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 (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 (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 | 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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0A7E9 | - |
- |
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 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 | 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 | Comment | Organism |
---|---|---|
hexamer | quaternary structure, overview | Escherichia coli |
homohexamer | crystal structure | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
UMP kinase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Escherichia coli |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Escherichia coli |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
7.4 | 8.5 | - |
Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |
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 |