Information on EC 3.6.4.8 - proteasome ATPase

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

EC NUMBER
COMMENTARY hide
3.6.4.8
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RECOMMENDED NAME
GeneOntology No.
proteasome ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O = ADP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (polypeptide-degrading)
Belongs to the AAA-type superfamily and, like EC 3.6.4.5 (minus-end-directed kinesin ATPase), is involved in channel gating and polypeptide unfolding before proteolysis in the proteasome. Six ATPase subunits are present in the regulatory particle (RP) of 26S proteasome.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
strain K12
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-
Manually annotated by BRENDA team
most lethal form of human malaria
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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
ATP + H2O
ADP + phosphate
show the reaction diagram
CTP + H2O
CDP + phosphate
show the reaction diagram
GTP + H2O
GDP + phosphate
show the reaction diagram
ITP + H2O
IDP + phosphate
show the reaction diagram
-
-
-
-
?
TTP + H2O
TDP + phosphate
show the reaction diagram
-
-
-
-
?
UTP + H2O
UDP + phosphate
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
ATP + H2O
ADP + phosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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increasing ATP beyond the optimal range is inhibitory
ATPgammaS
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inhibits 26S proteasome ATPase activity over the same concentration range in which it stimulates peptidase activity
Hemin
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inhibitor of proteasome 19S ATPases
lactacystin
N-ethylmaleimide
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O-GlcNac transferase
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inhibition of Rpt2 by O-GlcNAc leads to a decrease in proteasome activity
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p-chloromercuriphenyl-sulfonic acid
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PSI
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proteasome inhibitor
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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increasing ATP beyond the optimal range is inhibitory
casein
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1000fold molar excess, 5fold stimulation of ATPase activity
GFPssrA
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green fluoresence protein with C-terminus fused to the 11 residue peptide ssrA, 5fold stimulation of ATPase activity
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polyubiquitin
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modest but reproducible stimulatory effect on 26S proteasome ATPase activity
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RNA
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synthetic and mRNA-type RNAs stimulates activity efficiently
additional information
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Rip-1 does not alter ATPase activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03 - 0.55
ATP
0.307 - 0.493
CTP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.13
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artificial Rpt complex consisting of Rpt subunits Rpt4, proteasome ATPase is thought to hydrolyze ATP at the interface between neighboring subunits
0.88
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artificial Rpt complex consisting of Rpt subunits Rpt1 and Rpt2, proteasome ATPase is thought to hydrolyze ATP at the interface between neighboring subunits
additional information
comparative proteomic analysis of wild-type cells and those lacking proteasomal function through deletion of the proteasome-activating nucleotidase PanA, reduced growth rate and cell yield of mutant strain, enriched proteins exclusive to or more abundant in the panA mutant includes cell division, dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase and oxidoreductase homologs, differences in transcriptional regulation and signal transduction proteins, proteasome deficiency causes an up-regulation of stress responses, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems enriched in panA mutants
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
7.4
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in-gel proteasome activity assay
7.5
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ATPase activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
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full-length protein
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.3
sequence calculation
4.5
sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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LG-2 EBV B cell
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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embryonic lung cell
Manually annotated by BRENDA team
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cell-free reticulocyte lysate-based system
Manually annotated by BRENDA team
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
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)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6000
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C-terminal domain of Rpt3, Rpt3C, determined by SDS-PAGE
16600
6 * 20000, SDS-PAGE, 6 * 16600, predicted
20000
6 * 20000, SDS-PAGE, 6 * 16600, predicted
24000
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recombinant subunit, expressed in E. coli, 2D-PAGE
35000
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Nas6-Rpt3C complex, determined by SDS-PAGE
45160
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TBP-1, calculated from amino acid sequence
45230
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SUG1, calculated from amino acid sequence
45410
x * 45410, amino acid sequence calculation
45664
x * 45664, amino acid sequence calculation
45770
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p45, predicted from the open reading frame
47000
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SDS-PAGE
47970
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YTA2, calculated from amino acid sequence
48260
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YTA1, calculated from amino acid sequence
48630
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MSS1, calculated from amino acid sequence
48830
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YTA5, calculated from amino acid sequence
49240
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S4, calculated from amino acid sequence
51550
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TBP-7, calculated from amino acid sequence
51730
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CIM5, calculated from amino acid sequence
100000
gel filtration, suggesting that Mpa-ID oligomerized in solution as a hexamer
650000
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PAN, proteasome-activating nucleotidase, complex with ATPase activity, gel filtration
2000000
additional information
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650000 Da ATPase comlex
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
heterohexamer
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the base structure of the 19S regulatory particle consists of heterohexameric proteasomal ATPases, Rpt1-Rpt6 in yeast
hexamer
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the structure is solved by molecular replacement with the N-terminal actinobacterial proteasomal ATPase oligosaccharide-binding domain OB1 as the search model. Each PAN-N monomer consists of an N-terminal coiled-coil helix and a C-terminal OB domain. The helices form two-strands coiled coils, while the OB domains associate into hexameric rings. Thus the structure can be described as a trimer of dimers
crystal structure of the archaeal 20S proteasome in complex with the C-terminus of the archaeal proteasome regulatory ATPase, PAN is determined. This structure defines the detailed interactions between the critical C-terminal HbYX motif and the 20S a-subunits and indicates that the intersubunit pocket in the 20S undergoes an induced-fit conformational change on binding of the HbYX motif
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purified recombinant N-terminus of p27 (residues 1-128) in complex with the C-terminal ATPase domain of Rpt5 (residues 173-442), sitting drop method, mixing of 0.001 ml of 4 mg/ml protein in 10 mM HEPES pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, with 0.001 ml of precipitant solution containing 2.0 M NaCl, 10% PEG 6000, X-ray diffraction structure determination and analysis at 1.7 A and 4.0 A resolution, respectively
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characterization of the structural determinants of Pup (mybacterial ubiquitin-like protein) and its interaction witrh Mpa. The N-terminal coiled-coil domain of Mpa makes extensive contacts along the central region (residues 21-58) of Pup leaving its N-terminal uncontrained and available for other functional interactions
of the conserved interdomain shows a five stranded double beta barrel structure containing a Greek key motif, 2.0 A resolution. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. The central in the Mpa hexamer is involved in protein substrate translocation and degradation. Mpa is a multidomain structure, with an N-terminal coiled coil domain, a 150 amino acid interdomain (Mpa-ID) that is unique to the proteasome-associated ATPases, a canonical AAA (ATPase associated with various activities) domain, and a small C-terminal domain. The Mpa-ID forms a tightly packed ring-shaped hexamer in the crystal structure as well as in solution. In fact, two hexamers stack end to end, forming a dodecamer, being the packiung unit in the crystal structure
structure solved by multiple isomorphous replacement. One ARC-N monomer consists of two oligosaccharide-binding domains in tandem, the first from Ser80 to Tyr136 (OB1) and the second from Gly140 to Lys217 (OB2). The domains associate separately into hexameric rings
crystal structures of full-length Hsm3 and the complex with the C-terminal domain of Rpt1 is determined
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the structure of the complex between the non-ATPase subunit Nas6 and the C-terminal domain of the proteasomal ATPase Rpt3 is determined at a resolution of 2.2 A
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eukaryotic ATPases form a heterohexameric ring with the arrangement Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5 (Rp, regulatory particles) in fully assembled proteasomes. This quaternary organization clarifies the functional overlap of specific RP assembly chaperones and leads to the identification of a potential RP assembly intermediate that includes four ATPases (Rpt6-Rpt3-Rpt4-Rpt5) and their cognate chaperones Rpn14, Nas6, and Nas2
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
26S proteinase
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26Sproteasome
full length protein, the interdomain Mpa-ID and the mutant forms
glutathione Sepharose column chromatography and Ni-NTA resin column chromatography
Mpa fragments are purified as N-terminal fusions with His6-tagged maltose-binding protein followed by a TEV cleavage site, producing the folowing fragments: Mpa-wt (residues 1-609), Mpa-CC (residues 1-98), Mpa-ID (99-216), and Mpa-CC-ID (1-216)
proteasomes are prepared from rabbit reticulocyte lysates
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proteins are extracted from heart tissue
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proteins are extracted from pupae and larvae of Drosophila melanogaster
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recombinant expression of His-tagged C-terminal ATPase domain of Rpt5 (residues 173-442) from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, and gel filtration as protein complex with the N-terminus of p27
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography; recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant PAN, expressed in Escherichia coli Bl21(DE3)
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recombinant rat SuG1, produced in Escherichia coli
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recombinant Rpt subunits
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recombinant Rpt2
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the Nas6-Rpt3C complex, expressed in Escherichia coli Rosetta DE3 cells, is purified on a HisTrap HP, a HiTrapQ anion-exchange, and a gel-filtration column
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ultracentrifugation and MonoQ column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5 genes cloned encoding 26S proteasome components
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6 ATPase subunits of the 26S proteasome cloned
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cDNA clone encoding MS73
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cDNA encoding regulatory subunit p45
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cDNAs encoding 26Sproteasome subunits, genes SOPSC8, SOPSC1 and SOPRS7 from the 19/22S regulatory complex
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cloning of a cDNA encoding the proteasome subunit 4 ATPase, gene PSF4, PCR and DNA sequencing
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complete genome sequenced
expressed in a yeast two-hybrid system
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expressed in Escherichia coli
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli, strains DH5alpha and GM2163, pCR-BluntII-TOPO to generate plasmid pJAM636, transformed into Haloferax volcanii DS70, recombinant strain, plasmids pJAM650, pJAM1012
expression in Escherichia coli
gene panA, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3); gene panB, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
gene RPT1, phylogenetic analysis; gene RPT2, phylogenetic analysis; gene RPT3, phylogenetic analysis; gene RPT4, phylogenetic analysis; gene RPT5a, phylogenetic analysis; gene RPT5b, phylogenetic analysis; gene RPT6, phylogenetic analysis
gene rSUG1 expressed in Escherichia coli
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human ATPase subunit MSS1 gene used for screening
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Mouse Rpt2 rescued the expression of yeast Rpt2 with a weak growth defect
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pAMJHW03 amplified, putative ATPase cloning of S4 and expression of PAN in Escherichia coli Bl21(DE3)
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recombinant expression of FLAG-tagged wild-type and mutant subunits Rpt3 and Rpt6 in HeLa cells
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recombinant expression of His-tagged N-terminus of p27 (residues 1-128) and the C-terminal ATPase domain of Rpt5 (residues 173-442) in a mixed cultre of tranformed Escherichia coli strain BL21(DE3) cells
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several subunit genes of the 20S catalytic core and the 19/22S regulatory complex have been isolated
several subunit genes of the 20S catalytic core and the 19/22S regulatory complex have been isolated, MBP1 gene of the 19/22S regulatory complex cloned
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the cDNA encoding Nas6 and the cDNA encoding the C-terminal region of Rpt3, residues 348-428, are subcloned into the first and second cassette of pETDuet1, respectively
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the Rpt1/p48B gene is cloned into the pCaSpeR4-Ubi-promoter plasmid
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G158E
the mutation impairs the light-specific hypocotyl elongation response elicited by class I S(1)-b-methyl-a,b-diaminopropionic acid, the mutated amino acid site resides within the full AAA-ATPase domain
R173E/W187A/K235E
E365A/D367A
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mutant constructed for interaction studies between the C-terminal domain of Rpt3 and Nas6, the complex is completely disrupted for this mutant
G360A/G387A
site-directed mutagenesis, the Rpt6 mutant reduces the Rpn14 association with proteasome base and disrupts holoenzyme formation in vitro
K397A/R399A
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mutant constructed for interaction studies between the C-terminal domain of Rpt3 and Nas6, the complex is completely disrupted for this mutant
R403A
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assembly defect of the proteasome is not observed in the single mutant
R403A/R409A
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double mutation causes assembly defect of the 26 S proteasome, suggesting that both Arg residues of Rpt1 are responsible for the Hsm3 binding and the proteasome assembly
R409A
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assembly defect of the proteasome is not observed in the single mutant
T193C
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25% increase in N-ethylmaleimide sensitivity
T226C
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25% increase in N-ethylmaleimide sensitivity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine