Information on EC 2.7.4.25 - (d)CMP kinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.4.25
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RECOMMENDED NAME
GeneOntology No.
(d)CMP kinase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + (d)CMP = ADP + (d)CDP
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
CMP phosphorylation
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-
pyrimidine deoxyribonucleotide phosphorylation
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SYSTEMATIC NAME
IUBMB Comments
ATP:(d)CMP phosphotransferase
The prokaryotic cytidine monophosphate kinase specifically phosphorylates CMP (or dCMP), using ATP as the preferred phosphoryl donor. Unlike EC 2.7.4.14, a eukaryotic enzyme that phosphorylates UMP and CMP with similar efficiency, the prokaryotic enzyme phosphorylates UMP with very low rates, and this function is catalysed in prokaryotes by EC 2.7.4.22, UMP kinase. The enzyme phosphorylates dCMP nearly as well as it does CMP [1].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
in a strain deleted for cmk, the pools of CMP and dCMP are elevated approximately 30fold
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 + 1-beta-D-arabinofuranosylcytosine 5'-phosphate
ADP + 1-beta-D-arabinofuranosylcytosine 5'-diphosphate
show the reaction diagram
-
-
-
?
ATP + 2',3'-dideoxy-CMP
ADP + ?
show the reaction diagram
2,3-dideoxy-CMP is a poor substrate
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-
?
ATP + 2-thiouridine 5'-monophosphate
ADP + 2-thiouridine 5'-diphosphate
show the reaction diagram
2-thiouridine 5'-monophosphate is a poor substrate
-
-
?
ATP + ara-CMP
ADP + ara-CDP
show the reaction diagram
ATP + CMP
ADP + CDP
show the reaction diagram
ATP + CMP
ADP + CTP
show the reaction diagram
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cytidylate kinase
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-
?
ATP + dCMP
ADP + dCDP
show the reaction diagram
ATP + dCMP
ADP + dCTP
show the reaction diagram
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cytidylate kinase
-
-
?
ATP + dUMP
ADP + dUDP
show the reaction diagram
dUMP is a poor substrate
-
-
?
ATP + GTP
ADP + GDP
show the reaction diagram
-
GTP is a poor substrate with Bacillus subtilis CMP kinase
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-
?
ATP + UMP
ADP + UDP
show the reaction diagram
CTP + CMP
CDP + CDP
show the reaction diagram
the activity with CTP is still measurable but less than 0.05% of that with ATP
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-
?
dATP + CMP
dADP + CDP
show the reaction diagram
dCTP + CMP
dCDP + CDP
show the reaction diagram
the activity with dCTP is still measurable but less than 0.05% of that with ATP
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-
?
dUTP + CMP
dUDP + CDP
show the reaction diagram
the activity with dUTP is still measurable but less than 0.05% of that with ATP
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-
?
GTP + CMP
GDP + CDP
show the reaction diagram
ITP + CMP
IDP + CDP
show the reaction diagram
ITP is a poor substrate
-
-
?
UTP + CMP
UDP + CDP
show the reaction diagram
the activity with UTP is still measurable but less than 0.05% of that with ATP
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-
?
additional information
?
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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 + CMP
ADP + CDP
show the reaction diagram
ATP + dCMP
ADP + dCDP
show the reaction diagram
additional information
?
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bacterial CMP kinases are specific for CMP and dCMP, whereas the related eukaryotic NMP kinase phosphorylates CMP and UMP with similar efficiency. Bacterial CMP kinase has a narrower NMP-binding pocket and a hydrogen-bonding network involving the pyrimidine moiety specific for the cytosine nucleobase compared to eukaryotic UMP?CMP kinases
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
TMP, Ant-dCMP, 5-methyl-CMP, AMP, dAMP, GMP, and dGMP are no inhibitors of the enzyme
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.36
1-beta-D-arabinofuranosylcytosine 5'-phosphate
in 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 2 mM MgCl2, 1 mM phosphoenolpyruvate, 0.2 mM NADH, at 30C
0.15 - 0.65
2',3'-dideoxy-CMP
0.37 - 1
ara-CMP
0.36 - 0.37
araCMP
0.038 - 0.12
ATP
0.035 - 20.2
CMP
0.087
dATP
0.0394 - 7.3
dCMP
0.094
dGMP
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reaction with ATP
1.46
dUMP
in 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 2 mM MgCl2, 1 mM phosphoenolpyruvate, 0.2 mM NADH, at 30C
0.64
GTP
0.87
ITP
in 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 2 mM MgCl2, 1 mM phosphoenolpyruvate, 0.2 mM NADH, at 30C
0.93 - 13.85
UMP
additional information
additional information
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kinetics and substrate specificities of wild-type and mutant enzymes, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0083 - 0.65
2',3'-dideoxy-CMP
0.085 - 56
ara-CMP
0.069 - 103
CMP
0.04 - 109
dCMP
0.013 - 12.2
UMP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.083 - 105
ara-CMP
4641
0.0114 - 2940
CMP
100
0.0068 - 1160
dCMP
450
0.0033 - 1.25
UMP
133
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0023
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enzyme from cmk mutant strain KP1300, using CMP as substrate, at pH 8.4 and 37C
0.0033
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enzyme from cmk mutant strain JL924, using CMP as substrate, at pH 8.4 and 37C
0.0177
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enzyme from cmk wild type strain JL1278, using CMP as substrate, at pH 8.4 and 37C
additional information
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Mycobacterium abscessus (strain ATCC 19977 / DSM 44196 / CIP 104536 / JCM 13569 / NCTC 13031 / TMC 1543)
Mycobacterium abscessus (strain ATCC 19977 / DSM 44196 / CIP 104536 / JCM 13569 / NCTC 13031 / TMC 1543)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Yersinia pseudotuberculosis serotype O:3 (strain YPIII)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
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gel filtration
24060
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calculated from amino acid sequence
24620
electrospray ionization mass spectrometry
24700
calculated from amino acid sequence
30000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with CDP
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hanging drop vapor diffusion technique
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of the R188M mutant alone or with dCMP
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purified recombinant mutant R188M free or in complex with CMP, X-ray diffraction structure determination and analysis at 1.9 A and 2.8 A resolution, respectively
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hanging drop vapor diffusion method, using 0.4 M ammonium sulfate in 50 mM Tris-HCl buffer (pH 7.4), at 20C
in complex with substrates CMP, dCMP, ara-CMP and 2,3-dideoxy-CMP, hanging drop vapor diffusion method, using ammonium sulfate in a 50 mM Tris-HCl buffer (pH 7.4), at 20C
mutant enzyme R188M is crystallized either alone or in complex with dCMP, hanging drop vapor diffusion method, using ammonium sulfate as a precipitant (1.3 M in the case of enzyme alone, and 1.7 M for the R188M-dCMP complex)
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
43
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mutant D132A
45
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mutant R110M
47
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mutant D132N
48.5 - 57.8
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the melting temperature of CMP kinae in the absence of nucleotide substrates is at 48.5C, the melting temperature of CMP kinae in the presence of ATP is at 57.8C, the melting temperature of CMP kinae in the presence of CMP is at 50.5C
58
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midpoint denaturation temperature in presence of ATP
additional information
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thermal stability of CMP kinase genetic variants, overview
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
four key amino acids interacting with the pyrimidine ring of CMP, Ser36, Asp132, Arg110 and Arg188, contribute to the stability, catalysis and substrate specificity of Escherichia coli CMP kinase
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, no apparent loss of activity
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4C, up to 30 days, recombinant CMK remains stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anionic-exchange column chromatography, gel filtration, and anionic-exchange resin column chromatography
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blue Sepharose column chromatography and Ultrogel AcA54 gel filtration
Ni-NTA column chromatography
of the recombinant protein
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of the recombinant proteins
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression of wild-type and mutant enzymes
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hanging-drop method in 50 mM Tris-HCl buffer, pH 7.4, 20C, with ammonium sulfate as a precipitant
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overexpression in Escherichia coli
overexpression in Escherichia coli to produce CTP
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D132A
the mutant shows reduced activity compared to the wild type enzyme, the mutation has the most dramatic consequences on the protein stability as Tm decreases by 9C in comparison with the wild type protein, an increase in the specificity of the D132A variant for UMP over CMP and dCMP is observed
D132H
the mutant shows reduced activity compared to the wild type enzyme
D132N
the mutant shows reduced activity compared to the wild type enzyme
D132S
the substitution results in a moderate decrease in stability without significant changes in Km value for CMP and dCMP
D185A
the mutant shows severely decreased CMP and dCMP phosphoryation activity compared to the wild type enzyme
R110M
the mutant shows reduced activity compared to the wild type enzyme
R181M
the mutant shows severely decreased CMP and dCMP phosphoryation activity compared to the wild type enzyme
R188M
the mutation does not affect enzyme stability but dramatically decreases the kcat_Km ratio compared with wild type enzyme, the mutant has no activity towards UMP
S101A
the mutation reduces CMP phosphorylation only moderately, but dramatically reduces dCMP phosphorylation
S36A
the S36A substitution mainly changes the Km value for the two natural substrates, which increases by a factor of 70 (CMP) and 37 (dCMP) compared with the parent molecule and decreases kcat of 1.6fold (CMP) and 7.4fold (dCMP) with respect to the wild type enzyme
V164E
substitution of Val164 by a Glu residue apparently does not affect the catalytic properties of Escherichia coli CMP kinase
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
CMP kinase and actetate kinase in a whole cell-biocatalysis to obtain CTP
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