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Literature summary for 3.6.1.23 extracted from

  • Tchigvintsev, A.; Singer, A.U.; Flick, R.; Petit, P.; Brown, G.; Evdokimova, E.; Savchenko, A.; Yakunin, A.F.
    Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme (2011), Biochem. J., 437, 243-253.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21-Gold(DE3) cells Saccharomyces cerevisiae

Crystallization (Commentary)

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

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

Inhibitors Comment Organism Structure
EDTA less than 10% residual activity at 1 mM Saccharomyces cerevisiae

KM Value [mM]

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

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 [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

Organism UniProt Comment Textmining
Saccharomyces cerevisiae P33317
-
-

Purification (Commentary)

Purification (Comment) Organism
nickel affinity resin column chromatography and Superdex-200 gel filtration Saccharomyces cerevisiae

Substrates and Products (Substrate)

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

Subunits Comment Organism
homotrimer 3 * 18100, His tagged enzyme, calculated from amino acid sequence Saccharomyces cerevisiae

Synonyms

Synonyms Comment Organism
DUT1
-
Saccharomyces cerevisiae
dUTP pyrophosphatase
-
Saccharomyces cerevisiae
dUTPase
-
Saccharomyces cerevisiae

Turnover Number [1/s]

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 Range

pH Minimum pH Maximum Comment Organism
7 9
-
Saccharomyces cerevisiae

General Information

General Information Comment Organism
physiological function the enzyme plays a key role in preventing uracil incorporation into DNA Saccharomyces cerevisiae

kcat/KM [mM/s]

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