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Literature summary 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)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.6.1.25 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

EC Number Protein Variants Comment Organism
3.6.1.25 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
3.6.1.25 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
3.6.1.25 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

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.6.1.25 0.021
-
 tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.04
-
 tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.058
-
 tripolyphosphate His-tagged wild type enzyme, at pH 7.1 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.1
-
 tripolyphosphate untagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.191
-
 tripolyphosphate His-tagged mutant enzyme K52R, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.39
-
 tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.72
-
 tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.8
-
 ATP His-tagged wild type enzyme, at pH 8.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.6.1.25 1.2
-
 ATP His-tagged mutant enzyme K8A, at pH 10.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.6.1.25 2.6
-
 tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 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

EC Number Metals/Ions Comment Organism Structure
3.6.1.25 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
3.6.1.25 Mg2+ the enzyme hydrolyzes tripolyphosphate with high catalytic efficiency in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 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]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.6.1.25 19000
-
 2 * 19000, SDS-PAGE Nitrosomonas europaea

Organism

EC Number Organism UniProt Comment Textmining
3.6.1.25 Nitrosomonas europaea
-
-
-

Purification (Commentary)

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.6.1.25 ATP + H2O low affinity Nitrosomonas europaea ?
-
 ?
3.6.1.25 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 ?
-
 ?
3.6.1.25 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

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

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.6.1.25 50 55
-
 Nitrosomonas europaea

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.6.1.25 0.28
-
 tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 0.36
-
 ATP His-tagged wild type enzyme, at pH 8.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.6.1.25 0.98
-
 tripolyphosphate His-tagged mutant enzyme K52R, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 3.96
-
 ATP His-tagged mutant enzyme K8A, at pH 10.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.6.1.25 10
-
 tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 21
-
 tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 60
-
 tripolyphosphate His-tagged wild type enzyme, at pH 7.1 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 76
-
 tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 288
-
 tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 887
-
 tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 7900
-
 tripolyphosphate untagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.6.1.25 9.7
-
 around pH 9.7 Nitrosomonas europaea

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
3.6.1.25 0.0015
-
 His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea Zn2+
3.6.1.25 0.01
-
 His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea Cd2+
3.6.1.25 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.6.1.25 0.45
-
 ATP His-tagged wild type enzyme, at pH 8.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.6.1.25 3.3
-
 ATP His-tagged mutant enzyme K8A, at pH 10.1 and 50°C, in the presence of 10 mM Mn2+ Nitrosomonas europaea
3.6.1.25 4
-
 tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 5.1
-
 tripolyphosphate His-tagged mutant enzyme K52R, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 29
-
 tripolyphosphate His-tagged mutant enzyme K85A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 190
-
 tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 2300
-
 tripolyphosphate His-tagged mutant enzyme K8A, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 3300
-
 tripolyphosphate His-tagged wild type enzyme, at pH 7.1 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 3600
-
 tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 37°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 7200
-
 tripolyphosphate His-tagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea
3.6.1.25 79000
-
 tripolyphosphate untagged wild type enzyme, at pH 9.7 and 50°C, in the presence of 5 mM Mg2+ Nitrosomonas europaea