Cloned (Comment) | Organism |
---|---|
caspase-1 expression in Escherichi coli in insoluble inclusion body | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
active forms of caspase-1 bound to the active-site inhibitors Ac-WEHD-CHO and z-Val-Ala-Asp-fluoromethylketone, or active-site ligand-free enzyme, the active-site ligand-free and allosterically inhibited conformations are nearly identical, hanging-drop vapor diffusion at 4°C against a reservoir containing for mutant R286K: 0.1 M 1,4-piperazinediethanesulfonic acid, pH 6.0, 200 mM Li2SO4, 25% PEG 2000 MME, 10 mM DTT, 3 mM NaN3, and 2 mM MgCl2, or for mutant E390D: 0.1 M Pipes, pH 6.0, 350 mM (NH4)2SO4, 20% PEG 2000 MME, 10 mM DTT, 3 mM NaN3, and 2 mM MgCl2, or for mutant R286K/E390D: 0.1 M Pipes, pH 6.0, 175 mM (NH4)2SO4, 20% PEG 2000 MME, 10 mM DTT, 3 mM NaN3, and 2 mM MgCl, X-ray diffraction structure determination and analysis at 1.8-2.1 A resolution, molecular replacement | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
D336A | site-directed mutagenesis, the mutant shows an about 2fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
E390A | site-directed mutagenesis, the mutant shows an about 130fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
N337A | site-directed mutagenesis, the mutant shows an about 2fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
R286A | site-directed mutagenesis, the mutant shows an about 230fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
R286K | site-directed mutagenesis, the mutant shows an about 150fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
R286K/E390D | site-directed mutagenesis, the mutant shows an about 37fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
S332A | site-directed mutagenesis, the mutant shows an about 4fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
S333A | site-directed mutagenesis, the mutant shows an about 2fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
S339A | site-directed mutagenesis, the mutant shows an about 7fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
T334A | site-directed mutagenesis, the mutant shows an about 2fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
T388A | site-directed mutagenesis, the mutant shows an about 2fold loss of catalytic efficiency compared to the wild-type enzyme | Homo sapiens |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Ac-WEHD-CHO | active-site inhibitor | Homo sapiens | |
additional information | allosteric inhibitors in caspase-1 directly disrupt the hydrogen bond network by preventing the salt bridge between Arg286 and Glu390 from forming | Homo sapiens | |
N-carbobenzyloxy-Val-Ala-Asp-fluoromethylketone | i.e. Z-VAD-FMK, active-site inhibitor | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | wild-type and mutant enzyme kinetics, caspase-1 shows positive cooperativity, a network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state, an allosteric circuit promotes site-to-site coupling. Arg286 and Glu390, which form a salt bridge, have major effects, causing 100 to 200fold reductions in catalytic efficiency, kcat/Km. Two neighbors, Ser332 and Ser339, have minor effects, causing 4 to 7fold reductions, overview | Homo sapiens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant caspase-1 solubilized from insoluble inclusion bodies from Escherichi coli by cation exchange chromatography | Homo sapiens |
Renatured (Comment) | Organism |
---|---|
recombinant caspase-1 from Escherichi coli expressed in insoluble inclusion bodies | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
Ac-Trp-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin + H2O | - |
Homo sapiens | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | structure of the on-state and the off-state of caspase-1, four of the network H-bonding side chains, Ser332, Ser333, Ser339, and Thr388, form a cluster that lies behind loop 2, consisting of residues 285-290, which contains the catalytic Cys285 | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Homo sapiens |