Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21-Gold(DE3) cells | Saccharomyces cerevisiae |
Crystallization (Comment) | Organism |
---|---|
hanging drop vapor diffusion method, apo-enzyme using 0.1 M ammonium sulfate, 0.1 M Bis-Tris (pH 5.5), and 25% (w/v) PEG3350. In complex with dUMP using 0.2 M sodium acetate, 0.1 M Tris-HCl (pH 8.5), and 30% (w/v) PEG4000. In complex with alpha,beta-imido dUTP using 30% Jeffamine ED-2001 (pH 7.0), 0.1 M potassium-HEPES (pH 7.0) and 3 mM MgCl2 | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
D32A | the mutant exhibits negligible activity | Saccharomyces cerevisiae |
D85A | the mutant exhibits negligible activity | Saccharomyces cerevisiae |
D87A | the mutant exhibits negligible activity | Saccharomyces cerevisiae |
D87A/R137A/D85A | inactive | Saccharomyces cerevisiae |
F142A | the mutant exhibits very low activity | Saccharomyces cerevisiae |
Q114A | the mutant shows essentially the wild type affinity for dUTP and greatly reduced activity | Saccharomyces cerevisiae |
R111A | the mutant has reduced activity and lower substrate affinity (increased Km) compared to the wild type enzyme | Saccharomyces cerevisiae |
R137A | the mutant exhibits negligible activity | Saccharomyces cerevisiae |
R68A | the mutant exhibits very low activity | Saccharomyces cerevisiae |
S69A | the mutant exhibits negligible activity | Saccharomyces cerevisiae |
Y88A | the mutant protein is equally active against both dUTP and UTP and has reduced activity and lower substrate affinity (increased Km) compared to the wild type enzyme | Saccharomyces cerevisiae |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
EDTA | less than 10% residual activity at 1 mM | Saccharomyces cerevisiae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0132 | - |
dUTP | wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
0.0142 | - |
dUTP | mutant enzyme Q114A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
0.0315 | - |
dUTP | mutant enzyme R111A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
0.035 | - |
UTP | mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
0.041 | - |
dUTP | mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
0.0447 | - |
dITP | wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Co2+ | strictly dependent on a bivalent metal cation like Co2+ | Saccharomyces cerevisiae | |
Mg2+ | strictly dependent on a bivalent metal cation like Mg2+ which supports the highest rate of dUTP hydrolysis | Saccharomyces cerevisiae | |
Mn2+ | strictly dependent on a bivalent metal cation like Mn2+ | Saccharomyces cerevisiae | |
Ni2+ | strictly dependent on a bivalent metal cation like Ni2+ | Saccharomyces cerevisiae | |
Zn2+ | strictly dependent on a bivalent metal cation like Zn2+ | Saccharomyces cerevisiae |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
18100 | - |
3 * 18100, His tagged enzyme, calculated from amino acid sequence | Saccharomyces cerevisiae |
56600 | - |
gel filtration | Saccharomyces cerevisiae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | P33317 | - |
- |
Purification (Comment) | Organism |
---|---|
nickel affinity resin column chromatography and Superdex-200 gel filtration | Saccharomyces cerevisiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
dITP + H2O | shows also significant activity against dITP | Saccharomyces cerevisiae | dIMP + diphosphate | - |
? | |
dUTP + H2O | - |
Saccharomyces cerevisiae | dUMP + diphosphate | - |
? | |
additional information | alpha,beta-imido dUTP is a non-hydrolysable substrate | Saccharomyces cerevisiae | ? | - |
? | |
UTP + H2O | substrate for mutant enzyme Y88A | Saccharomyces cerevisiae | UMP + diphosphate | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotrimer | 3 * 18100, His tagged enzyme, calculated from amino acid sequence | Saccharomyces cerevisiae |
Synonyms | Comment | Organism |
---|---|---|
DUT1 | - |
Saccharomyces cerevisiae |
dUTP pyrophosphatase | - |
Saccharomyces cerevisiae |
dUTPase | - |
Saccharomyces cerevisiae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.3 | - |
dUTP | mutant enzyme Q114A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
1.3 | - |
dITP | wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
3.3 | - |
dUTP | mutant enzyme R111A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
5.3 | - |
UTP | mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
7.4 | - |
dUTP | mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
9.6 | - |
dUTP | wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
7 | 9 | - |
Saccharomyces cerevisiae |
General Information | Comment | Organism |
---|---|---|
physiological function | the enzyme plays a key role in preventing uracil incorporation into DNA | Saccharomyces cerevisiae |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
29 | - |
dITP | wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
90 | - |
dUTP | mutant enzyme Q114A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
100 | - |
dUTP | mutant enzyme R111A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
150 | - |
UTP | mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
180 | - |
dUTP | mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae | |
740 | - |
dUTP | wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C | Saccharomyces cerevisiae |