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Literature summary for 3.4.21.B48 extracted from

  • Okubo, A.; Li, M.; Ashida, M.; Oyama, H.; Gustchina, A.; Oda, K.; Dunn, B.M.; Wlodawer, A.; Nakayama, T.
    Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site (2006), FEBS J., 273, 2563-2576.
    View publication on PubMed
Search for more literature for 3.4.21.B48

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutant enzymes as soluble proteins in Escherichia coli Alicyclobacillus sendaiensis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant wild-type enzyme and mutants D164N and E78H/D164N, crystallization buffer contains 0.2 M ammonium sulfate and 30% PEG 8000, pH 4.6, X-ray diffraction structure determination and analysis at 2.0-2.3 A resolution Alicyclobacillus sendaiensis

Protein Variants

Protein Variants Comment Organism
D164N site-directed mutagenesis, replacement of the catalytic residue of kumamolisin-As with that of subtilisin, disrupted interaction of Ser278 with residue 164, the mutant shows 1.3% of the wild-type turnover Alicyclobacillus sendaiensis
E78A/D164N site-directed mutagenesis, completely inactive mutant which stays unprocessed Alicyclobacillus sendaiensis
E78H/D164N site-directed mutagenesis, replacement of the catalytic residues of kumamolisin-As with those of subtilisin, disruption of the catalytic triad, the mutant shows 0.0001% of the wild-type turnover Alicyclobacillus sendaiensis
E78Q/D164N site-directed mutagenesis, completely inactive mutant which stays unprocessed Alicyclobacillus sendaiensis

Inhibitors

Inhibitors Comment Organism Structure
N-acetyl-Ile-Pro-Phe
-
 Alicyclobacillus sendaiensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics, wild-type and mutant enzymes, and pH-dependency Alicyclobacillus sendaiensis
0.0007
-
 IQF peptide pH 4.0, 40°C, recombinant mutant E78H Alicyclobacillus sendaiensis
0.0008
-
 IQF peptide pH 4.0, 40°C, recombinant mutant D164N Alicyclobacillus sendaiensis
0.001
-
 IQF peptide pH 4.0, 40°C, recombinant wild-type enzyme Alicyclobacillus sendaiensis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
19000
-
 x * 38000, active recombinant enzyme, SDS-PAGE, x * 19000, recombinant isolated propeptide, SDS-PAGE, x * 57000, inactive recombinant precursor enzyme, SDS-PAGE Alicyclobacillus sendaiensis
38000
-
 x * 38000, active recombinant enzyme, SDS-PAGE, x * 19000, recombinant isolated propeptide, SDS-PAGE, x * 57000, inactive recombinant precursor enzyme, SDS-PAGE Alicyclobacillus sendaiensis
57000
-
 x * 38000, active recombinant enzyme, SDS-PAGE, x * 19000, recombinant isolated propeptide, SDS-PAGE, x * 57000, inactive recombinant precursor enzyme, SDS-PAGE Alicyclobacillus sendaiensis

Organism

Organism UniProt Comment Textmining
Alicyclobacillus sendaiensis
-
 gene scpA
-
Alicyclobacillus sendaiensis NTAP-1
-
 gene scpA
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification the enzyme needs to be cleaved in to the mature enzyme and the N-terminal prepropeptide, autocatalytic cleavage Alicyclobacillus sendaiensis

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli by heat treatment and anion exchange chromatography at pH 7.0, to homogeneity, separation of the propeptide from the mature enzyme by hydroxyapatite chromatography Alicyclobacillus sendaiensis

Reaction

Reaction Comment Organism Reaction ID
specifically hydrolyzes Leu15-Tyr16 peptide bond in oxidized insulin B chain, additional cleavage at Phe25-Tyr26 at a considerably lower rate. Good cleavage of the Phe-I-(p-nitrophenylalanine) bond in synthetic peptides. The enzyme preferentially hydrolyzes peptides having an Ala or Pro residue at P2 position and prefers such charged amino acid residues as Glu or Arg at the P2' position. No cleavage: Asp-Pro-Ala-Lys-Phe-(p-nitrophenylalanine)-Arg-Leu mechanism, active site structure, the catalytic triad is formed by Glu78, Asp82, and Ser278, Ser278 forms direct hydrogen bonds with the side chain of Asn164 Alicyclobacillus sendaiensis
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Benzyloxycarbonyl-L-Ala-L-Ala-L-Leu 4-nitroanilide + H2O
-
 Alicyclobacillus sendaiensis ?
-
 ?
Benzyloxycarbonyl-L-Ala-L-Ala-L-Leu 4-nitroanilide + H2O
-
 Alicyclobacillus sendaiensis NTAP-1 ?
-
 ?
IQF peptide + H2O IQF fluorogenic peptide substrates Alicyclobacillus sendaiensis ?
-
 ?
IQF peptide + H2O IQF fluorogenic peptide substrates Alicyclobacillus sendaiensis NTAP-1 ?
-
 ?

Subunits

Subunits Comment Organism
? x * 38000, active recombinant enzyme, SDS-PAGE, x * 19000, recombinant isolated propeptide, SDS-PAGE, x * 57000, inactive recombinant precursor enzyme, SDS-PAGE Alicyclobacillus sendaiensis
More the enzyme consists of an N-terminal prepropeptide and the mature enzyme part Alicyclobacillus sendaiensis

Synonyms

Synonyms Comment Organism
kumamolisin-AS
-
 Alicyclobacillus sendaiensis
SCPA
-
 Alicyclobacillus sendaiensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
55
-
 assay at Alicyclobacillus sendaiensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
 additional information
-
 Alicyclobacillus sendaiensis
0.00045
-
 IQF peptide pH 4.0, 40°C, recombinant mutant E78H/D164N Alicyclobacillus sendaiensis
0.033
-
 IQF peptide pH 4.0, 40°C, recombinant mutant E78H Alicyclobacillus sendaiensis
5.3
-
 IQF peptide pH 4.0, 40°C, recombinant mutant D164N Alicyclobacillus sendaiensis
395
-
 IQF peptide pH 4.0, 40°C, recombinant wild-type enzyme Alicyclobacillus sendaiensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4
-
 assay at Alicyclobacillus sendaiensis