Information on EC 2.7.4.22 - UMP kinase

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

EC NUMBER
COMMENTARY hide
2.7.4.22
-
RECOMMENDED NAME
GeneOntology No.
UMP kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + UMP = ADP + UDP
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
Pyrimidine metabolism
-
-
UTP and CTP de novo biosynthesis
-
-
pyrimidine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:UMP phosphotransferase
This enzyme is strictly specific for UMP as substrate and is used by prokaryotes in the de novo synthesis of pyrimidines, in contrast to eukaryotes, which use the dual-specificity enzyme UMP/CMP kinase (EC 2.7.4.14) for the same purpose [2]. This enzyme is the subject of feedback regulation, being inhibited by UTP and activated by GTP [1].
CAS REGISTRY NUMBER
COMMENTARY hide
9036-23-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
strain KUR1244
SwissProt
Manually annotated by BRENDA team
strain KUR1244/K12 pyrH (high dose); strain KUR1244/K12 pyrH (low dose)
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain JL1019 - uracil, genotype: pyrC1502; strain JL1019 + uracil, genotype: pyrC1502
SwissProt
Manually annotated by BRENDA team
strain JL1219, genotype: pyrH1608, udk+
SwissProt
Manually annotated by BRENDA team
strain JL1220, genotype: pyrH1609, udk-10
SwissProt
Manually annotated by BRENDA team
strain JL1269, genotype: pyrH1609, udk+
SwissProt
Manually annotated by BRENDA team
strain KP1217, genotype: pyrH+, udk+
SwissProt
Manually annotated by BRENDA team
gene pyrH; plasmid pET-14b containing cDNA for UMPK, expression in Escherichia coli BL21 (DE3), overexpression by induction with isopropyl-b-d-thiogalactoside
Uniprot
Manually annotated by BRENDA team
strain MO6-24/O and CMM1474
-
-
Manually annotated by BRENDA team
strain MO6-24/O and CMM1474
-
-
Manually annotated by BRENDA team
pathovar campestris, strain 17, gene XC1936
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
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-fluoro-UMP
ADP + 5-fluoro-UDP
show the reaction diagram
ATP + 6-aza-UMP
ADP + 6-aza-UDP
show the reaction diagram
ATP + dUMP
ADP + dUDP
show the reaction diagram
3.7% of the activity detected with UMP
-
-
?
ATP + UMP
ADP + UDP
show the reaction diagram
GTP + UMP
GDP + UDP
show the reaction diagram
-
reaction rate is 3-5% of that with ATP
-
-
r
MgATP2- + UMP
MgADP- + UDP
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 + UMP
ADP + UDP
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cd2+
causes severe precipitation problems in the phosphate buffer; precipitates in phosphate buffer
MgCl2
in the presence of MgCl2 the saturation kinetics of recombinant purified UMP kinase are hyperbolic for UMP and sigmoidal for ATP
MnCl2
in the presence of MnCl2 the saturation kinetics of recombinant purified UMP kinase are hyperbolic for UMP and sigmoidal for ATP
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-[([5-oxo-4-[(2R)-tetrahydrofuran-2-ylmethyl]-4,5-dihydro-1H-1,2,4-triazol-3-yl]sulfanyl)acetyl]piperidine-4-carboxylic acid
-
1-[2-[(2-amino-6-phenylpyrimidin-4-yl)amino]phenyl]ethanone
-
-
2-(4-acetyl-3,5-dimethyl-1H-pyrazol-1-yl)-N-[1-(2-methoxybenzyl)-1H-pyrazol-5-yl]acetamide
-
2-amino-8-(2-oxo-2-phenylethoxy)-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 75% inhibition, crystal enzyme complex structure, overview
2-amino-8-hydroxy-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 18% inhibition
2-amino-8-[2-(2-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 48% inhibition
2-amino-8-[2-(3-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 77% inhibition
2-amino-8-[2-(4-fluorophenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 73% inhibition
2-amino-8-[2-(4-hydroxyphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 77% inhibition
2-amino-8-[2-(4-methoxyphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 74% inhibition
2-amino-8-[2-(4-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
10 micromol, 60 min, room temperature, 80% inhibition
4-[(2-amino-6-oxo-4,5,6,9-tetrahydro-1H-purin-8-yl)methyl]benzonitrile
-
10 micromol, 60 min, room temperature, 53% inhibition, crystal enzyme complex structure, overview
4-[2-amino-4-(4-chlorophenyl)-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl]butanoic acid
-
4-[2-amino-4-(4-methoxyphenyl)-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl]butanoic acid
-
4-[[(2-amino-6-oxo-4,5,6,9-tetrahydro-1H-purin-8-yl)oxy]acetyl]benzonitrile
-
10 micromol, 60 min, room temperature, 68% inhibition
5-bromo-UMP
5-Bromo-UTP
5-fluoro-UTP
5-iodo-UMP
5-iodo-UTP
5-[(9H-[1,2,4]triazolo[4,3-a]benzimidazol-3-ylsulfanyl)methyl]furan-2-carboxylic acid
hydrogen bonding interactions of ZINC12561276 molecule with the active site residues of Mtb-UMPK homology model, overview
dUMP
weak competitive inhibitor
guanylyl imidodiphosphate
-
mutant D201N
N-benzyl-2-[(2S,3R,4S,5R)-3,4-dihydroxy-5-[[(methylsulfonyl)amino]methyl]tetrahydrofuran-2-yl]-N-methylacetamide
-
N4,6-diphenylpyrimidine-2,4-diamine
-
-
N4-(2-methoxyphenyl)-6-phenylpyrimidine-2,4-diamine
-
-
N4-(2-methylphenyl)-6-phenylpyrimidine-2,4-diamine
-
-
N4-(3-methylphenyl)-6-phenylpyrimidine-2,4-diamine
-
-
N4-(4-methoxyphenyl)-6-phenylpyrimidine-2,4-diamine
-
-
N4-(4-methylphenyl)-6-phenylpyrimidine-2,4-diamine
-
-
N4-ethyl-N4,6-diphenylpyrimidine-2,4-diamine
-
-
-
N6-[(4-nitrophenyl)methyl]-N2-[[3-(trifluoromethyl)phenyl]methyl]-1H-purine-2,6-diamine
-
-
phosphate
phosphate-inhibited UpUMPK activity with an IC50 value of 1 mM, the molar content of phosphate in the soluble UMPK is 4%
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate
-
-
3'-anthraniloyl-dGTP
5'-guanylyl-imidodiphosphate
-
-
5-fluoro-UTP
below 0.15 mM 5-fluoro-UTP increases the activity of the enzyme by 80%
7-deaza-dGTP
cGMP
-
lower affinity or extent of activation than GTP
dialdehyde-GTP
-
slight stimulatory effect, 30% maximal increase in UMP-kinase activity
GMP-4-nitrophenol
much weaker activator (only 50% increase in activity at 1 mM)
GMP-PNP
guanosine
-
lower affinity or extent of activation than GTP
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11 - 0.12
5-fluoro-UMP
0.14 - 0.71
6-aza-UMP
0.03 - 30
ATP
1
GTP
-
wild-type
0.19 - 0.77
MgATP2-
0.0023 - 22.2
UMP
0.023 - 0.046
UTP
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
120 - 436.2
ATP
19 - 545.2
UMP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
623.1 - 1250
ATP
4
2181 - 4362
UMP
133
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1
5-bromo-UMP
Ki above 1 mM, wild type enzyme, at pH 7.4 and 30°C
0.097
ATP
in presence of 0.1 mM UMP
0.14 - 0.21
GTP
0.014 - 10.9
UMP
0.0009 - 1.2
UTP
additional information
additional information
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6
1-[2-[(2-amino-6-phenylpyrimidin-4-yl)amino]phenyl]ethanone
Staphylococcus aureus
-
pH 7.5, 22°C
139
2-amino-8-(2-oxo-2-phenylethoxy)-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
243
2-amino-8-[2-(2-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
51
2-amino-8-[2-(3-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
79
2-amino-8-[2-(4-fluorophenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
82
2-amino-8-[2-(4-hydroxyphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
44
2-amino-8-[2-(4-methoxyphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
64
2-amino-8-[2-(4-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
Staphylococcus aureus
-
pH 8.5, 23°C
254
4-[(2-amino-6-oxo-4,5,6,9-tetrahydro-1H-purin-8-yl)methyl]benzonitrile
Staphylococcus aureus
-
pH 8.5, 23°C
88
4-[[(2-amino-6-oxo-4,5,6,9-tetrahydro-1H-purin-8-yl)oxy]acetyl]benzonitrile
Staphylococcus aureus
-
pH 8.5, 23°C
0.02
5-iodo-UTP
Mycobacterium tuberculosis
P9WHK5
wild type enzyme, at pH 7.4 and 30°C
0.4
dTTP
Mycobacterium tuberculosis
P9WHK5
wild type enzyme, at pH 7.4 and 30°C
0.24
dUTP
Mycobacterium tuberculosis
P9WHK5
wild type enzyme, at pH 7.4 and 30°C
0.0156
N4,6-diphenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
pH 7.5, 22°C
32.9
N4-(2-methoxyphenyl)-6-phenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
pH 7.5, 22°C
53.8
N4-(2-methylphenyl)-6-phenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
pH 7.5, 22°C
0.1
N4-(3-methylphenyl)-6-phenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
above, pH 7.5, 22°C
0.1
N4-(4-methoxyphenyl)-6-phenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
above, pH 7.5, 22°C
0.1
N4-(4-methylphenyl)-6-phenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
above, pH 7.5, 22°C
0.025
N4-ethyl-N4,6-diphenylpyrimidine-2,4-diamine
Staphylococcus aureus
-
pH 7.5, 22°C
-
1
phosphate
Ureaplasma parvum
Q9PPX6
phosphate ion is found in the active site
0.01 - 0.45
UTP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00014
-
R62H/D77N mutant, without UTP, no activity with 1 mM UTP
0.0002
-
R62H mutant, without UTP, no activity with 1 mM UTP
0.0003
-
D77N mutant, without UTP, no activity with 1 mM UTP
0.00613
-
wild-type, with 1 mM UTP
0.011
0-45% ammonium sulfate precipitate
0.01238
-
wild-type, without UTP
0.033
45-55% ammonium sulfate precipitate
0.053
55-80% ammonium sulfate precipitate
0.13
-
mutant D201N, rate of the reverse reaction
0.15
-
mutant D146N, at pH 6
0.32
-
mutant D146N, at pH 8
0.6
-
with 6-aza-UMP as substrate
1.04
-
mutant D201N, at pH 6
1.5
-
mutant R62H, at pH 6
1.7
-
at 2.0 mM ATP, without GTP
1.9
-
mutant D77N, at pH 6
2.7
-
at 2.0 mM ATP, without GTP
3.6
-
mutant D201N, in the presence of 1 mM ATP and 1 mM UMP
3.7
-
at 2.0 mM ATP, without GTP
4.4
-
mutant R62H, at pH 8
4.6
-
at 2.0 mM ATP, without GTP
5.1
activity dependent of concentrations of ATP, at 2.0 mM ATP, without GTP
8.2
-
mutant D201N, at pH 8; mutant D77N, at pH 8
9.7
-
at 2.0 mM ATP, with 0.5 mM GTP
10.2
at 8.0 mM ATP, with 0.5 mM GTP
11.3
at 8.0 mM ATP, without GTP
11.8
-
at 8.0 mM ATP, without GTP
12.8
-
at 8.0 mM ATP, without GTP
18.1
-
at 8.0 mM ATP, without GTP
19.4
-
at 8.0 mM ATP, with 0.5 mM GTP
23.5
-
at 2.0 mM ATP, with 0.5 mM GTP
23.6
-
at 8.0 mM ATP, with 0.5 mM GTP
23.8
-
D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP
24
-
with 5-fluoro-UMP as substrate
25
-
His-tagged protein
26
-
native protein
26.1
at 2.0 mM ATP, without GTP
28.1
at 8.0 mM ATP, without GTP
28.6
-
at 2.0 mM ATP, with 0.5 mM GTP
30.4
-
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP
32
-
mutant D174N, at pH 6
36.3
-
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP
36.8
-
D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP
38.1
-
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP
38.4
-
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP
38.9
-
at 8.0 mM ATP, without GTP
39.1
-
at 8.0 mM ATP, with 0.5 mM GTP
40.7
-
D93A/D159N mutant protein, 0.2 mM ATP
45
-
mutant D174N, at pH 8
46.1
-
D159N mutant protein, 0.2 mM ATP
46.6
-
D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP
46.8
-
activity independent of concentrations of ATP, at 2.0 mM ATP, without GTP
48.9
-
N140A/D159N mutant protein, 0.2 mM ATP
51.4
-
N72A/D93A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP
51.9
-
D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP
52
-
mutant N140A
52.7
-
N72A/D93A/D159N mutant protein, 2 mM ATP
52.8
-
at 2.0 mM ATP, with 0.5 mM GTP
55.7
-
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP
57.6
-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP
57.7
at 2.0 mM ATP, with 0.5 mM GTP
58.2
-
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP
60.5
-
at 8.0 mM ATP, with 0.5 mM GTP
60.8
-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP
61.5
-
D93A/D159N mutant protein, 0.3 mM UMP
61.6
-
D159N mutant protein, 0.3 mM UMP; N72A/D159N mutant protein, 0.3 mM UMP
62.3
-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP
62.4
-
D159N mutant protein, 0.3 mM UMP
63
-
mutant D168N, at pH 6
65.9
at 2.0 mM ATP, with 0.5 mM GTP
66
with MgCl2 and ATP as phosphate donor
67
-
with 6-aza-UMP as substrate
68.5
-
at 2.0 mM ATP, with 0.5 mM GTP
71.1
at 8.0 mM ATP, with 0.5 mM GTP
76.1
-
N72A/D93A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP
77.1
-
D93A/D159N mutant protein, 2 mM ATP
78.3
-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP
78.7
-
N72A/D93A/D159N mutant protein, 0.3 mM UMP
81.9
-
N140A/D159N mutant protein, 0.3 mM UMP
83.4
-
D93A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP
84.2
-
D159N mutant protein, 1 mM UMP, 0.5 mM GTP; N72A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP
86.6
-
mutant D168N, at pH 8
87.4
-
at 8.0 mM ATP, with 0.5 mM GTP
88
-
mutant T138A
90
-
mutant T138A/N140A
90.7
-
D93A/D159N mutant protein, 0.3 mM UMP
91.7
-
D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP
92.5
-
D159N mutant protein, 2 mM ATP; N72A/D159N mutant protein, 2 mM ATP
93.9
-
D93A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP
96
-
wild-type
98.2
-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP
99.6
-
D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP
100.3
-
D159N mutant protein, 2 mM ATP, 0.5 mM GTP; N72A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP
110.5
-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP
124.2
-
at 8.0 mM ATP, with 0.5 mM GTP
126
-
with UMP as substrate
130
with MnCl2 and ATP as phosphate donor
134.3
-
at 2.0 mM ATP, with 0.5 mM GTP
135
at pH 6.5, in the presence of MnCl2
160
-
wild-type, in the presence of ATP and GTP
162
-
with 5-fluoro-UMP as substrate
182
at pH 6.5, in the presence of MgCl2
274
at pH 7.5, in the presence of MnCl2
399
at pH 7.5, in the presence of MgCl2
666
at pH 8.5, in the presence of MnCl2
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
with 4 mM ATP, in the presence of MgCl2
6.8
; purified recombinant enzyme
7
pH variation influences only turnover rate, at 60°C; variation of pH only influences the turnover number, at 60°C, 50 mM succinate/phosphate buffer, phosphate buffer, glycine buffer, assays are performed with 100 microM UMP, 500 microM ATP, and 10 mM MgCl2
7.1
with 4 mM ATP, in the presence of MnCl2
7.5
-
assay at
7.8
with 10 mM ATP, in the presence of MnCl2
9
-
when Mg2+ serves as the cofactor, maximal enzyme activity is found at pH 9.0
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 10.7
various buffers, at 60°C
7.4 - 8.5
-
-
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
assay at room temperature
30
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.24
-
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
near the bacterial membranes
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Xanthomonas campestris pv. campestris (strain ATCC 33913 / DSM 3586 / NCPPB 528 / LMG 568 / P 25)
Xanthomonas campestris pv. campestris (strain ATCC 33913 / DSM 3586 / NCPPB 528 / LMG 568 / P 25)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25000
6 * 25000, SDS-PAGE
26000
-
x * 26000, SDS-PAGE
26080
-
monomer, calculated from amino acid sequence; monomer, mass spectroscopy
26300
mass spectroscopy, N-terminal sequencing
27500
-
x * 27500, His6-tagged enzyme, SDS-PAGE
28120
-
His-tagged protein, monomer, mass spectroscopy
28250
-
His-tagged protein, monomer, calculated from amino acid sequence
32100
-
mutant D159N, monomer, sedimentation velocity at pH 9.0
32800
-
wild-type, monomer, sedimentation velocity at pH 9.0
35300
-
mutant D159N, monomer, sedimentation velocity at pH 7.4
59400
-
wild-type, dimer, sedimentation velocity at pH 6.0
87500
minor form, gel filtration
140000
gel filtration; gel flitration
145300
-
wild-type, hexamer, sedimentation velocity at pH 9.0
146400
-
mutant D159N, hexamer, sedimentation velocity at pH 9.0
147400
-
mutant D159N, hexamer, sedimentation velocity at pH 7.4
150000
154000
-
gel filtration
156000
-
sedimentation equilibrium
157000
-
wild-type, hexamer, sedimentation velocity at pH 6.0
173200
sedimentation equilibrium ultracentrifugation
189000
major form, representing two-thirds of the protein population, gel filtration
297400
-
wild-type, oligomer, sedimentation velocity at pH 9.0
482900
-
wild-type, oligomer, sedimentation velocity at pH 6.0
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homohexamer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme in complex with ATP, 300 nl sitting drops containing 1.5 mg/mL purified protein are mixed with 3.3 mM ATP, 0.33 mM MgCl2, 66 mM Li2SO4, 3% PEG 3000, 33 mM imidazole, pH 8.0, and 20 mM spermine tetrahydrochloride, equilibration against a reservoir solution of 200 mM Li2SO4, 10% PEG 3000, and 100 mM imidazole, pH 8.0, cryoprotection in 70% reservoir solution with 30% glycerol, 2 days, X-ray diffraction structure determination and analysis at 2.82 A resolution; UMP kinase in complex with ATP and Mg2+ at 2.82 A resolution, 0.2 M lithium sulfate, 10% polyethylene glycol 3000, 0.1 M imidazole, pH 8.0, space group P 61 2 2, allosteric nucleotide-binding site identified, a structural model for the allosteric regulation presented
bound to the UMP substrate, resolved at 2.3 A resolution, bound to the UDP product, resolved at 2.6 A resolution, bound to UTP, resolved at 2.45 A resolution
-
D159N, hanging drop vapor diffusion method; purified recombinant UMP kinase mutant D159N, bound to GTP, by hanging drop vapour diffusion method, 0.004 ml of protein solution containing 4 mg/ml UMPK mutant in 50 mM Tris-HCl, pH 8.0, and 100 mM NaCl, mixed with 0.004 ml precipitant solution containing 22.5 mM GTP and 21.5% w/v PEG 400 in 100 mM sodium acetate, pH 4.6, equilibration against reservoir solution containing 43% PEG 400, 20°C, X-ray diffraction structure determination and analysis at 2.8-3.0 A resolution, molecular replacement and structure modelling
-
complexed with GTP, hanging drop vapor diffusion method, using 150 mM sodium acetate pH 4.8, 270 mM ammonium sulfate, 18% (w/v) PEG MME 550. Complexed with UDP, hanging drop vapor diffusion method, using 0.15 M sodium acetate pH 4.8, 0.35 M ammonium sulfate, 28% (w/v)PEG MME 550
-
using sodium/potassium tartrate (1.2 M) at pH 7.4 and 10 mM GTP
resolved at 2.4 A resolution. Complexed with AMP-PNP, resolved at 3 A resolution. Complexed with AMP-PNP and UMP, resolved at 2.55 A resolution
ternary complex with UMP and the nonhydrolyzable ATP analogue alpha,beta-methylene-ATP (SsUMPK-UMP-AMPPCP), resolution 2.1 A, a complex with UMP (SsUMPK-UMP), resolution 2.2 A, a complex with UTP (SsUMPK-UTP), resolution 2.8 A, hanging drop vapor diffusion, protein solution (2UL) in 10 mM TRIS/Cl pH 7.6 with 4.6 mg/ml SSUMPK and 2 mM UMP and 5 mM MgCl2 mixed with 2 UL mother solution (0.65 M sodium acetate, 100 mM CdCl2, 0.1 M HEPES), pH 7.5; with ATP and UMP, with UMP, with UTP
purified recombinant enzyme, X-ray diffraction structure determination and analysis at 2.5 A resolution; UMPK with N-terminal His-tag, in complex with a phosphate ion, co-crystallized with UMP, and UTP, cross-talk region between two subunits of UpUMPK is identified, vapor diffusion, hanging drop, 0.2 m ammonium fluoride and 20% (w/v) poly(ethylene glycol) 3350, 15°C, enzyme concentration is 1.8 mg/ml, 5 mM GTP, resolution of 2.5 A, space group P 1 21 1, cell dimensions a =79.8, b = 96.6, c = 96.3 A, beta = 105.8
purified recombinant SeMet-substituted apo-enzyme XC1936, crystallization in a strong magnetic field, protein in 5 mM 2-mercaptoethanol, 100 mM N-(2-acetamido)-2-iminodiacetic acid, pH 6.8, and 0.02% NaN3, optimization of the reservoir solution, 25°C, X-ray diffraction structure determination and analysis at 2.35 A resolution
-
apo-form UMPK, 2.35 A resolution, crystallisation is significantly improved in a strong magnetic field, buffer-screening procedure, 20 mM N-(2-acetamido)-2-iminodiacetic acid (ADA) pH 6.8, 5 mM beta-mercaptoethanol and 0.02% NaN3, sitting-drop vapour diffusion in 96-well plates, 100 K, monoclinic space group P212121, unit-cell paramerters a = 111.45, b = 120.07, c = 125.79A
-
of the apo-form, crystallization improves by a strong magnetic field, optimum buffer for solubilization is 20 mM N-(2-acetamido)iminodiacetic acid, pH 6.8, 0.02% NaN3 and 5 mM 2-mercaptoethanol
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
-
-
663998
7.4
-
the UMP kinase activity of the HP0777 protein is lost within a week when preserved at pH 9.0, but can be maintained for at least 2 weeks in 50 mM Tris-HCl, pH 7.4
721458
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42
-
half-maximal inactivation
43
-
mutant T138A/N140A, temperature of half-inactivation
48
-
Tm for mutant D201N
51
-
mutant D201N is half-inactivated
52
-
Tm for mutant D77N
55
Tm of mutant R11H
56
-
mutant T138A, temperature of half-inactivation
57
Tm of mutant G232D
59 - 62
-
mutants R62H, D146N, D168N and D174N are half inactivated between 59 and 62°C
59
-
mutant N140A, temperature of half-inactivation
60 - 75
the enzyme shows Tm values of 60°C in the absence of nucleotides and 65°C and 75°C with 1 mM GTP and UTP, respectively
62.7
-
Tm in the presence of 0.1 mM GTP
63.7
-
Tm in the presence of 1 mM ATP
64
-
wild-type protein is half-inactivated
65.8
-
Tm in the presence of 1 mM GTP
68
-
wild-type, temperature of half-inactivation
69.8
-
Tm in the presence of 5 mM GTP
70
-
half-maximal inactivation in the presence of 1 mM UTP
73
-
Tm for mutant D159N
74.6
-
Tm in the presence of 0.1 mM UTP
75.6
-
Tm in the presence of 1 mM UMP
82.4
-
Tm in the presence of 1 mM UTP
86.5
-
Tm in the presence of 5 mM UTP
additional information
-
thermal stability of the N72A/D159N variant is close to that of the D159N variant, temperature of half-inactivation of the D93A/D159N variant is 10°C lower than that of the D159N variant, temperature of half-inactivation of the D93A/D159N variant is 15°C lower than that of the D159N variant,
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 12 months storage, less than 10% loss of activity
0°C, 6 h, less than 10% loss of activity
4°C, 50 mM Tris-HCl (pH 7.4) as insoluble proteins, after solubilization with 0.1 M borate (pH 9) or with 1 mM UTP in 50 mM Tris-HCl (pH 7.4), the enzyme is fully active
-
4°C, 50 mM Tris-HCl (pH 7.4), several months, no significant loss of activity. An exception is mutant D174N, which loses two-thirds of its activity after 3 months at 4°C
-
4°C, in 20 mM phosphate (pH 7.0) and 100 mM NaCl, about 2 months, no apparent loss of activity
room temperature, 0.1 M borate buffer (pH 8.5), 2 months, no loss of activity
-
room temperature, 50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 2 mM UTP, 2 weeks, no loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
-
ammonium sulfate precipitation, DEAE cellulose column chromatography, Sephadex G-200 gel filtration, and nickel metal chelate affinity chromatography
heating at 70°C, ammonium sulfate precipitation, and column chromatography on Dyematrex Gel Red A and the anion exchange materials DE52 and Q6. Minor amounts (around 2%) of a smaller 15 kDa protein, presumably an SsUMPK degradation product, co-purified with SsUMP.; of the recombinant protein
immobilized metal-affinity chromatography (IMAC) on a nickel column, SDS-PAGE, further purified by FPLC on an anion-exchange column, greater than 99% purity
-
metal affinity column using the gravity flow procedure, purity determined by SDS-PAGE
Ni-NTA affinity chromatography and gel filtration; recombinant His-tagged enzyme from Escherichia coli strain B834(DE3) by nickel affinity chromatography and gel filtration
Ni-NTA column chromatography
-
of the recombinant protein
-
of the recombinant protein by affinity chromatography
of the recombinant protein by affinity chromatography, no difference in activity compared to the wild type
of the recombinant proteins
-
recombinant N-terminally His6-tagged SeMet-substituted XC1936 apo-form from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis, the His-tag is cleaved off by tobacco etch virus protease
-
TALON resin column chromatography and Superdex 200 gel filtration
the recombinant enzyme from Escherichia coli
to homogeneity
-
wild-type and mutant D159N
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3)/pDIA17 cells
expressed in Escherichia coli DH5alpha cells
expressed in Escherichia coli strain SG13009
-
expression in Escherichia coli
expression in Escherichia coli BL21 (DE3)
expression in Escherichia coli BL21 (DE3)-pLysS, overexpression of the Hig6-tagged target protein is induced by the addition of 0.5 mM IPTG at 20°C for 21 h, SeMet-substituted XC1936 is expressed in a similar way
-
expression in Escherichia coli BL21(DE3)/pDIA17
-
expression in Escherichia coli strain BL21(DE3)/pDIA17
-
expression in Escherichia coli, site directed mutagenesis
expression in Escherichia coli; plasmid vector pUHE23-2, expression in Escherichia coli strain NF1830
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3); His-tagged version expressed in Escherichia coli BL21(DE3)
-
expression of the His-tagged enzyme in Escherichia coli strain B834(DE3); vector pDEST14 is transformed into Escherichia coli B834(DE3)
gene pyrH or Rv2883c, sequence comparisons and phylogenetic analysis and tree
gene pyrH, DNA and amino acid sequence determination, analysis, and comparisons; the mutants UpUMPKF133N and UpUMPK-F133A are constructed by site-directed mutagenesis using the plasmid pET-14b containing cDNA for UMPK, expression in Escherichia coli BL21 (DE3), overexpression by induction with isopropyl-beta-D-thiogalactoside
gene pyrH, recombinant expression in Escherichia coli strain BL21 (DE3) from vector pT73.3
-
His-tagged version expressed in Escherichia coli BL21(DE3)
-
overexpression in Escherichia coli wild type and SeMet-substituted protein
-
overexpression in Escherichia coli, site directed mutagenesis
-
overexpression of N-terminally His6-tagged SeMet-substituted XC1936 apo-form in Escherichia coli strain BL21(DE3)
-
overproduced in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N137A
-
Tm 10°C lower than wild type, loss of cooperativity with ATP, sensitive to activation by GTP
T135A
-
Tm 10°C lower than wild type, loss of cooperativity with ATP, increase in Km for UMP, sensitive to activation by GTP
T135A/N137A
-
Tm 10°C lower than wild type, at pH 7.4 in 50 mM Tris irreversible inactivated within hours
D115A
-
little detrimental effect, activated by 5'-guanylyl-imidodiphosphate within a concentration range that is roughly similar to that of wild type enzyme
D146N
-
84% of wild-type activity in the pellet of the sonicated bacterial extract
D93A
-
site-directed mutagenesis, inhibition by UTP appears significantly altered in the case of the D93A mutant as compared to the wild-type enzyme, the D93A substitution completely suppresses the related subunit-subunit hydrogen bonds between its main chain and Asp93, no inhibition by UTP in presence or absence of GTP. The Tm of the D93A variant is 10°C lower than that of the wild-type enzyme
D93A/D159N
-
D93 involved in hydrogen bond between the subunits of a dimer, mutation decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition
G232D
resistance to heat denaturation is altered, catalytic avtivity is reduced to 17% of the wild-type
H96A
-
involved in GTP activation, abolishing GTP activation
L226Q
-
more insoluble than wild-type, impairs the stability of the enzyme
N111A
-
little detrimental effect, activated by 5'-guanylyl-imidodiphosphate within a concentration range that is roughly similar to that of wild type enzyme
N140A/D159N
-
in N140A mutant protein is the cooperativity of inhibition caused by UTP suppressed
N72A
-
site-directed mutagenesis, no inhibition by UTP in presence or absence of GTP
N72A/D159N
-
N72 involved in hydrogen bond between the subunits
N72A/D93A
-
site-directed mutagenesis, the N72A mutation has less severe effects on enzyme activity regulation than the D93A substitution, reduced inhibition by UTP in absence of GTP, no inhibition in presence of GTP. The Tm of the mutant variant is 15°C lower than that of the wild-type enzyme
N72A/D93A/D159N
-
N72, D93 involved in hydrogen bonds between the subunits
P141L
-
affects enzyme activity and especially the allosteric regulation
P141Q
-
more soluble than wild-type
R103A
-
involved in GTP activation, abolishing GTP activation
R11H
lowered catalytic activity, 45% of the wild-type, resistance to heat denaturation is impaired
R127A
-
decreasing affinity for GTP
R130A
-
involved in GTP activation, abolishing GTP activation
R92A
-
involved in GTP activation, abolishing GTP activation
S124A
-
decreasing affinity for GTP
T138A
-
decreases half-denaturation temperature of UMP kinase by around 10°C, results in 4times higher Km for UMP, moderate loss of sensitivity to UTP inhibition, important loss in activation by GTP
T138A/N140A
-
decreases half-denaturation temperature of UMP kinase by around 25°C, increases the apparant Km for ATP and UMP by a factor of 2.6 and 12, respectively
W119A
-
involved in GTP activation, abolishing GTP activation
G232D
-
resistance to heat denaturation is altered, catalytic avtivity is reduced to 17% of the wild-type
-
R11H
-
lowered catalytic activity, 45% of the wild-type, resistance to heat denaturation is impaired
-
G232D
-
resistance to heat denaturation is altered, catalytic avtivity is reduced to 17% of the wild-type
-
R11H
-
lowered catalytic activity, 45% of the wild-type, resistance to heat denaturation is impaired
-
D113A
the mutant shows an increased Km value for UMP compared to the wild type enzyme
F81W
the mutant shows an increased Km value for UMP compared to the wild type enzyme
F81W/S96A
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
P139A
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
P139H
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
P139W
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
R150A
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
R82H
the mutant shows an increased Km value for UMP compared to the wild type enzyme and 0.7% of the wild-type specific activity
D113A
-
the mutant shows an increased Km value for UMP compared to the wild type enzyme
-
F81W
-
the mutant shows an increased Km value for UMP compared to the wild type enzyme
-
F81W/S96A
-
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
-
R150A
-
the mutant shows a decreased Km value for UMP compared to the wild type enzyme
-
R82H
-
the mutant shows an increased Km value for UMP compared to the wild type enzyme and 0.7% of the wild-type specific activity
-
A122T
activity decreased to 26% of the wild-type
D201G
activity decreased to 16% of the wild-type, significant loss of sensitivity to activation by GTP
D201N/E241D
activity decreased to 1% of the wild-type
F133A
site-directed mutagenesis, activity is only 20% of the wild-type, still no activation by GTP, with variable UMP concentration and fixed ATP concentration exhibits negative cooperativity with UMP, Hill coefficient 0.85; site-directed mutagenesis, the mutant enzyme is not activated by GTP and exhibits negative cooperativity with UMP
F133N
site-directed mutagenesis, activity is only 50% of the wild-type, still no activation by GTP, demonstrating that F133N is involved in subunit interactions but apparently not in GTP activation, with variable UMP concentration and fixed ATP concentration exhibits negative cooperativity with UMP, Hill coefficient 0.65, marked decrease in activity; site-directed mutagenesis, the mutant enzyme is not activated by GTP and exhibits negative cooperativity with UMP
D77N
-
no activity
R62H
-
no activity
R62H/D77N
-
strain CMM1474, reduced virulence
D77N
-
no activity
-
R62H
-
no activity
-
R62H/D77N
-
strain CMM1474, reduced virulence
-
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
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