Information on EC 2.7.4.13 - (deoxy)nucleoside-phosphate kinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.4.13
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RECOMMENDED NAME
GeneOntology No.
(deoxy)nucleoside-phosphate kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + deoxynucleoside phosphate = ADP + deoxynucleoside diphosphate
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
purine deoxyribonucleosides salvage
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pyrimidine deoxyribonucleotide phosphorylation
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pyrimidine deoxyribonucleotides biosynthesis from CTP
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pyrimidine deoxyribonucleotides de novo biosynthesis I
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pyrimidine deoxyribonucleotides de novo biosynthesis II
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pyrimidine deoxyribonucleotides de novo biosynthesis III
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pyrimidine deoxyribonucleotides de novo biosynthesis IV
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superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli)
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purine metabolism
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SYSTEMATIC NAME
IUBMB Comments
ATP:deoxynucleoside-phosphate phosphotransferase
dATP can substitute for ATP.
CAS REGISTRY NUMBER
COMMENTARY hide
37278-20-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
from infected Escherichia coli cells; maturation defective phage mutant T4amBL292, phage-coded activity which is a component of T4 dNTP-synthezising enzyme complex
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Manually annotated by BRENDA team
no activity in Escherichia coli
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Manually annotated by BRENDA team
ATCC 2610
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
essential catalytic site residue, active site modelling, overview. The charged amino acid residues of the NMP binding domain and the presence of an OH-group at position 17 are important for the catalytic activity. Arginine residues at positions 130 and 172 are involved in the binding to the donor gamma-phosphoryl and acceptor alpha-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. The NMPK-P loop, or a glycine enriched loop, forms an anion hole interacting with donor phosphoryl groups. It contains a GX1X2X3X4GKX5T(S) consensus called the Walker A motif
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + 5-bromo-dUMP
ADP + 5-bromo-dUDP
show the reaction diagram
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phosphorylated at 45% the rate of dAMP
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r
ATP + dAMP
ADP + dADP
show the reaction diagram
ATP + dCMP
ADP + dCDP
show the reaction diagram
ATP + deoxynucleoside monophosphate
ADP + deoxynucleoside diphosphate
show the reaction diagram
ATP + deoxynucleoside phosphate
ADP + deoxynucleoside diphosphate
show the reaction diagram
ATP + dGMP
ADP + dGDP
show the reaction diagram
ATP + dTMP
ADP + dTDP
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 + dAMP
ADP + dADP
show the reaction diagram
Q6QGP4
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-
-
?
ATP + dCMP
ADP + dCDP
show the reaction diagram
Q6QGP4
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-
-
?
ATP + deoxynucleoside monophosphate
ADP + deoxynucleoside diphosphate
show the reaction diagram
ATP + deoxynucleoside phosphate
ADP + deoxynucleoside diphosphate
show the reaction diagram
ATP + dGMP
ADP + dGDP
show the reaction diagram
Q6QGP4
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-
-
?
ATP + dTMP
ADP + dTDP
show the reaction diagram
Q6QGP4
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-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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requirement, can replace Mg2+
Fe2+
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activation, less effective than Mg2+, Co2+, Mn2+
Mn2+
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requirement, can replace Co2+ or Mg2+
additional information
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no activation by Ba2+, Ca2+, Cd2+, Cr3+, Cu2+, Fe3+, Hg2+, Ni2+, Zn2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dAMP
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competitive, dTMP as substrate
dCMP
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competitive, dTMP as substrate
dGMP
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competitive, dTMP as substrate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11 - 0.67
ATP
0.22 - 1.1
dAMP
0.034 - 0.53
dCMP
0.22 - 0.33
dGMP
0.173 - 0.89
dTMP
2.8
dUMP
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pH 7.5, 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.15
dAMP
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dTMP as substrate, pH 7.6, 37°C
0.032
dCMP
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dTMP as substrate, pH 7.6, 37°C
0.15
dGMP
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dTMP as substrate, pH 7.6, 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14.7
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purified enzyme
25.78
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dCMP
29
recombinant protein in sonicated cell exctracts, with dGMP as substrate
36
recombinant protein after Toyopearl 650 M flow-through, with dGMP as substrate
36.54
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dGMP
40
recombinant protein after ammonium sulphate precipitation, with dGMP as substrate
41.24
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dTMP
73.17
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dAMP
84.2
homogeneous recombinant enzyme with dCMP as substrate
153.6
homogeneous recombinant enzyme with dGMP as substrate
155
affinity chromatography eluant of the recombinant protein, with dGMP as substrate; ion-exchange chromatography MonoQ eluant of the recombinant protein, with dGMP as substrate
additional information
specific activties of wild-type enzyme and enzyme mutants with different dNMP acceptor substrates, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8.5
all enzyme mutants but T17S
7.6
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substrate dTMP
additional information
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between pH 7-8
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 9
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about 80% of maximal activity at pH 6.2 and about 65% of maximal activity at pH 9 with substrate dGMP
6.2 - 9.4
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about 65% of maximal activity at pH 6.2 and about 70% of maximal activity at pH 9.4 with substrate dAMP
6.3 - 9
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at pH 6.3: about 70% of maximal activity with substrate dTMP, about 85% of maximal activity with substrate dCMP, at pH 9.0: about 50% of maximal activity with substrate dTMP, 65% of maximal activity with substrate dCMP
additional information
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active over a broad range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.2
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determined under partially denaturing condiions
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20640
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calculated from amino acid sequence
22930
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calculated from amino acid sequence
24260
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calculated from amino acid sequence
24690
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calculated from amino acid sequence
28670
calculated from nucleotid sequence
29000
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1 * 29000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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60 min, stable
42
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10% loss of activity after 30 min
47
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50% loss of activity after 30 min
52
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rapid inactivation, t1/2: 5 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, purified enzyme, 15% glycerol, only loss of a small part of activity after 6 months
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0°C, 25% loss of activity per day in crude cell extract
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4°C, several months, ionic strength 0.1 or above
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1190fold to homogeneity
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recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and affinity chromatography
two alternative methods, the first involves ion-exchange chromatography on Mono Q, the second involves affinity chromatography on Toyopearl AF-Red, the protein is purified 5.34fold in both cases
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli strain BL21 (DE3); expression in Escherichia coli strain BL21 (DE3), CK expression at 37°C forms insoluble inclusion bodoes, therefore expression was performed at 25°C
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gene dnk, recombinant expression of wild-type and mutant enzymes in Escherichia coli strains BL21(DE3) and XL10Gold
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D16N
site-directed mutagenesis of the P loop-related residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
D170N
site-directed mutagenesis of the core residue, almost inactive mutant
E176Q
site-directed mutagenesis of the core residue, almost inactive mutant
G137A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type enzyme
K131E
site-directed mutagenesis, of the dNMP-binding residue, the mutant shows slightly increased activity and altered substrate specificity compared to the wild-type enzyme
Q134A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
R130K
site-directed mutagenesis of the dNMP-binding residue, almost inactive mutant
R172I
site-directed mutagenesis of the core residue, almost inactive mutant
S13A
site-directed mutagenesis of the P loop-related residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
T138A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
T17N
site-directed mutagenesis of the P loop-related residue, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type enzyme
T17S
site-directed mutagenesis, of the P loop-related residue, the mutant shows reduced activity compared to the wild-type enzyme
W150A
site-directed mutagenesis of the core residue
W150F
site-directed mutagenesis of the conserved residue, the mutant shows unaltered activity compared to the wild-type enzyme
additional information
all enzyme mutants but T17S show an increased pH optimum compared to the wild-type enzyme