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Literature summary for 3.6.1.11 extracted from

  • Beassoni, P.R.; Gallarato, L.A.; Boetsch, C.; Garrido, M.N.; Lisa, A.T.
    Pseudomonas aeruginosa exopolyphosphatase is also a polyphosphate ADP phosphotransferase (2015), Enzyme Res., 2015, 404607 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.6.1.11

Activating Compound

Activating Compound Comment Organism Structure
Dimethylamine 9% activation compared to NH4+ Pseudomonas aeruginosa
methylamine 28% activation compared to NH4+ Pseudomonas aeruginosa
trimethylamine 5% activation compared to NH4+ Pseudomonas aeruginosa

Cloned(Commentary)

Cloned (Comment) Organism
gene ppx, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strains XL10-Gold and BL21-CodonPlus Pseudomonas aeruginosa

Protein Variants

Protein Variants Comment Organism
additional information construction of a truncated enzyme mutants comprising amino acid residues 1-314 of 506 (N-paPpx(1-314)), or residues 1-303 (N-paPpx(1-303)), or residues 315-506 (C-paPpx(315-506)) of paPpx, amplified from Pseudomonas aeruginosa wild-type strain PAO1 chromosomal DNA through PCR. Only paPpx(1-506) and N-paPpx(1-314) are enzymatically active, while C-paPpx(315-506) lacks enzymatic activity Pseudomonas aeruginosa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetic analysis of recombinant wild-type and truncated mutant enzymes, exopolyphosphatase activity and polyphosphate:ADP phosphotransferase activity (EC 2.7.4.), overview Pseudomonas aeruginosa
0.0013
-
 polyphosphate75 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
0.00329
-
 polyphosphate65 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
0.00714
-
 polyphosphate45 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
0.01103
-
 polyphosphate25 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
0.02083
-
 polyphosphate25 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
0.0236
-
 polyphosphate45 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
0.02517
-
 polyphosphate65 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
0.03067
-
 polyphosphate75 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa

Metals/Ions

Metals/Ions Comment Organism Structure
Cs+ activates wild-type, full-length enzyme paPpx(1-506) Pseudomonas aeruginosa
K+ a nonessential activator of paPpx, presence of K+ does not affect the affinity of the enzyme for Mg2+, activates wild-type, full-length enzyme paPpx(1-506). The activity curve obtained with K+ is sigmoid and reaches its maximum activity at concentrations of 80 mM. Km(app)K+ is 42 mM Pseudomonas aeruginosa
Mg2+ required, values of 0.5(app)Mg2+ in paPpx(1-506) and NpaPpx(1-314) are 0.30 mM and 0.28 mM, respectively. The interaction between paPpx(1-506) and Mg2+ occurs in the N-terminal domain Pseudomonas aeruginosa
additional information behavior of the full-length paPpx(1-506) and N-paPpx(1-314) against different concentration of divalent ions such as Mg2+, Zn2+, Ca2+, and Mn2+ as effectors, in presence of a saturating concentration (0.008 mM) for the substrate polyphosphate65. The activation of both enzyme variants by Mg2+ is similar and shows no inhibition at high concentrations of this ion. The activation by Ca2+ and Mn2+ is negligible. Li+ and Na+ have no effects on enzyme activity, while NH4+, K+, Rb+, and Cs+ are activators of paPpx(1-506). Tetramethylammonium is not an activator of paPpx(1-506) Pseudomonas aeruginosa
NH4+ activates wild-type, full-length enzyme paPpx(1-506). The activity curve obtained with NH4+ is sigmoid and reaches its maximum activity at concentrations of 30 mM. Km(app)NH4+ is 10 mM Pseudomonas aeruginosa
Rb+ activates wild-type, full-length enzyme paPpx(1-506) Pseudomonas aeruginosa
Zn2+ Zn2+ is able to activate the enzyme only 20% compared to Mg2+ Pseudomonas aeruginosa

Organism

Organism UniProt Comment Textmining
Pseudomonas aeruginosa
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-CodonPlus by nickel affinity chromatography, dialysis, tag cleavage thriugh thrombin, and again dialysis Pseudomonas aeruginosa

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information Pseudomonas aeruginosa exopolyphosphatase catalyzes the hydrolysis of polyphosphates (polyP), producing polyphosphate_n-1 plus inorganic phosphate, but the exopolyphosphatase is also a polyphosphate:ADP phosphotransferase. 0.1 ml of enzyme and polyphosphate substrate in 50 mM Tris-HCl, pH 8.0, 80 mM KCl, and 5 mM MgCl2 are mixed with 0.4 ml of a solution with 2.5% (NH4)6Mo7O24 x (H2O)4 in 3 NH2SO4 and 0.4 ml of 2% ascorbic acid/2% hydrazine in 0.1 NH2SO4, and the solution is brought to a final volume of 1.2 ml with triple glass-distilled water. Quantification of free phosphate is performed after 30 min of incubation at 37°C through measurement of the absorbance at 820 nm Pseudomonas aeruginosa ?
-
-
polyphosphate25 + H2O
-
 Pseudomonas aeruginosa polyphosphate24 + phosphate
-
 ?
polyphosphate45 + H2O
-
 Pseudomonas aeruginosa polyphosphate44 + phosphate
-
 ?
polyphosphate65 + H2O
-
 Pseudomonas aeruginosa polyphosphate64 + phosphate
-
 ?
polyphosphate75 + H2O
-
 Pseudomonas aeruginosa polyphosphate74 + phosphate
-
 ?

Subunits

Subunits Comment Organism
? x * 56419, sequence calculation, wild-type, full-length enzyme Pseudomonas aeruginosa

Synonyms

Synonyms Comment Organism
paPpx
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Pseudomonas aeruginosa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.29
-
 polyphosphate45 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
6.69
-
 polyphosphate75 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
6.86
-
 polyphosphate65 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
7.26
-
 polyphosphate25 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
40.2
-
 polyphosphate25 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
41.23
-
 polyphosphate45 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
53.03
-
 polyphosphate65 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
57.02
-
 polyphosphate75 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Pseudomonas aeruginosa

General Information

General Information Comment Organism
additional information a homology model of paPpx in a closed conformation is constructed by comparative modeling, molecular dynamic simulations, overview. Docking study with bound metals and/or ADP defining the N-paPpx(1-314) model in open conformation as receptor. Enzyme electrostatic potential calculations Pseudomonas aeruginosa

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
218.13
-
 polyphosphate75 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
224.15
-
 polyphosphate45 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
272.55
-
 polyphosphate65 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
348.54
-
 polyphosphate25 pH 8.0, 37°C, truncated enzyme Pseudomonas aeruginosa
3644.6
-
 polyphosphate25 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
5774.5
-
 polyphosphate45 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
16118.5
-
 polyphosphate65 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa
43861.5
-
 polyphosphate75 pH 8.0, 37°C, full-length enzyme Pseudomonas aeruginosa