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Literature summary for 3.4.22.36 extracted from

  • Datta, D.; Scheer, J.M.; Romanowski, M.J.; Wells, J.A.
    An allosteric circuit in caspase-1 (2008), J. Mol. Biol., 381, 1157-1167.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
caspase-1 expression in Escherichi coli in insoluble inclusion body Homo sapiens

Crystallization (Commentary)

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

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

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 [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

Organism UniProt Comment Textmining
Homo sapiens
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant caspase-1 solubilized from insoluble inclusion bodies from Escherichi coli by cation exchange chromatography Homo sapiens

Renatured (Commentary)

Renatured (Comment) Organism
recombinant caspase-1 from Escherichi coli expressed in insoluble inclusion bodies Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Ac-Trp-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin + H2O
-
Homo sapiens ?
-
?

Subunits

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Homo sapiens