARN mutants are generated from the wild-type clone (pGEX-6P-1 PARN445-540) using a Site-Directed Mutagenesis Kit. SeMet-containing PARN-RNA-recognition motif is expressed in Escherichia coli.
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D478A
-
generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.0 microM
D489A
-
site-directed mutagenesis, active site mutant, inactive mutant
E240A
-
site-directed mutagenesis, active site mutant, inactive mutant
E455/W456/W475A
-
severely defective in cap binding, active deadenylase
E455A
-
no defect in its cap binding
F484A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
H529A
-
site-directed mutagenesis, active site mutant, inactive mutant
K454A
-
generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.03 microM
L197A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
L216A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
L291A
-
site-directed mutagenesis, activity and kinetics with RNA substrates compared to the wild-type enzyme
L414A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
M425A
-
site-directed mutagenesis, activity and kinetics with RNA substrates compared to the wild-type enzyme
N412A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
P365A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
P365A/F484A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
P365A/N412A/F484A
-
site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
T458A
-
generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 30.58 microM
W456A
-
cap binding slightly affected
W475A
-
severely defective in cap binding
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
leukemic cells from patients with acute lymphoblastic leukemia and acute myeloid leukemia display altered expression for CNOT6, CNOT6L, CNOT7 deadenylase and poly(A)-specific ribonuclease with most significant alterations for poly(A)-specific ribonuclease and CNOT7 mRNA levels. In acute lymphoblastic leukemia, a significant amount of poly(A)-specific ribonuclease is phosphorylated
additional information