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Literature summary for 3.6.1.25 extracted from

  • Delvaux, D.; Murty, M.R.; Gabelica, V.; Lakaye, B.; Lunin, V.V.; Skarina, T.; Onopriyenko, O.; Kohn, G.; Wins, P.; De Pauw, E.; Bettendorff, L.
    A specific inorganic triphosphatase from Nitrosomonas europaea: structure and catalytic mechanism (2011), J. Biol. Chem., 286, 34023-34035.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Nitrosomonas europaea

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method, selenomethionine-substituted protein, using 3.5 M sodium formate as a precipitation agent and 0.1 M Bis-Tris propane buffer, pH 7.0, at 20°C Nitrosomonas europaea

Protein Variants

Protein Variants Comment Organism
K52R the His-tagged mutant loses 90-99% of its activity (catalytic efficiency is at least 1000times lower) compared to the wild type enzyme. Mn2+ does not induce a significant activation of this mutant Nitrosomonas europaea
K85A the His-tagged mutant is about 10times less active than the wild type enzyme, but the optimal conditions for activity are essentially the same. The mutant is more strongly activated by Mn2+ than by Mg2+, and the inhibitory effects of Ca2+ and Zn2+ are less pronounced Nitrosomonas europaea
K8A the His-tagged mutant remains highly active with tripolyphosphate as substrate and Mg2+ as activator, with a catalytic efficiency close to that of the recombinant wild type enzyme Nitrosomonas europaea

Inhibitors

Inhibitors Comment Organism Structure
Cd2+
-
Nitrosomonas europaea
Cu2+
-
Nitrosomonas europaea
Zn2+ complete inhibition at 0.005 mM Nitrosomonas europaea

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.021
-
tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.04
-
tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.058
-
tripolyphosphate His-tagged wild type enzyme, at pH 7.1 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.1
-
tripolyphosphate untagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.191
-
tripolyphosphate His-tagged mutant enzyme K52R, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.39
-
tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.72
-
tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.8
-
ATP His-tagged wild type enzyme, at pH 8.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
1.2
-
ATP His-tagged mutant enzyme K8A, at pH 10.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
2.6
-
tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
74
-
tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ Co2+ is a better activator than Mn2+, although the maximum activity is less than 10% that measured in the presence of 5 mM Mg2+ Nitrosomonas europaea
Mg2+ the enzyme hydrolyzes tripolyphosphate with high catalytic efficiency in the presence of 5 mM Mg2+ Nitrosomonas europaea
Mn2+ very poor substituent for Mg2+ in the pH range 7.0-10, but there is a significant Mn2+-dependent activity at pH 10.0-10.5. Half-maximum activation is obtained at 0.4 mM Nitrosomonas europaea

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
19000
-
2 * 19000, SDS-PAGE Nitrosomonas europaea

Organism

Organism UniProt Comment Textmining
Nitrosomonas europaea
-
-
-

Purification (Commentary)

Purification (Comment) Organism
His-Trap column chromatography, gel filtration Nitrosomonas europaea

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + H2O low affinity Nitrosomonas europaea ?
-
?
additional information the enzyme has low affinity for CTP, ATP or thiamine triphosphate. Nucleoside triphosphatase activity is negligible in the presence of 5 mM Mg2+, but a small activity is observed in the presence of 1 mM Mn2+, in particular with GTP. Guanosine 5’-tetraphosphate and long chain polyphosphate (containing about 65 phosphate residues) are not hydrolyzed. The enzyme has no adenylyl cyclase activity Nitrosomonas europaea ?
-
?
tripolyphosphate + H2O very good substrate with Mg2+ as activator. The enzyme has a strong preference for linear tripolyphosphate compared with cyclic trimetaphosphate and to the linear tetraphosphate Nitrosomonas europaea diphosphate + phosphate
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 19000, SDS-PAGE Nitrosomonas europaea

Synonyms

Synonyms Comment Organism
inorganic triphosphatase
-
Nitrosomonas europaea
triphosphate tunnel metalloenzyme
-
Nitrosomonas europaea
TTM
-
Nitrosomonas europaea

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50 55
-
Nitrosomonas europaea

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.28
-
tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
0.36
-
ATP His-tagged wild type enzyme, at pH 8.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
0.98
-
tripolyphosphate His-tagged mutant enzyme K52R, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.96
-
ATP His-tagged mutant enzyme K8A, at pH 10.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
10
-
tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
21
-
tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
60
-
tripolyphosphate His-tagged wild type enzyme, at pH 7.1 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
76
-
tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
288
-
tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
887
-
tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
7900
-
tripolyphosphate untagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9.7
-
around pH 9.7 Nitrosomonas europaea

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.0015
-
His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea Zn2+
0.01
-
His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea Cd2+
0.02
-
His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea Cu2+

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.45
-
ATP His-tagged wild type enzyme, at pH 8.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.3
-
ATP His-tagged mutant enzyme K8A, at pH 10.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
4
-
tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
5.1
-
tripolyphosphate His-tagged mutant enzyme K52R, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
29
-
tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
190
-
tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
2300
-
tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3300
-
tripolyphosphate His-tagged wild type enzyme, at pH 7.1 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3600
-
tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
7200
-
tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
79000
-
tripolyphosphate untagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea