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Literature summary for 3.4.21.4 extracted from

  • Uesugi, Y.; Usuki, H.; Arima, J.; Iwabuchi, M.; Hatanaka, T.
    Molecular dissection of Streptomyces trypsin on substrate recognition (2011), Biochim. Biophys. Acta, 1814, 1295-1304.
    View publication on PubMed
Search for more literature for 3.4.21.4

Cloned(Commentary)

Cloned (Comment) Organism
recombinant enzyme expression from expression vector pTONA5a, which includes a promoter from Streptomyces metalloendopeptidase, in Streptomyces lividans strain 1326 Streptomyces griseus
recombinant enzyme expression from expression vector pTONA5a, which includes a promoter from Streptomyces metalloendopeptidase, in Streptomyces lividans strain 1326 Streptomyces omiyaensis

Protein Variants

Protein Variants Comment Organism
L71R/S172A site-directed mutagenesis, inactive active site mutant Streptomyces griseus
L71Y site-directed mutagenesis, the mutant shows a change in topological specificity and high hydrolytic activity toward type IV collagen. The active site mutant shows slightly lower activities toward type I collagen than the wild-type Streptomyces griseus
L71Y/Q72R site-directed mutagenesis, the mutant shows a change in topological specificity and high hydrolytic activity toward type IV collagen. The active site mutant shows slightly lower activities toward type I collagen than the wild-type Streptomyces griseus
Q72R site-directed mutagenesis, the mutant shows a change in topological specificity and high hydrolytic activity toward type IV collagen. The mutant shows slightly lower activities toward type I collagen than the wild-type Streptomyces griseus
S172A site-directed mutagenesis, inactive active site mutant Streptomyces griseus
S172A site-directed mutagenesis, inactive active site mutant Streptomyces omiyaensis
Y71L site-directed mutagenesis, the mutant displays high activity toward type I collagen similar to the wild-type enzyme Streptomyces omiyaensis
Y71L/S172A site-directed mutagenesis, inactive active site mutant Streptomyces omiyaensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0061
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide wild-type enzyme, pH 8.0, 37°C Streptomyces omiyaensis
0.012
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant L71Y/Q72R, pH 8.0, 37°C Streptomyces griseus
0.0172
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant L71Y, pH 8.0, 37°C Streptomyces griseus
0.0175
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant Y71L, pH 8.0, 37°C Streptomyces omiyaensis
0.0177
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide wild-type enzyme, pH 8.0, 37°C Streptomyces griseus

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ required Streptomyces griseus
Ca2+ required Streptomyces omiyaensis

Organism

Organism UniProt Comment Textmining
Streptomyces griseus
-
-
-
Streptomyces omiyaensis
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme from Streptomyces lividans strain 1326 culture supernatant by cation exchange chromatography and dialysis Streptomyces griseus
recombinant enzyme from Streptomyces lividans strain 1326 culture supernatant by cation exchange chromatography and diialysis Streptomyces omiyaensis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
66.1
-
 wild-type enzyme, pH 8.0, 37°C, substrate benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide Streptomyces griseus
90.3
-
 mutant L71Y, pH 8.0, 37°C, substrate benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide Streptomyces griseus
110.2
-
 mutant L71Y/Q72R, pH 8.0, 37°C, substrate benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide Streptomyces griseus
190.2
-
 mutant Y71L, pH 8.0, 37°C, substrate benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide Streptomyces omiyaensis
501.5
-
 wild-type enzyme, pH 8.0, 37°C, substrate benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide Streptomyces omiyaensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide + H2O
-
 Streptomyces griseus benzyloxycarbonylglycyl-L-prolyl-L-arginine + 7-amino-4-methylcoumarin
-
 ?
benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide + H2O
-
 Streptomyces omiyaensis benzyloxycarbonylglycyl-L-prolyl-L-arginine + 7-amino-4-methylcoumarin
-
 ?
bovine skin DQ-collagen type I + H2O
-
 Streptomyces griseus ?
-
 ?
bovine skin DQ-collagen type I + H2O
-
 Streptomyces omiyaensis ?
-
 ?
human placenta DQ-collagen type IV + H2O
-
 Streptomyces griseus ?
-
 ?
human placenta DQ-collagen type IV + H2O
-
 Streptomyces omiyaensis ?
-
 ?
additional information three-dimensional structure and topological specificities, substrate recognition in which residue 71 plays a crucial role, overview. Identification of amino acid residue(s) related to collagen binding of wild-type and mutant enzymes Streptomyces omiyaensis ?
-
 ?
additional information three-dimensional structure and topological specificities, substrate recognition, overview. The neighboring residues Leu71 and Gln72 in the N-terminal beta-barrel domain of the enzyme synergistically play an important role in substrate recognition. Identification of amino acid residue(s) related to collagen binding for wild-type and mutant enzymes Streptomyces griseus ?
-
 ?

Subunits

Subunits Comment Organism
More three-dimensional structure and topological specificities, substrate recognition, overview Streptomyces griseus
More three-dimensional structure and topological specificities, substrate recognition, overview Streptomyces omiyaensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Streptomyces griseus
37
-
 assay at Streptomyces omiyaensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
33
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide wild-type enzyme, pH 8.0, 37°C Streptomyces griseus
51.3
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant L71Y, pH 8.0, 37°C Streptomyces griseus
57.3
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant L71Y/Q72R, pH 8.0, 37°C Streptomyces griseus
93
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant Y71L, pH 8.0, 37°C Streptomyces omiyaensis
279.5
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide wild-type enzyme, pH 8.0, 37°C Streptomyces omiyaensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Streptomyces griseus
8
-
 assay at Streptomyces omiyaensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0019
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide wild-type enzyme, pH 8.0, 37°C Streptomyces griseus
0.0032
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant L71Y, pH 8.0, 37°C Streptomyces griseus
0.0048
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant L71Y/Q72R, pH 8.0, 37°C Streptomyces griseus
0.0054
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide mutant Y71L, pH 8.0, 37°C Streptomyces omiyaensis
0.0419
-
 benzyloxycarbonylglycyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide wild-type enzyme, pH 8.0, 37°C Streptomyces omiyaensis