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Literature summary for 2.7.4.22 extracted from

  • Meyer, P.; Evrin, C.; Briozzo, P.; Joly, N.; Barzu, O.; Gilles, A.M.
    Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP (2008), J. Biol. Chem., 283, 36011-36018.
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
GTP
-
Escherichia coli
GTP allosteric activator, the GTP-binding site, situated at 15 A from the UMP-binding site and at 24 A from the ATP-binding site, is delineated by two contiguous dimers. movements induced by binding GTP, overview. UTP and GTP are tightly coupled in both wild-type enzyme and N140A variant Escherichia coli

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli
His-tagged version expressed in Escherichia coli BL21(DE3) Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
D159N, hanging drop vapor diffusion method Escherichia coli
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 Escherichia coli

Protein Variants

Protein Variants Comment Organism
D159N site-directed mutagenesis Escherichia coli
D159N used as reference enzyme Escherichia coli
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 Escherichia coli
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 Escherichia coli
N140A site-directed mutagenesis, UTP and GTP are tightly coupled in both wild-type enzyme and N140A variant. The Tm of the mutant variant is 10°C lower than that of the wild-type enzyme Escherichia coli
N140A/D159N in N140A mutant protein is the cooperativity of inhibition caused by UTP suppressed Escherichia coli
N72A site-directed mutagenesis, no inhibition by UTP in presence or absence of GTP Escherichia coli
N72A/D159N N72 involved in hydrogen bond between the subunits Escherichia coli
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 Escherichia coli
N72A/D93A/D159N N72, D93 involved in hydrogen bonds between the subunits Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
UMP substrate inhibition Escherichia coli
UTP Mg-free competitive cooperative inhibitor, the inhibition is reversible by GTP. UTP and GTP are tightly coupled in both wild-type enzyme and N140A variant Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information reaction kinetics Escherichia coli
0.0238
-
UMP pH 7.4, 30°C, mutant D93A Escherichia coli
0.0238
-
UMP D93A/D159N mutant protein, 2 mM ATP Escherichia coli
0.024
-
UMP pH 7.4, 30°C, mutant N72A/D93A Escherichia coli
0.024
-
UMP N72A/D93A/D159N mutant protein, 2 mM ATP Escherichia coli
0.0244
-
UMP D93A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
0.0246
-
UMP N72A/D93A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
0.0275
-
UMP D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP Escherichia coli
0.0321
-
UMP pH 7.4, 30°C, mutant N72A Escherichia coli
0.0321
-
UMP N72A/D159N mutant protein, 2 mM ATP Escherichia coli
0.0357
-
UMP D93A/D159N mutant protein, 0.2 mM ATP Escherichia coli
0.046
-
UMP pH 7.4, 30°C, wild-type enzyme Escherichia coli
0.046
-
UMP D159N mutant protein, 2 mM ATP Escherichia coli
0.0478
-
UMP N72A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
0.05
-
UMP D159N mutant protein, 0.2 mM ATP Escherichia coli
0.05
-
UMP N140A/D159N mutant protein, 0.2 mM ATP Escherichia coli
0.051
-
UMP D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
0.0593
-
UMP D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP Escherichia coli
0.0739
-
UMP N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP Escherichia coli
0.0755
-
UMP D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
0.0788
-
UMP D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP Escherichia coli
0.1086
-
UMP N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
0.1812
-
UMP N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP Escherichia coli
0.19
-
ATP pH 7.4, 30°C, wild-type enzyme Escherichia coli
0.19
-
MgATP2- D159N mutant protein, 0.3 mM UMP Escherichia coli
0.2
-
MgATP2- D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
0.207
-
UMP D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
0.21
-
MgATP2- D159N mutant protein, 0.3 mM UMP Escherichia coli
0.21
-
MgATP2- N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP Escherichia coli
0.23
-
MgATP2- N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
0.24
-
MgATP2- D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP Escherichia coli
0.25
-
MgATP2- N140A/D159N mutant protein, 0.3 mM UMP Escherichia coli
0.26
-
MgATP2- D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
0.27
-
MgATP2- D93A/D159N mutant protein, 0.3 mM UMP Escherichia coli
0.27
-
MgATP2- D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP Escherichia coli
0.36
-
ATP pH 7.4, 30°C, mutant D93A Escherichia coli
0.36
-
MgATP2- D93A/D159N mutant protein, 0.3 mM UMP Escherichia coli
0.4
-
MgATP2- N72A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
0.41
-
MgATP2- D93A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
0.53
-
MgATP2- N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP Escherichia coli
0.59
-
ATP pH 7.4, 30°C, mutants N72A and N72A/D93A Escherichia coli
0.59
-
MgATP2- D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
0.59
-
MgATP2- N72A/D159N mutant protein, 0.3 mM UMP Escherichia coli
0.59
-
MgATP2- N72A/D93A/D159N mutant protein, 0.3 mM UMP Escherichia coli
0.64
-
MgATP2- D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP Escherichia coli
0.66
-
MgATP2- N72A/D93A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
0.77
-
MgATP2- D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP Escherichia coli
1.69
-
UMP D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + UMP Escherichia coli the enzyme regulatory mechanisms involve GTP and UTP, overview ADP + UDP
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli P0A7E9
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Escherichia coli
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Escherichia coli

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
23.8
-
D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP Escherichia coli
30.4
-
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP Escherichia coli
36.3
-
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
36.8
-
D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP Escherichia coli
38.1
-
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM UTP Escherichia coli
38.4
-
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP Escherichia coli
40.7
-
D93A/D159N mutant protein, 0.2 mM ATP Escherichia coli
46.1
-
D159N mutant protein, 0.2 mM ATP Escherichia coli
46.6
-
D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
48.9
-
N140A/D159N mutant protein, 0.2 mM ATP Escherichia coli
51.4
-
N72A/D93A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
51.9
-
D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP Escherichia coli
52.7
-
N72A/D93A/D159N mutant protein, 2 mM ATP Escherichia coli
55.7
-
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
57.6
-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
58.2
-
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP Escherichia coli
60.8
-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP Escherichia coli
61.5
-
D93A/D159N mutant protein, 0.3 mM UMP Escherichia coli
61.6
-
D159N mutant protein, 0.3 mM UMP Escherichia coli
61.6
-
N72A/D159N mutant protein, 0.3 mM UMP Escherichia coli
62.3
-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP Escherichia coli
62.4
-
D159N mutant protein, 0.3 mM UMP Escherichia coli
76.1
-
N72A/D93A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
77.1
-
D93A/D159N mutant protein, 2 mM ATP Escherichia coli
78.3
-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM UTP Escherichia coli
78.7
-
N72A/D93A/D159N mutant protein, 0.3 mM UMP Escherichia coli
81.9
-
N140A/D159N mutant protein, 0.3 mM UMP Escherichia coli
83.4
-
D93A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
84.2
-
D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
84.2
-
N72A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
90.7
-
D93A/D159N mutant protein, 0.3 mM UMP Escherichia coli
91.7
-
D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
92.5
-
D159N mutant protein, 2 mM ATP Escherichia coli
92.5
-
N72A/D159N mutant protein, 2 mM ATP Escherichia coli
93.9
-
D93A/D159N mutant protein, 1 mM UMP, 0.5 mM GTP Escherichia coli
98.2
-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP Escherichia coli
99.6
-
D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP Escherichia coli
100.3
-
D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
100.3
-
N72A/D159N mutant protein, 2 mM ATP, 0.5 mM GTP Escherichia coli
110.5
-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + UMP the enzyme regulatory mechanisms involve GTP and UTP, overview Escherichia coli ADP + UDP
-
?
ATP + UMP movements induced by binding uridine nucleotides, GTP and UTP interaction mechanism, analysis, overview Escherichia coli ADP + UDP
-
?
MgATP2- + UMP
-
Escherichia coli MgADP- + UDP
-
?
additional information Asp93 appears as one of the key amino acids in the transfer of information from the regulatory site to the active site Escherichia coli ?
-
?

Subunits

Subunits Comment Organism
hexamer quaternary structure, overview Escherichia coli
homohexamer crystal structure Escherichia coli

Synonyms

Synonyms Comment Organism
UMP kinase
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Escherichia coli

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
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, Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Escherichia coli

pH Range

pH Minimum pH Maximum Comment Organism
7.4 8.5
-
Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
Escherichia coli

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.44
-
UMP pH 7.4, 30°C, wild-type enzyme, in absence of GTP Escherichia coli
0.44
-
UMP D159N mutant protein, 2 mM ATP Escherichia coli
10.9
-
UMP pH 7.4, 30°C, mutant N72A/D93A, in absence of GTP Escherichia coli
10.9
-
UMP N72A/D93A/D159N mutant protein, 2 mM ATP Escherichia coli