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Literature summary for 3.4.21.5 extracted from

  • Marino, F.; Pelc, L.A.; Vogt, A.; Gandhi, P.S.; Di Cera, E.
    Engineering thrombin for selective specificity toward protein C and PAR1 (2010), J. Biol. Chem., 285, 19145-19152.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of wild-type and mutant enzymes in baby hamster kidney cells, only mutants W215M and W215T, and also the wild-type enzyme are expressed in Escherichia coli strain BL21(DE3) Homo sapiens

Protein Variants

Protein Variants Comment Organism
C191A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
C220A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D100A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D14A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D14lA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D178A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D186aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D189A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D1aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D221A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D222A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
D60eA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E14cA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E14eA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E14hA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E186bA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E192A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E1cA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E217A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E39A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E77A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E80A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E8A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
E97aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
F227A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
F245A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
F34A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
F60hA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
G193A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
G223a site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
G226A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
H71A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
I174A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
I24A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
I82A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K109A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K10A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K110A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K169A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K186dA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K224A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K235A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K236A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K240A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K36A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K60fA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K70A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K81A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
K9A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
L60A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
L65A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
L99A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
M84A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
additional information engineering thrombin for selective specificity toward protein C and PAR1 by site-directed mutagenesis, overview Homo sapiens
additional information when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1 Homo sapiens
N60gA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
P186A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
P198A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
P37A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
P60bA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
P60cA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Q38A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R101A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R126A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R14dA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R165A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R173A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R175A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R187A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R221aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R233A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R35A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R4A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R67A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R73A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R75A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R77aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R93A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
R97A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
S171A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
S214A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
S36aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
T172A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
T60iA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
T74A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
V163A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
V200A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215A/E217A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme, the mutant shows significant perturbation of fibrinogen and PAR1 cleavage over protein C activation Homo sapiens
W215A/E217K site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme, the mutant features a less pronounced anticoagulant/antithrombotic profile compared with the wild-type Homo sapiens
W215D site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215E site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme, mutant W215E is 10fold more specific for protein C than fibrinogen and PAR1 Homo sapiens
W215F site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215H site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215I site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme, mutant W215I features a kcat/Km value for cleavage of fibrinogen that is about 100000fold lower than that of wild-type. The kcat/Km value for PAR1 activation is 10,000fold lower compared with wild-type, but the kcat/Km value for activation of protein C in the presence of thrombomodulin is perturbed Homo sapiens
W215L site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215M site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215P site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215R site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215T site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215V site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W215Y site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W237A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W29A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W60dA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
W96A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y117A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y184aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y225A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y228A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y60aA site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y76A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
Y89A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
Hirudin
-
Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ required Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
fibrinogen + H2O Homo sapiens
-
fibrin + fibrinopeptide A + fibrinopeptide B
-
?
protease-activated receptor 1 + H2O Homo sapiens activation ?
-
?
protein kinase C + H2O Homo sapiens activation ?
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens
-
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification recombinant mutant prothrombins are activated through proteolytic cleavage using crude venom from Echis carinatus and 1:100 dilution of venom from Agkistrodon acutus in the presence of 10 mM benzamidine and 10 mM CaCl2 Homo sapiens

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type enzyme and mutants from baby hamster kidney cells. Recombinant enzyme mutants W215M and W215T, and wild-type enzyme from Escherichia coli including unfolding and refolding of the pelleted enzymes, dialysis, heparin affinity hcromatography Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-Phe-Pro-Arg-4-nitroanilide + H2O
-
Homo sapiens D-Phe-Pro-Arg + 4-nitroaniline
-
?
fibrinogen + H2O
-
Homo sapiens fibrin + fibrinopeptide A + fibrinopeptide B
-
?
protease-activated receptor 1 + H2O activation Homo sapiens ?
-
?
protein kinase C + H2O activation Homo sapiens ?
-
?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Homo sapiens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3 7.43 fibrinogen pH 7.0, 25°C, Kcat/KM values for diverse recombinant thrombin mutants, overview Homo sapiens
3 7.43 Protein kinase C pH 7.0, 25°C, Kcat/KM values for diverse recombinant thrombin mutants, overview Homo sapiens
3 7.43 protease-activated receptor 1 pH 7.0, 25°C, Kcat/KM values for diverse recombinant thrombin mutants, overview Homo sapiens
7.23
-
fibrinogen pH 7.0, 25°C, recombinant wild-type enzyme Homo sapiens
7.23
-
Protein kinase C pH 7.0, 25°C, recombinant wild-type enzyme Homo sapiens
7.23
-
protease-activated receptor 1 pH 7.0, 25°C, recombinant wild-type enzyme Homo sapiens