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 | 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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics, recombinant wild-tyype and mutant enzymes, overview | Pyrococcus horikoshii |
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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus horikoshii | O59413 | - |
- |
Pyrococcus horikoshii DSM 12428 | O59413 | - |
- |
Pyrococcus horikoshii OT-3 | O59413 | - |
- |
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 | 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 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 | 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 | Comment | Organism |
---|---|---|
dodecamer | - |
Pyrococcus horikoshii |
dodecamer | 12 * 20000, about, recombinant enzyme, SDS-PAGE | Pyrococcus horikoshii |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
80 | - |
- |
Pyrococcus horikoshii |
85 | - |
assay at | Pyrococcus horikoshii |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Pyrococcus horikoshii |
8.5 | - |
- |
Pyrococcus horikoshii |
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 | 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 |