Information on EC 3.4.22.B78 - endopepidase PH1704

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The expected taxonomic range for this enzyme is: Pyrococcus horikoshii

EC NUMBER
COMMENTARY hide
3.4.22.B78
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
endopepidase PH1704
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
the enzyme prefers substrates with an Arg residue at the P1 site
show the reaction diagram
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Ala-Ala-Leu-Arg-7-amido-4-methylcoumarin + H2O
Ala-Ala-Leu-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
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?
Ala-Ala-Phe-7-amido-4-methylcoumarin + H2O
Ala-Ala-Phe + 7-amino-4-methylcoumarin
show the reaction diagram
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-
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?
Ala-Ala-Phe-Arg-7-amido-4-methylcoumarin + H2O
Ala-Ala-Phe-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
Ala-Arg-7-amido-4-methylcoumarin + H2O
Ala-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
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?
Ala-Phe-7-amido-4-methylcoumarin + H2O
Ala-Phe + 7-amino-4-methylcoumarin
show the reaction diagram
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?
Gelatin + H2O
?
show the reaction diagram
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?
Leu-Arg-7-amido-4-methylcoumarin + H2O
Leu-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
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?
additional information
?
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the enzyme has both aminopeptidase and endopeptidase activities, substrate specificity, overview. Ala-Phe-7-amido-4-methylcoumarin, and Ala-Ala-Phe-7-amido-4-methylcoumarin are poor substrates. Phe-7-amido-4-methylcoumarin, Ala-7-amido-4-methylcoumarin, Val-7-amido-4-methylcoumarin, Asp-7-amido-4-methylcoumarin, and Ser-7-amido-4-methylcoumarin are no substrates
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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5 mM, 29% inhibition of protease activity
Co2+
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0.2 mM, 75% inhibition of protease activity
Cu2+
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0.2 mM, 57% inhibition of protease activity
Fe3+
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0.2 mM, 27% inhibition of protease activity
K+
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5 mM, 19% inhibition of protease activity
Mg2+
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5 mM, 10% inhibition of protease activity
Mn2+
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5 mM, 81% inhibition of protease activity
Na+
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5 mM, 28% inhibition of protease activity
Ni2+
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0.2 mM, 84% inhibition of protease activity
Zn2+
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0.2 mM, 63% inhibition of protease activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
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inhibits 29% at 5 mM, 19% at 1 mM
Co2+
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inhibits 74% at 0.2 mM and precipitates the enzyme at 1 mM
Cu2+
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inhibits 57% at 0.2 mM, 81% at 1 mM, and precipitates the enzyme at 5 mM
Fe3+
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inhibits 28% at 0.2 mM, 68% at 1 mM, and precipitates the enzyme at 5 mM
iodoacetamide
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strong inhibition; strongly inhibits the peptidase activity, confirming that Cys100 is a nucleophilic residue
K+
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inhibits 19% at 5 mM
Mg2+
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inhibits 10% at 5 mM
Mn2+
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inhibits 81% at 5 mM, 42% at 1 mM
Na+
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inhibits 28% at 5 mM
Ni2+
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inhibits 85% at 0.2 mM and precipitates the enzyme at 1 mM
Zn2+
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inhibits 64% at 0.2 mM, 77% at 1 mM, and precipitates the enzyme at 5 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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steady-state kinetics, recombinant wild-tyype and mutant enzymes, overview
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
19.87
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purified recombinant wild-type enzyme, pH 7.5, 85C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9
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pH 6.5: about 45% of maximal activity, pH 9.0: about 55% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 95
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50C: about 60% of maximal activity, 95C: about 60% of maximal activity
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
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12 * 20000, about, recombinant enzyme, SDS-PAGE
240000
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about, recombinant enzyme, native PAGE; non-denaturing PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by 8fold heat treatment at 80C for 15 min, and gel filtration. Affinity chromatography is not suitable for this enzyme because recombinant PH1704 can be precipitated in the present of nickel
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, phylogenetic tree, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3); expression in Escherichia coli
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expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E12T
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the mutant enzyme is more stable than the wild-type enzyme
Y120P
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site-directed mutagenesis, the mutant shows highly increased activity compared to the wild-type enzyme; with Ala-Ala-Phe-Arg-7-amido-4-methylcoumarin as substrate, kcat and kcat/Km is 10- and 21fold higher than that of the wild-type enzyme
E12T
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the mutant enzyme is more stable than the wild-type enzyme
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Y120P
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site-directed mutagenesis, the mutant shows highly increased activity compared to the wild-type enzyme; with Ala-Ala-Phe-Arg-7-amido-4-methylcoumarin as substrate, kcat and kcat/Km is 10- and 21fold higher than that of the wild-type enzyme
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