Information on EC 3.6.1.39 - thymidine-triphosphatase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.1.39
-
RECOMMENDED NAME
GeneOntology No.
thymidine-triphosphatase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
dTTP + H2O = dTDP + phosphate
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphorous acid anhydride hydrolysis
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NIL
-
-
Pyrimidine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
dTTP nucleotidohydrolase
Also acts, more slowly, on dUTP and UTP.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
dCTP + H2O
dCDP + phosphate
show the reaction diagram
dTTP + H2O
dTDP + phosphate
show the reaction diagram
dTTP + H2O
dTMP + diphosphate
show the reaction diagram
dUTP + H2O
dUDP + phosphate
show the reaction diagram
dUTP + H2O
dUMP + diphosphate
show the reaction diagram
UTP + H2O
UDP + phosphate
show the reaction diagram
additional information
?
-
-
wild type gp4 helicase unwinds the duplex DNA
-
-
-
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
dTTP + H2O
dTDP + phosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
-
weak activator
K+
-
weak activator
Li+
-
weak activator
Na+
-
weak activator
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AgCl
-
Ki: 4 mM
dCTP
-
competitive inhibitor with dTTP as substrate, Ki: 0.25 mM
diphosphate
-
with dTTP or dUDP as substrate
dTDP
-
with dTTP or dUTP as substrate
dUDP
-
with dTTP or dUTP as substrate
dUTP
-
with dTTP as substrate
HgCl2
-
Ki: 0.6 mM
NaVO4
-
Ki: 0.8 mM
para-hydroxy-mercuribenzoate
-
no inhibition in presence of 25 mM beta-mercaptoethanol
Tartrate
-
Ki: 1.5 mM
additional information
-
different purin- and pyrimidinnucleotide tested
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
single-stranded DNA
-
the presence of single-stranded DNA stimulates the rate of dTTP hydrolysis about 100fold in the case of wild type enzyme
sulfhydryl compound
-
required for activity of the partially purified enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.091
dCTP
-
-
0.038 - 2.6
dTTP
0.125
dTTP/dCTP
-
equimolar
0.011
dUTP
-
-
additional information
additional information
-
dTTP hydrolysis kinetics are measured in real time using the phosphate-binding protein that acts as a sensitive sensor for fast detection of low concentrations of phosphate, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05 - 20
dTTP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.8 - 7.7
dTTP
145
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4
AgCl
-
-
0.25
dCTP
-
competitive inhibitor with dTTP as substrate
0.6
HgCl2
-
-
0.8
NaVO4
-
-
1.5
Tartrate
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
-
-
7.4 - 7.6
-
-
7.5
-
DNA binding and dTTPase assays at
7.5 - 8.5
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 11
-
-
6 - 9
-
no activity at pH 6, very low activity at pH 9
7 - 10.5
-
-
7.5 - 10
-
measured in Tris-buffer and glycine-buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
25% of activity compared with activity at 37C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
associated
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24000
-
SDS-PAGE
46000
-
gel filtration
48500
-
gel filtration and sucrose density gradient centrifugation
100000
-
gel filtration in presence of 1% Triton
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer or heptamer
-
in the presence of dTTP Gp4 oligomerizes to form hexamers and heptamers
additional information
-
all subunits are catalytic
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
-
loss of activity above pH 9
210043
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12 - 23
-
no loss of activity within 1 h
30 - 37
-
stability starts to decline above 30C and breaks down at 37C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 10 mM Tris/HCl, pH 7,8, 5 mM mercaptoethanol, stable on freezing and thawing two times
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial purification
purified to homogenity
-
use of preparative SDS-PAGE, for small amounts of pure protein
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli HMS-174(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E343D
-
the mutant does not hydrolyze dTTP
E343N
-
the mutant does not hydrolyze dTTP
N468A
-
the mutant hydrolyzes dTTP at rates comparable with wild type enzyme. N468A hydrolyzes nearly 4fold less efficiently than the wild type enzyme in the absence of single-stranded DNA and is not stimulated by the presence of single-stranded DNA
R493A/N468A
-
the mutant hydrolyzes dTTP at rates comparable with wild type enzyme and is not stimulated by the presence of single-stranded DNA
R493Q
-
the mutant hydrolyzes dTTP at rates comparable with wild type enzyme and is not stimulated by the presence of single-stranded DNA; the mutant maintains only the basal level of dTTPase activity but does not exhibit activation in the dTTP hydrolysis activity in the presence of single-stranded DNA
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
renaturation after preparative SDS-PAGE
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
potential prognostic marker for different diseases e.g. leukemia