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Literature summary for 3.5.1.87 extracted from

  • Pozo-Dengra, J.; Martinez-Gomez, A.I.; Martinez-Rodriguez, S.; Clemente-Jimenez, J.M.; Rodriguez-Vico, F.; Las Heras-Vazquez, F.J.
    Evaluation of substrate promiscuity of an L-carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43 (2010), Biotechnol. Prog., 26, 954-959.
    View publication on PubMed
Search for more literature for 3.5.1.87

Activating Compound

Activating Compound Comment Organism Structure
Co2+ 663.89% relative activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme, 536.91% relative activity of HQSA pretreated enzyme, consequently, the enzyme is incubated with Co2+ before activity assays Geobacillus stearothermophilus
Mn2+ 286.93% relative activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme, 197% relative activity of HQSA pretreated enzyme Geobacillus stearothermophilus
Ni2+ 341.94% relative activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme, 333.96% relative activity of HQSA pretreated enzyme Geobacillus stearothermophilus

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21 Geobacillus stearothermophilus

Inhibitors

Inhibitors Comment Organism Structure
2-mercaptoethanol 10 mM causes 29% inhibition, suggesting that no cysteine residue is crucial for enzyme activity, although they might have a structural role Geobacillus stearothermophilus
5,5'-dithiobis(2-nitrobenzoic acid) 2 mM, slight inhibition Geobacillus stearothermophilus
8-Hydroxyquinoline-5-sulfonic acid 10 mM, decreases the activity to 13% Geobacillus stearothermophilus
Cs+ 2 mM, slight inhibition Geobacillus stearothermophilus
dithiothreitol 10 mM causes 41% inhibition, suggesting that no cysteine residue is crucial for enzyme activity, although they might have a structural role Geobacillus stearothermophilus
EDTA 10 mM, decreases the activity to 15% Geobacillus stearothermophilus
Fe2+ 2 mM, strong inhibition Geobacillus stearothermophilus
Fe3+ 2 mM, strong inhibition Geobacillus stearothermophilus
Hg2+ 2 mM, strong inhibition Geobacillus stearothermophilus
Mg2+ slightly inhibited activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme and HQSA pretreated enzyme Geobacillus stearothermophilus
Na+ 2 mM, slight inhibition Geobacillus stearothermophilus
Pb2+ 2 mM, slight inhibition Geobacillus stearothermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.9
-
 N-carbamoyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
3.29
-
 N-carbamoyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
4.19
-
 N-carbamoyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
5
-
 N-formyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
5.82
-
 N-carbamoyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
6.75
-
 N-formyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
9.77
-
 N-formyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
12.66
-
 N-acetyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
12.9
-
 N-acetyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
12.99
-
 N-carbamoyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
13.82
-
 N-formyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
13.92
-
 N-acetyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
23.78
-
 N-Formyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
36.12
-
 N-acetyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
116
-
 N-acetyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ similar enzyme activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme and HQSA pretreated enzyme compared to activity without addition of any effectors Geobacillus stearothermophilus
Cu2+ slightly activated activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme and HQSA pretreated enzyme compared to activity without addition of any effectors Geobacillus stearothermophilus
additional information the enzymatic function is not affected by the sulfhydryl reagent iodoacetamide and is only slightly affected by 5,5'-dithiobis(2-nitrobenzoic acid) Geobacillus stearothermophilus
Zn2+ slightly activated activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme, similar activity of 8-hydroxyquinoline-5-sulfonic acid pretreated enzyme compared to activity of non 8-hydroxyquinoline-5-sulfonic acid treated enzyme without addition of any effectors Geobacillus stearothermophilus

Organism

Organism UniProt Comment Textmining
Geobacillus stearothermophilus
-
 CECT43, a thermophilic bacterium
-

Purification (Commentary)

Purification (Comment) Organism
-
 Geobacillus stearothermophilus

Reaction

Reaction Comment Organism Reaction ID
an N-carbamoyl-L-2-amino acid (a 2-ureido carboxylate) + H2O = an L-2-amino acid + NH3 + CO2 recombinant BsLcar is able to hydrolyze N-acetyl and N-formyl-amino acids in addition to N-carbamoyl-amino acids, showing strict L-enantioselectivity for all the three. Additionally, the enzyme shows the fastest amidohydrolase activity on the N-formyl-amino acid followed by N-carbamoyl and N-acetyl ones Geobacillus stearothermophilus
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 overview of effect of metal ions and other chemical agents on the enzyme activity of 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme and the HQSA pretreated enzyme Geobacillus stearothermophilus
33.34
-
 activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme, pH 7.5, 65°C Geobacillus stearothermophilus
40.2
-
 120% relative activity of 8-hydroxyquinoline-5-sulfonic acid (HQSA) pretreated enzyme compared to activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme, pH 7.5, 65°C Geobacillus stearothermophilus
65.69
-
 Mn2+, 197% relative activity of 8-hydroxyquinoline-5-sulfonic acid (HQSA) pretreated enzyme compared to activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme without addition of any effectors, pH 7.5, 65°C Geobacillus stearothermophilus
95.67
-
 Mn2+, 286.93% relative activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme compared to activity without addition of any effectors, pH 7.5, 65°C Geobacillus stearothermophilus
111.3
-
 Ni2+, 333.96% relative activity of 8-hydroxyquinoline-5-sulfonic acid (HQSA) pretreated enzyme compared to activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme without addition of any effectors, pH 7.5, 65°C Geobacillus stearothermophilus
114
-
 Ni2+, 341.94% relative activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme compared to activity without addition of any effectors, pH 7.5, 65°C Geobacillus stearothermophilus
179
-
 Co2+, 536.91% relative activity of 8-hydroxyquinoline-5-sulfonic acid (HQSA) pretreated enzyme pretreated enzyme compared to activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme without addition of any effectors, pH 7.5, 65°C Geobacillus stearothermophilus
221.4
-
 Co2+, 663.89% relative activity of non 8-hydroxyquinoline-5-sulfonic acid (HQSA) treated enzyme compared to activity without addition of any effectors, pH 7.5, 65°C Geobacillus stearothermophilus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-acetyl-L-alanine + H2O
-
 Geobacillus stearothermophilus L-alanine + acetate
-
 ?
N-acetyl-L-methionine + H2O
-
 Geobacillus stearothermophilus L-methionine + acetate
-
 ?
N-acetyl-L-phenylalanine + H2O
-
 Geobacillus stearothermophilus L-phenylalanine + acetate
-
 ?
N-acetyl-L-tyrosine + H2O
-
 Geobacillus stearothermophilus L-tyrosine + acetate
-
 ?
N-acetyl-L-valine + H2O
-
 Geobacillus stearothermophilus L-valine + acetate
-
 ?
N-carbamoyl-L-alanine + H2O
-
 Geobacillus stearothermophilus L-alanine + CO2 + NH3
-
 ?
N-carbamoyl-L-methionine + H2O
-
 Geobacillus stearothermophilus L-methionine + CO2 + NH3
-
 ?
N-carbamoyl-L-phenylalanine + H2O
-
 Geobacillus stearothermophilus L-phenylalanine + CO2 + NH3
-
 ?
N-carbamoyl-L-tyrosine + H2O
-
 Geobacillus stearothermophilus L-tyrosine + CO2 + NH3
-
 ?
N-carbamoyl-L-valine + H2O
-
 Geobacillus stearothermophilus L-valine + CO2 + NH3
-
 ?
N-formyl-L-alanine + H2O
-
 Geobacillus stearothermophilus L-alanine + CO2
-
 ?
N-formyl-L-methionine + H2O
-
 Geobacillus stearothermophilus L-methionine + HCOOH
-
 ?
N-formyl-L-phenylalanine + H2O
-
 Geobacillus stearothermophilus L-phenylalanine + CO2
-
 ?
N-formyl-L-tyrosine + H2O
-
 Geobacillus stearothermophilus L-tyrosine + CO2
-
 ?
N-formyl-L-valine + H2O
-
 Geobacillus stearothermophilus L-valine + CO2
-
 ?

Synonyms

Synonyms Comment Organism
BsLcar
-
 Geobacillus stearothermophilus
N-carbamoyl-amino-acid amidohydrolase
-
 Geobacillus stearothermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
65
-
 maximal activity for hydrolysis of N-acetyl and N-formyl-amino acids and N-carbamoyl-amino acids Geobacillus stearothermophilus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
65 70 thermal stability studies performed on BsLcar show that maximum activity gradually decreases at temperatures over 65°C for 60 min, with 60% activity remaining after incubation at 70°C for 60 min Geobacillus stearothermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.01
-
 N-acetyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
0.08
-
 N-acetyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
0.08
-
 N-acetyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
0.16
-
 N-carbamoyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
0.18
-
 N-carbamoyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
0.84
-
 N-carbamoyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
1.46
-
 N-Formyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
2.26
-
 N-acetyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
5.32
-
 N-carbamoyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
12.12
-
 N-formyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
27.26
-
 N-formyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
27.86
-
 N-acetyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
48.96
-
 N-carbamoyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
293.5
-
 N-formyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
839.1
-
 N-formyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 maximal activity for hydrolysis of N-acetyl and N-formyl-amino acids and N-carbamoyl-amino acids, in sodium phosphate, 100 mM Geobacillus stearothermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.39
-
 N-acetyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
0.69
-
 N-acetyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
5.96
-
 N-acetyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
13.86
-
 N-carbamoyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
27.49
-
 N-carbamoyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
61.4
-
 N-Formyl-L-tyrosine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
178.5
-
 N-acetyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
255.3
-
 N-carbamoyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
771.3
-
 N-acetyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
877
-
 N-formyl-L-phenylalanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
1834
-
 N-carbamoyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
4039
-
 N-formyl-L-valine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
11680
-
 N-carbamoyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
58700
-
 N-formyl-L-alanine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus
85880
-
 N-formyl-L-methionine pH 7.5, 65°C, preincubation with CoCl2 Geobacillus stearothermophilus