Information on EC 3.6.5.6 - tubulin GTPase

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

EC NUMBER
COMMENTARY hide
3.6.5.6
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RECOMMENDED NAME
GeneOntology No.
tubulin GTPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GTP + H2O = GDP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
phosphoric ester hydrolysis
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NIL
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-
SYSTEMATIC NAME
IUBMB Comments
GTP phosphohydrolase (microtubule-releasing)
An intrinsic activity of alpha-tubulin involved in tubulin folding, division plane formation in prokaryotic cells and others.
CAS REGISTRY NUMBER
COMMENTARY hide
9059-32-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Bacillus thuringiensis subsp. israelensis
-
-
Manually annotated by BRENDA team
gene ftsZ or DR 0631
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Prosthecobacter sp.
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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
-
hydrolyzed at a rate about half as fast like GTP
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-
?
GTP + H2O
GDP + phosphate
show the reaction diagram
ITP + H2O
IDP + phosphate
show the reaction diagram
-
hydrolyzed at equivalent rates like GTP
-
-
?
XTP + H2O
XDP + phosphate
show the reaction diagram
-
hydrolyzed at a rate about half as fast like GTP
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-
?
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
GTP + H2O
GDP + phosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
induction of the GTPase activity of brain tubulin, inhibition of tubulin assembly, prevents tubulin polymerization and induces rapid depolymerization of preformed microtubules
Co2+
stimulates, can partially substitute for Mg2+
Gd3+
-
stimulates the GTPase activity of brain tubulin, inhibits tubulin polymerization and induce microtubule depolymerization
K+
K+ ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues D235 and D238, K+ ion stabilizes dimer (molecular dynamics simulations)
Mn2+
stimulates slightly, can partially substitute for Mg2+
Nd3+
-
stimulates the GTP hydrolysis, inhibits tubulin polymerization and induces microtubule depolymerization
Ni2+
stimulates slightly, can partially substitute for Mg2+
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Br-
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Cl-
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enhancement of tubulin polymerization by Cl--induced blockade of intrinsic GTPase. GTPase activity of tubulin is lower (2/3-fold) in Cl--containing solutions than that in Cl--free solutions containing Br- or NO3-
curcumin
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diethyldicarbonate
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inhibits GTP hydrolysis, but not the assembly reaction
EDTA
strong inhibition at 2 mM
GTP
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inhibitory effect produced at concentrations above 3 mM, above 5 mM leads to depolymerization of microtubules
guanosine 5'-(gamma-fluorotriphosphate)
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reversible competitive dead-end inhibitor
guanosine 5'-(gamma-thiotriphosphate)
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inhibits both tubulin polymerization and GTP hydrolysis
N-ethylmaleimide
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inhibits GTP hydrolysis, but not the assembly reaction
N-terminal part of stathimin like-domains
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-
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NO3-
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rotenone
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rotenone inhibits both the assembly and the GTP hydrolysis rate of microtubules in vitro
tubulin
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unassembled tubulin denatures relatively easily, and the denatured tubulin can inhibit the assembly itself
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vinblastine
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additional information
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the phosphorylated protein shows hardly any activity
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,3,4-trimethoxy-4'-(carbomethoxy)-1,1'-biphenyl
allocolchicine
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ATP
ATP interacts with FtsZDr and stimulates its GTPase activity by about 2fold possibly by increasing both substrate affinity and rate of reaction
Colchicine
dimethyl sulfoxide
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cosolvent stabilizes tubulin
glycerol
Gsalpha
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Gsalpha facilitates GTP hydrolysis on tubulin, which leads to microtubule depolymerization by increased GTPase activation on tubulin
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nocodazole
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induced by this antimitotic drug, GTP hydrolysis rate stimulated four-to fivefold
quercetin
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0.05 mM, stimulates GTPase activity about 8fold, but inhibits polymerization of microtubules and specifically inhibits colchicine binding to tubulin
sodium glutamate
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cosolvent stabilizes tubulin
stathmin-like domains
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Sucrose
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cosolvent stabilizes tubulin
tubulin folding cofactor
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GTPase activating proteins A, B, C and D
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.008 - 4.8
GTP
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000037 - 1500
GTP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 20
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5.6
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in the presence of 50 microM curcumin
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6 - 7.2
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7.4
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assay at
8
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GTPase activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 37
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-
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
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predicted pI-value
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
54400
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calculated molecular mass, verified by SDS-PAGE and Western blotting
101000
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1 * 101000 + 1 * 106000 + 1 * 115000 + 1 * 128000, bands are probably tubulin dimers formed by cross-linking 2 alpha, 2 beta and an alpha and beta subunit
106000
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1 * 101000 + 1 * 106000 + 1 * 115000 + 1 * 128000, bands are probably tubulin dimers formed by cross-linking 2 alpha, 2 beta and an alpha and beta subunit
110000
115000
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1 * 101000 + 1 * 106000 + 1 * 115000 + 1 * 128000, bands are probably tubulin dimers formed by cross-linking 2 alpha, 2 beta and an alpha and beta subunit
128000
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1 * 101000 + 1 * 106000 + 1 * 115000 + 1 * 128000, bands are probably tubulin dimers formed by cross-linking 2 alpha, 2 beta and an alpha and beta subunit
220000
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gel filtration in absence of glycerol
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
tetramer
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1 * 101000 + 1 * 106000 + 1 * 115000 + 1 * 128000, bands are probably tubulin dimers formed by cross-linking 2 alpha, 2 beta and an alpha and beta subunit
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6 - 7.4
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tubulin stable in this range
644203
additional information
protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer
699587
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, frozen as pellets in liquid nitrogen
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-80°C, 50 mM Mes-K buffer, pH 6.8, 33% glycerol, 0.25 mM MgCl2, 0.5 mM EGTA, 0.1 mM GTP
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-80°C, in MES-Na buffer pH 6.4, 1 mM EGTA, 0.5 mM MgCl2, 1 mM GTP, 8 M glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bovine brain tubulin is purified by two cycles of polymerization and depolymerization
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goat brain microtubule protein is isolated by two cycles of polymerization and depolymerization, MAP-free tubulin is purified from the microtubule protein by phosphocellulose chromatography
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MAP-free tubulin is purified from the microtubule protein of goat brain by phosphocellulose chromatography
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microtubules are prepared from freshly isolated lens epithelial fractions from mice
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ovine brain tubulin is prepared from freshly-killed animals using phosphocellulose chromatography
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purified to ca. 35%
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recombinant His-tagged beta-tubulin mutant T238A from Saccharomyces cerevisiae by nickel affinity chromatography
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recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by dialysis and nickel affinity chromatography, tag cleavage by thrombin, and dialysis
using a glutathione-Sepharose affinity column
using a Ni-nitrilotriacetic acid resin column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
analysis of interaction between tublin and PN2-3 domain of centrosomal P4.1-associated protein
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beta-tubulin gene cloned and used for transformation
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demonstration of the several different stages of FtsZ-ring formation and their timing in relation to the stages of DNA replication
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expression in Escherichia coli
Prosthecobacter sp.
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gene ftsZ or DR 0631, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3) , FtsZDr-GFP expressing in Deinococcus radiodurans produces typical Z ring perpendicular to the plane of first cell division
into pBAD/myc-HisB for expression in Escherichia TOP10 cells
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into the pUC19 vector, and in the pGEX-KG vector for expression in Escherichia coli BL21DE3 cells
overexpression of His-tagged beta-tubulin mutant T238A in Saccharomyces cerevisiae, the transformed cells shows no mutant and wild-type microtubule dynamics
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Rac1 cDNA cloned and expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C354A
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site-directed mutagenesis of beta-tubulin, the mutation dramatically reduces the rate of microtubule shrinking and the frequency of catastrophe
C354S
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site-directed mutagenesis of beta-tubulin, the mutation dramatically reduces the rate of microtubule shrinking and the frequency of catastrophe
T143G
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mutation in tubulin signature motif of beta-tubulin, both GTP-binding affinity and microtubule-dependent GTPase activity are reduced at least 15 fold, mutant cells have a delay in mitosis
T238A
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naturally occuring mutation, the buried mutation T238A in alphabeta-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency, the mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized alphabetab-tubulin. The mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules. The buried T238A mutation in beta-tubulin hyperstablizes microtubules in vivo and in vitro. Mutant-induced changes in polymerization dynamics do not result from defective GTPase activity. The T238A alphabeta-tubulin undergoes spontaneous nucleation more readily than wild-type, even in the presence of a nonhydrolyzable GTP analog, GTPgammaS, phenotype, overview