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Literature summary for 3.1.8.1 extracted from

  • Carletti, E.; Jacquamet, L.; Loiodice, M.; Rochu, D.; Masson, P.; Nachon, F.
    Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase (2009), J. Enzyme Inhib. Med. Chem., 24, 1045-1055.
    View publication on PubMed
Search for more literature for 3.1.8.1

Activating Compound

Activating Compound Comment Organism Structure
beta-Lactoglobulin maximum activity in the presence of 0.1% (w/v) beta-lactoglobulin. There are neither additives nor synergistic effect of ZnCl2 and beta-lactoglobulin Brevundimonas diminuta
paraoxon stabilizes Brevundimonas diminuta

Application

Application Comment Organism
biotechnology future biotechnological developments of PTE as a detoxifying enzyme Brevundimonas diminuta

Cloned(Commentary)

Cloned (Comment) Organism
PTEm gene without the 29-amino acid leader cloned into pET17b vector, expressed in Escherichia coli HMS 174 (DE3) pLysS. PCR fragments of mutants cloned into the expression plasmid pET17b and expressed in Escherichia coli BL21 (DE3) pLysS Brevundimonas diminuta

Protein Variants

Protein Variants Comment Organism
H123A KM remains unaffected, whereas kcat decreases 2.5fold. Alters the bacterial expression yield of Co2+-PTE and Ki for Zn(OH)2 inhibition Brevundimonas diminuta
H123I KM remains unaffected, whereas kcat decreases 10fold. Alters the bacterial expression yield of Co2+-PTE and Ki for Zn(OH)2 inhibition Brevundimonas diminuta
H123N KM remains unaffected, whereas kcat decreases 80fold. Alters the bacterial expression yield of Co2+-PTE and Ki for Zn(OH)2 inhibition Brevundimonas diminuta

Inhibitors

Inhibitors Comment Organism Structure
additional information without additives, both Zn2+- and Co-PTE dramatically loose about 90% of their activity upon dilution. Activity of both Zn2+- and Co-PTE remains unchanged upon addition of CoCl2, CdCl2, FeSO4 and MgCl2 up to 2 mM Brevundimonas diminuta
NiCl2 is a non-competitive inhibitor of Co-PTE Brevundimonas diminuta
ZnCl2 induces a non-competitive partial inhibition of Zn2+-PTE and Co2+-PTE at pH 8.5. Inhibition of hydrolysis of O,O-(3-chloro-4-methyl-2-oxo-2H-chromen-7-yl)ethylmethylphosphonate is one order of magnitude higher than for paraoxon. Inhibition results from interactions with colloidal Zn(OH)2 formed in alkaline buffer that alters the catalytic machinery. ZnCl2 has an inhibitory effect when a protein environment stabilizes PTE. Adding ZnCl2 to the enzyme stabilized by beta-lactoglobulin results in 10% and 20% decrease in activity for Co-PTE and Zn-PTE, respectively Brevundimonas diminuta

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.26
-
 paraoxon Zn2+-PTE, at pH 8.5 and 30°C Brevundimonas diminuta
0.34
-
 paraoxon Co2+-PTE mutant H123N, at pH 8.5 and 30°C Brevundimonas diminuta
0.36
-
 paraoxon Co2+-PTE mutant H123I, at pH 8.5 and 30°C Brevundimonas diminuta
0.39
-
 paraoxon Co2+-PTE mutant H123A, at pH 8.5 and 30°C Brevundimonas diminuta
0.4
-
 paraoxon Co2+-PTE, at pH 8.5 and 30°C Brevundimonas diminuta

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ Co-PTE has Zn2+ and Co2+ in equimolar amount, the later being located most likely in the labile beta-site. A single Co2+ in the active site already provides a significant enhancement of the catalytic efficiency compared to Zn-PTE Brevundimonas diminuta
Fe2+ trace Brevundimonas diminuta
Zn2+ recombinant Zn-PTE contains only Zn2+ whereas Co-PTE has Zn2+ and Co2+ in equimolar amount, the later being located most likely in the labile beta-site. 0.05 mM ZnCl2 has a modest stabilizing effect, as Zn-PTE and Co-PTE retain respectively 38% and 27% of their activity 2 min after dilution. There are neither additives nor synergistic effect of ZnCl2 and beta-lactoglobulin Brevundimonas diminuta

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
36000
-
 gel filtration Brevundimonas diminuta
36290
-
 sequence analysis Brevundimonas diminuta
36290
-
 mass spectrometry under acidic conditions Brevundimonas diminuta

Organism

Organism UniProt Comment Textmining
Brevundimonas diminuta P0A434
-
-

Purification (Commentary)

Purification (Comment) Organism
by ion exchange chromatography, affinity chromatography and ultrafiltration Brevundimonas diminuta

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-chloro-4-methyl-2-oxo-2H-chromen-7-yl ethyl methylphosphonate + H2O
-
 Brevundimonas diminuta 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one + ethyl methylphosphonate
-
 ?
paraoxon + H2O
-
 Brevundimonas diminuta 4-nitrophenol + di-ethyl phosphate
-
 ?

Subunits

Subunits Comment Organism
monomer Zn- and Co-PTE, gel filtration Brevundimonas diminuta

Synonyms

Synonyms Comment Organism
phosphotriesterase
-
 Brevundimonas diminuta
PTE
-
 Brevundimonas diminuta

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
50
-
 paraoxon Co2+-PTE mutant H123I, at pH 8.5 and 30°C Brevundimonas diminuta
390
-
 paraoxon Co2+-PTE mutant H123A, at pH 8.5 and 30°C Brevundimonas diminuta
1290
-
 paraoxon Zn2+-PTE, at pH 8.5 and 30°C Brevundimonas diminuta
1610
-
 paraoxon Co2+-PTE mutant H123N, at pH 8.5 and 30°C Brevundimonas diminuta
4010
-
 paraoxon Co2+-PTE, at pH 8.5 and 30°C Brevundimonas diminuta

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.052
-
 ZnCl2 Co2+-PTE, at pH 8.5 and 30°C, with paraoxon as substrate Brevundimonas diminuta
0.155
-
 ZnCl2 Zn2+-PTE, at pH 8.5 and 30°C, with paraoxon as substrate Brevundimonas diminuta
0.23
-
 ZnCl2 Co2+-PTE mutant H123N, at pH 8.5 and 30°C, with paraoxon as substrate Brevundimonas diminuta
0.3
-
 ZnCl2 Co2+-PTE mutant H123A, at pH 8.5 and 30°C, with paraoxon as substrate Brevundimonas diminuta
0.63
-
 ZnCl2 Co2+-PTE mutant H123I, at pH 8.5 and 30°C, with paraoxon as substrate Brevundimonas diminuta
3
-
 NiCl2 Co2+-PTE, at pH 8.5 and 30°C, with paraoxon as substrate Brevundimonas diminuta

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.53
-
 Co2+-PTE, at pH 8.5 and 30°C, with O,O-(3-chloro-4-methyl-2-oxo-2H-chromen-7-yl)ethylmethylphosphonate as substrate Brevundimonas diminuta ZnCl2

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
140
-
 paraoxon Co2+-PTE mutant H123I, at pH 8.5 and 30°C Brevundimonas diminuta
1000
-
 paraoxon Co2+-PTE mutant H123A, at pH 8.5 and 30°C Brevundimonas diminuta
4700
-
 paraoxon Co2+-PTE mutant H123N, at pH 8.5 and 30°C Brevundimonas diminuta
5000
-
 paraoxon Zn2+-PTE, at pH 8.5 and 30°C Brevundimonas diminuta
10000
-
 paraoxon Co2+-PTE, at pH 8.5 and 30°C Brevundimonas diminuta