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Literature summary for 3.4.22.B78 extracted from

  • Zhan, D.; Bai, A.; Yu, L.; Han, W.; Feng, Y.
    Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120 (2014), PLoS One, 9, e103902.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, phylogenetic tree, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pyrococcus horikoshii
expression in Escherichia coli Pyrococcus horikoshii

Protein Variants

Protein Variants Comment Organism
Y120P 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 Pyrococcus horikoshii
Y120P site-directed mutagenesis, the mutant shows highly increased activity compared to the wild-type enzyme Pyrococcus horikoshii

Inhibitors

Inhibitors Comment Organism Structure
Ca2+ inhibits 29% at 5 mM, 19% at 1 mM Pyrococcus horikoshii
Co2+ inhibits 74% at 0.2 mM and precipitates the enzyme at 1 mM Pyrococcus horikoshii
Cu2+ inhibits 57% at 0.2 mM, 81% at 1 mM, and precipitates the enzyme at 5 mM Pyrococcus horikoshii
Fe3+ inhibits 28% at 0.2 mM, 68% at 1 mM, and precipitates the enzyme at 5 mM Pyrococcus horikoshii
iodoacetamide strong inhibition; strongly inhibits the peptidase activity, confirming that Cys100 is a nucleophilic residue Pyrococcus horikoshii
K+ inhibits 19% at 5 mM Pyrococcus horikoshii
Mg2+ inhibits 10% at 5 mM Pyrococcus horikoshii
Mn2+ inhibits 81% at 5 mM, 42% at 1 mM Pyrococcus horikoshii
Na+ inhibits 28% at 5 mM Pyrococcus horikoshii
Ni2+ inhibits 85% at 0.2 mM and precipitates the enzyme at 1 mM Pyrococcus horikoshii
Zn2+ inhibits 64% at 0.2 mM, 77% at 1 mM, and precipitates the enzyme at 5 mM Pyrococcus horikoshii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetics, recombinant wild-tyype and mutant enzymes, overview Pyrococcus horikoshii

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ 5 mM, 29% inhibition of protease activity Pyrococcus horikoshii
Co2+ 0.2 mM, 75% inhibition of protease activity Pyrococcus horikoshii
Cu2+ 0.2 mM, 57% inhibition of protease activity Pyrococcus horikoshii
Fe3+ 0.2 mM, 27% inhibition of protease activity Pyrococcus horikoshii
K+ 5 mM, 19% inhibition of protease activity Pyrococcus horikoshii
Mg2+ 5 mM, 10% inhibition of protease activity Pyrococcus horikoshii
Mn2+ 5 mM, 81% inhibition of protease activity Pyrococcus horikoshii
Na+ 5 mM, 28% inhibition of protease activity Pyrococcus horikoshii
Ni2+ 0.2 mM, 84% inhibition of protease activity Pyrococcus horikoshii
Zn2+ 0.2 mM, 63% inhibition of protease activity Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
20000
-
12 * 20000, about, recombinant enzyme, SDS-PAGE Pyrococcus horikoshii
240000
-
non-denaturing PAGE Pyrococcus horikoshii
240000
-
about, recombinant enzyme, native PAGE Pyrococcus horikoshii

Organism

Organism UniProt Comment Textmining
Pyrococcus horikoshii O59413
-
-
Pyrococcus horikoshii DSM 12428 O59413
-
-
Pyrococcus horikoshii OT-3 O59413
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Pyrococcus horikoshii
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by 8fold heat treatment at 80°C 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 Pyrococcus horikoshii

Reaction

Reaction Comment Organism Reaction ID
the enzyme prefers substrates with an Arg residue at the P1 site it also acts as arginyl aminopeptidase with higher efficiency Pyrococcus horikoshii

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
19.87
-
purified recombinant wild-type enzyme, pH 7.5, 85°C Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Ala-Ala-Leu-Arg-7-amido-4-methylcoumarin + H2O
-
Pyrococcus horikoshii Ala-Ala-Leu-Arg + 7-amino-4-methylcoumarin
-
?
Ala-Ala-Phe-7-amido-4-methylcoumarin + H2O
-
Pyrococcus horikoshii Ala-Ala-Phe + 7-amino-4-methylcoumarin
-
?
Ala-Ala-Phe-Arg-7-amido-4-methylcoumarin + H2O low activity Pyrococcus horikoshii Ala-Ala-Phe-Arg + 7-amino-4-methylcoumarin
-
?
Ala-Ala-Phe-Arg-7-amido-4-methylcoumarin + H2O the enzyme is also identified as an aminopeptidase. Activity with the endopeptidase substrate Ala-Ala-Phe-Arg-7-amido-4-methylcoumarin is 10% compared to the activity with the aminopeptidase substrate L-Arg-7-amido-4-methylcoumarin Pyrococcus horikoshii Ala-Ala-Phe-Arg + 7-amino-4-methylcoumarin
-
?
Ala-Arg-7-amido-4-methylcoumarin + H2O
-
Pyrococcus horikoshii Ala-Arg + 7-amino-4-methylcoumarin
-
?
Ala-Phe-7-amido-4-methylcoumarin + H2O
-
Pyrococcus horikoshii Ala-Phe + 7-amino-4-methylcoumarin
-
?
Gelatin + H2O
-
Pyrococcus horikoshii ?
-
?
Leu-Arg-7-amido-4-methylcoumarin + H2O
-
Pyrococcus horikoshii Leu-Arg + 7-amino-4-methylcoumarin
-
?
additional information 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 Pyrococcus horikoshii ?
-
?

Subunits

Subunits Comment Organism
dodecamer
-
Pyrococcus horikoshii
dodecamer 12 * 20000, about, recombinant enzyme, SDS-PAGE Pyrococcus horikoshii

Synonyms

Synonyms Comment Organism
PH1704
-
Pyrococcus horikoshii
PH1704 protease
-
Pyrococcus horikoshii
PH1704 protease ambiguous, the recombinant protein is also identified as an aminopeptidase Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
80
-
-
Pyrococcus horikoshii
85
-
assay at Pyrococcus horikoshii

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
50 95 50°C: about 60% of maximal activity, 95°C: about 60% of maximal activity Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Pyrococcus horikoshii
8.5
-
-
Pyrococcus horikoshii

pH Range

pH Minimum pH Maximum Comment Organism
6.5 9 pH 6.5: about 45% of maximal activity, pH 9.0: about 55% of maximal activity Pyrococcus horikoshii

General Information

General Information Comment Organism
evolution the enzyme is a member of DJ-1/ThiJ/PfpI superfamily that has diverse functional subclasses Pyrococcus horikoshii
additional information the enzyme has both aminopeptidase and endopeptidase activities, Cys100 is the catalytic nucleophilic residue, residue Tyr120 is important in substrate binding and is involved in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43, active site pocket structure, molecular docking study, overview. Cys100, His101, and Glu474 function as a catalytic triad. Active residues are Glu12, Glu15, Lys43, Gly70, Arg71, Cys100, His101, Tyr120, Val150, Arg471, Glu474, and Arg475 Pyrococcus horikoshii