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Literature summary for 3.5.1.14 extracted from

  • Ishikawa, K.; Ishida, H.; Matsui, I.; Kawarabayasi, Y.; Kikuchi, H.
    Novel bifunctional hyperthermostable carboxypeptidase/aminoacylase from Pyrococcus horikoshii OT3 (2001), Appl. Environ. Microbiol., 67, 673-679.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.5.1.14

Application

Application Comment Organism
synthesis because of its thermostability, the enzyme is expected to be useful for the production of L-amino acid derivatives from racemates at temperatures over 90°C Pyrococcus horikoshii

Cloned(Commentary)

Cloned (Comment) Organism
-
 Pyrococcus horikoshii

Protein Variants

Protein Variants Comment Organism
C102A no hydrolytic activity Pyrococcus horikoshii
C102S Km-values are similar to those of wild-type enzyme Pyrococcus horikoshii
E367Q the kcat-value increases slightly, whereas the Km value does not change for N-acetyl-L-phenylalanine as substrates. Substrate inhibition of aminoacylase activity is also observed. The temperature-dependent activity and pH profile of the activity of E367Q are similar to those of wild-type enzyme Pyrococcus horikoshii

Inhibitors

Inhibitors Comment Organism Structure
benzyloxycarbonyl-Gly-Gly-Phe
-
 Pyrococcus horikoshii
L-benzylsuccinate
-
 Pyrococcus horikoshii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.18
-
 N-acetyl-L-methionine pH 7.5, 85°C, wild-type enzyme Pyrococcus horikoshii
5.2
-
 N-acetyl-L-phenylalanine pH 7.5, 85°C, wild-type enzyme Pyrococcus horikoshii
6.2
-
 N-acetyl-L-phenylalanine pH 7.5, 85°C, mutant enzyme C102S Pyrococcus horikoshii
6.64
-
 N-acetyl-L-phenylalanine pH 7.5, 85°C, mutant enzyme E367Q Pyrococcus horikoshii

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ the enzyme contains 1.22 mol of Zn2+ and 0.17 mol of Ca2+ per mol of monomer enzyme protein Pyrococcus horikoshii
Co2+ when the enzyme is dialyzed against 50 mM EDTA (pH 7.5) for 10 h at 4°C and then against 50 mM sodium phosphate buffer (pH 7.5), the aminoacylase activity is decreased to less than 1.0%. The activity is restored by incubation in 50 mM sodium phosphate buffer (pH 7.5) containing ZnCl2, MnCl2, or CoCl2 at 4°C for 1 h. The restorative effect is dependent on the concentration of the metal ions. 88%, 23%, and 20% of activities are restored by adding 1.0 mM ZnCl2, 1.0 mM MnCl2, and 0.1 mM CoCl2, respectively Pyrococcus horikoshii
Mn2+ when the enzyme is dialyzed against 50 mM EDTA (pH 7.5) for 10 h at 4°C and then against 50 mM sodium phosphate buffer (pH 7.5), the aminoacylase activity is decreased to less than 1.0%. The activity is restored by incubation in 50 mM sodium phosphate buffer (pH 7.5) containing ZnCl2, MnCl2, or CoCl2 at 4°C for 1 h. The restorative effect is dependent on the concentration of the metal ions. 88%, 23%, and 20% of activities are restored by adding 1.0 mM ZnCl2, 1.0 mM MnCl2, and 0.1 mM CoCl2, respectively Pyrococcus horikoshii
Zn2+ the enzyme contains 1.22 mol of Zn2+ and 0.17 mol of Ca2+ per mol of monomer enzyme protein. When the enzyme is dialyzed against 50 mM EDTA (pH 7.5) for 10 h at 4°C and then against 50 mM sodium phosphate buffer (pH 7.5), the aminoacylase activity is decreased to less than 1.0%. The activity is restored by incubation in 50 mM sodium phosphate buffer (pH 7.5) containing ZnCl2, MnCl2, or CoCl2 at 4°C for 1 h. The restorative effect is dependent on the concentration of the metal ions. 88%, 23%, and 20% of activities are restored by adding 1.0 mM ZnCl2, 1.0 mM MnCl2, and 0.1 mM CoCl2, respectively Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
43000
-
 x * 43000, SDS-PAGE Pyrococcus horikoshii

Organism

Organism UniProt Comment Textmining
Pyrococcus horikoshii O58754
-
-
Pyrococcus horikoshii OT-3 O58754
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information N-acetyl-D-amino acids are not hydrolyzed Pyrococcus horikoshii ?
-
 ?
additional information N-acetyl-D-amino acids are not hydrolyzed Pyrococcus horikoshii OT-3 ?
-
 ?
N-acetyl-L-alanine + H2O
-
 Pyrococcus horikoshii acetate + L-alanine
-
 ?
N-acetyl-L-methionine + H2O
-
 Pyrococcus horikoshii acetate + L-methionine
-
 ?
N-acetyl-L-methionine + H2O
-
 Pyrococcus horikoshii OT-3 acetate + L-methionine
-
 ?
N-acetyl-L-phenylalanine + H2O
-
 Pyrococcus horikoshii acetate + L-phenylalanine
-
 ?
N-acetyl-L-phenylalanine + H2O
-
 Pyrococcus horikoshii OT-3 acetate + L-phenylalanine
-
 ?
N-acetyl-L-tryptophan + H2O weak activity Pyrococcus horikoshii acetate + L-tryptophan
-
 ?
N-acetyl-L-tryptophan + H2O weak activity Pyrococcus horikoshii OT-3 acetate + L-tryptophan
-
 ?
N-acetyl-L-tyrosine + H2O
-
 Pyrococcus horikoshii acetate + L-tyrosine
-
 ?
N-acetylglycine + H2O weak activity Pyrococcus horikoshii acetate + glycine
-
 ?
N-acetylglycine + H2O weak activity Pyrococcus horikoshii OT-3 acetate + glycine
-
 ?

Subunits

Subunits Comment Organism
? x * 43000, SDS-PAGE Pyrococcus horikoshii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0208
-
 N-acetyl-L-phenylalanine pH 7.5, 85°C, mutant enzyme C102S Pyrococcus horikoshii
34
-
 N-acetyl-L-phenylalanine pH 7.5, 85°C, wild-type enzyme Pyrococcus horikoshii
37.3
-
 N-acetyl-L-phenylalanine pH 7.5, 85°C, mutant enzyme E367Q Pyrococcus horikoshii
45.6
-
 N-acetyl-L-methionine pH 7.5, 85°C, wild-type enzyme Pyrococcus horikoshii

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.001
-
 L-benzylsuccinate pH 7.5, 85°C Pyrococcus horikoshii
2.93
-
 benzyloxycarbonyl-Gly-Gly-Phe pH 7.5, 85°C Pyrococcus horikoshii