Information on EC 3.6.4.6 - vesicle-fusing ATPase

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

EC NUMBER
COMMENTARY hide
3.6.4.6
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RECOMMENDED NAME
GeneOntology No.
vesicle-fusing 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
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NIL
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (vesicle-fusing)
A large family of ATP-hydrolysing enzymes involved in the heterotypic fusion of membrane vesicles with target membranes and the homotypic fusion of various membrane compartments. They belong to the AAA-type (_A_TPase _a_ssociated with a variety of cell _a_ctivities) ATPase superfamily. They include peroxin, which apparently is involved in Zellweger's syndrome.
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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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
long-tailed hamster
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-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
protozoa
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
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vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase N-ethylmaleimide-sensitive factor, NSF, and the adaptor protein soluble NSF attachment protein, alpha-SNAP, disassemble all SNARE complexes formed throughout different pathways
physiological function
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
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
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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NSF is a Ca2+-binding protein, GluR2-NSF interactions are inhibited by the presence of 15 micromol/l Ca2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
bafilomycin A
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N-ethylmaleimide
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TAT-NSF700
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-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
LIP5F388A
a weak SKD1 interactor, complements the lip5 mutant for heat and salt stress tolerance, most likely due to its overexpression driven by the strong CaMV 35S promoter in the transgenic plants
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Lyst-interacting protein5
LIP5, interacts strongly with enzyme SKD1 and stimulates the ATPase activity of the enzyme by 4 to 5fold. LIP5 is a positive regulator of enzyme SKD1 in multivesicular bodies biogenesis and is a critical target of the mitogen-activated protein kinases MPK3 and MPK6 and plays an important role in the plant immune system. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates and NaCl under heat and salt stresses, respectively. LIP5 is regulated by phosphorylation, overview. Mutation of MPK phosphorylation sites in LIP5 does not affect its interaction with SKD1 but reduces its stability and, as a result, compromises its ability to complement the basal resistance of the lip5 mutants
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p47
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cofactor to ATPase p97
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SNARE complex
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SNARE-stimulated ATP hydrolysis, all four SNARE complexes similarly stimulate the steady-state rate of NSF-catalyzed ATP hydrolysis activity, suggesting that the stimulation of ATPase activity is independent of SNARE primary sequence variation and N-terminal domain architecture
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Vps2p helix 5 peptide C
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stimulates Vps4p ATPase activity and stabilizes the hexamer
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additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.62
MgATP2-
-
-
additional information
additional information
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Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.85
ATP
Saccharomyces cerevisiae
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higher order oligomer of the enzyme, pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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ATPase activity assay
8
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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ATPase activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21
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ATPase activity assay
30
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ATPase activity assay
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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adult, CDC-48.1:GFP fusion construct is expressed from embryos through to adult worms
Manually annotated by BRENDA team
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CDC-48.1:GFP fusion construct is expressed from embryos through to adult worms; CDC-48.2:GFP fusion is mainly expressed in embryos
Manually annotated by BRENDA team
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from PBD patients
Manually annotated by BRENDA team
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subesophageal or other
Manually annotated by BRENDA team
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; HsPex6p
Manually annotated by BRENDA team
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larval brain
Manually annotated by BRENDA team
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MIN-6 beta cell
Manually annotated by BRENDA team
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N-ethylmaleimide sensitive factor is required for fusion of the Caenorhabditis elegans uterine anchor cell
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Metallosphaera sedula (strain ATCC 51363 / DSM 5348)
Metallosphaera sedula (strain ATCC 51363 / DSM 5348)
Metallosphaera sedula (strain ATCC 51363 / DSM 5348)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28000
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NSF D1 ATPase domain, determined by SDS-PAGE
39000
apo-form, Guinier analysis
48000
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predicted from amino acid sequence
48100
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predicted from amino acid sequence
48500
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predicted molecular mass
60000
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recombinant enzyme, purified from E. coli, gel filtration
85000
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mutant Vps4pdeltaCC/E233Q, gel filtration
95000
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Vps4p, gel filtration
97000
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monomer form of p97, SDS-PAGE
143000
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Pex1p
148000
ATP-bound form, Guinier analysis
245000
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gel filtration
300000
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high-molecular weight form of p97, SDS-PAGE
320000 - 400000
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heterodimeric PpPex1p-PpPex6p complex
400000
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mutant Vps4pdeltaCC/E233Q, gel filtration
440000
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mutant Vps4pE233Q, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
decamer
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mutants Vps4pE233Q and Vps4pdeltaCC/E233Q, SDS-PAGE
hexamer
homohexamer
monomer
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1 * 60000, SDS-PAGE
oligomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method, using 5% (w/v) polyethylene glycol 3350 and 100 mM MES (pH 6.0)
hanging-drop vapour-diffusion method. Preparation of crystals of the N-terminal domain of PEX1. The crystals belong to space group P3(1) or P3(2) with unit-cell parameters a = b = 63.5 A, c = 33.5 A, and contain one protein molecule per crystallographic asymmetric unit. 2.05 A resolution
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sitting drop vapour diffusion method, at 19C against a reservoir containing 0.1 M MES buffer (pH 5.9-6.5) and 0.8-1.9 M MgSO4
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Vps4p complexes with and without their MIT N-terminal domains and Vta1p cofactors, hanging drop vapour diffusion method, using
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sitting-drop vapor diffusion at 20C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
HiTrap Ni2+-chelating column chromatography
Ni-chelating column chromatography and Superdex gel filtration
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Ni-NTA column chromatography, DEAE Sepharose column chromatography, and Superdex 75 gel filtration
Ni2+ Sepharose column chromatography, Q-Sepharose column chromatography, and Superdex 200 gel filtration
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protein extracts from cells and brain tissues are prepared
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recombinant GST- and His6-tagged Vps4p and Vps4101437 from Saccharomyces cerevisiae strain BY4741
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recombinant His6-tagged enzyme NSF from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant NSF is purified by nickel-nitrilotriacetic acid-agarose affinity chromatography
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recombinant protein is purified using a NTA-Ni2+-agarose cartridge
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recombinant protein, expressed in Escherichia coli XL1-blue
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recombinant wild-type and mutant Vps4ps
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA containing the NSF D1 ATPase domain is inserted into the pET28a vector for expression in Escherichia coli BL21 cells
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cloning of PEX2, PEX6, PEX12 and PEX1 using yeast genes for homology search
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constructing of a genomic library from the haploid yeast strain SD228-7 using the Escherichia coli-yeast shuttle vector pCS19
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) pLysE cells
expressed in Escherichia coli Rosetta 2 (DE3) cells
expression in Escherichia coli
expression in rat 3Y1B fibroblast cells
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expression in U2OS TRex cells
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full-length, partial cDNA fragments and point mutations of mouse NSF are amplified by PCR and subcloned into different expression vectors including pGEX-6P-2, pETH-32, pcDNA3-His6, and pcDNA3-3HA
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His6-NSF for expression in Escherichia coli BL21 cells
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isolation of PEX2 and PEX6 cDNA, Pex1p expressed in wild-type chinese hamster ovary cells, CHO-K1
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N-terminal domain of PEX1
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recombinant expression of GST- and His6-tagged Vps4p and Vps4101437 in Saccharomyces cerevisiae strain BY4741
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recombinant expression of His6-tagged enzyme NSF in Escherichia coli strain BL21(DE3), coexpression of alpha-SNAP, VAMP2(1-96), syntaxin1A(1-265)-C145S/S249C/K253C, syntaxin1A(191-265)-C145S/S249C/K253C, and SNAP25A
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recombinant expression of wild-type and mutant Vps4ps
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SKD1 and GFP5 PCR-amplified fragments are cloned in pBSII KS+ and subsequently subcloned into pMZ215S, and in the binary vectors pCambia 1300 and pbinSRNACatN
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SKD1 cDNA amplified by reverse transcription-PCR
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the vectors YCplac111, pLexA, pET11d and pB42AD are used for cloning and expression of wild-type protein, single domains and various mutants
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VPS24 gene cloned from a yeast genomic library
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VPS4 gene cloned and overexpressed in Escherichia coli XL1-blue, using a GST fusion vector
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E232Q
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ATPase-deficient mutant
E329Q
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in contrast to wild-type NFS E329Q mutant is enriched on various structures throughout the cell and is only minimally released by saponin permeabilization
E209K
site-directed mutagenesis
E329Q
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no ATPase activity
K266A
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no nucleotide binding
K549A
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decrease in NSF vesicular transport activity
D100A
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mutant to study the Ca2+ sensitivity of the interaction between NSF and the glutamate receptor subunit 2 of AMPAR
D142A
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mutant to study the Ca2+ sensitivity of the interaction between NSF and the glutamate receptor subunit 2 of AMPAR
E153A
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mutant to study the Ca2+ sensitivity of the interaction between NSF and the glutamate receptor subunit 2 of AMPAR
G282E
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mutant protein has no ATPase activity
E235Q
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no ATPase activity, mutation leads to perturbation of various membrane transports via endosomes
E305Q
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mutant assembles into oligomer like wild-type, similar ATPase activity as wild-type
E305Q/E578Q
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no ATPase activity
E578Q
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mutant assembles into oligomer like wild-type, severely impaired ATPase activty
S569A
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mutation of serine 569 to alanine abolishs the phosphorylation of D2 domain by active Pctaire1
K251A
the p97 mutant carrying mutations in the D2 domain shows little ATPase activity when compared with wild type enzyme; the Walker A mutation to the D1 domain alone shows a moderate decrease in ATPase activity, has reduced affinity for nucleotide in the D1 domain and so contains little prebound ADP
K524A
the p97 mutant carrying mutations in the D2 domain shows little ATPase activity when compared with wild type enzyme
R359A
the p97 mutant carrying mutations in the D2 domain shows little ATPase activity when compared with wild type enzyme
R635A
the p97 mutant carrying mutations in the D2 domain shows little ATPase activity when compared with wild type enzyme
S569A
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mutation of serine 569 to alanine abolishs the phosphorylation of D2 domain by active Pctaire1
A505L
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lethal mutation
E243A
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site-directed mutagenesis, a pore loop mutant, displays essentially unchanged oligomerization, introducing E243A point mutation increases the affinity of peptide C binding by 3fold, shows reduced ATPase activity compared to the wild-type
E243A/E247A
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site-directed mutagenesis, pore loop mutant, displays essentially unchanged oligomerization, the double mutant binds peptides 4fold (peptide C) or 10fold (peptide B) more tightly than wild-type Vps4p, shows reduced ATPase activity compared to the wild-type
E247A
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site-directed mutagenesis, a pore loop mutant, displays essentially unchanged oligomerization, introducing E247A point mutation increases the affinity of peptide C binding by 4fold, shows reduced ATPase activity compared to the wild-type
E329Q
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hydrolysis mutant
G89D
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higher intrinsic ATPase activity than wild-type, no stimulation by Sec17
K179A
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no ATP binding, enhanced binding to Vps20p and Vta1p in vitro
K266A
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ATP-binding mutant
Q216A
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monomeric mutant form
R241A
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site-directed mutagenesis, a pore loop 2 mutant, does not bind peptides with appreciable affinity
R251A
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site-directed mutagenesis, a pore loop 2 adjacent mutant, does not bind peptides with appreciable affinity
T240A
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site-directed mutagenesis, a pore loop mutant, displays essentially unchanged oligomerization
T240F
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site-directed mutagenesis, a pore loop 2 mutant
T240K
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site-directed mutagenesis, a pore loop 2 mutant
T240V
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site-directed mutagenesis, a pore loop 2 mutant
W206A
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site-directed mutagenesis, a pore loop 1 mutant, does not bind peptides with appreciable affinity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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