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 | 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 | Comment | Organism | Structure |
---|---|---|---|
Hirudin | - |
Homo sapiens |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | required | Homo sapiens |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | - |
- |
- |
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 (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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Homo sapiens |
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 |