Cloned (Comment) | Organism |
---|---|
recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes, with a secretion tag, in Escherichia coli strain BL21(DE3) | Severe acute respiratory syndrome-related coronavirus |
Crystallization (Comment) | Organism |
---|---|
purified enzyme mutant C112S in complex with ubiquitin, sitting drop vapour diffusion method, mixing of enzyme and ubiquitin in a 1:1 molar ratio giving 8 mg/ml and 2 mg/ml, respectively, addition of a reservoir solution consisting of 18% PEG 3000, 0.1 M CHES, pH 9.5, 22°C, X-ray diffraction structure determination and analysis at 1.4 A resolution | Severe acute respiratory syndrome-related coronavirus |
Protein Variants | Comment | Organism |
---|---|---|
C112S | site-directed mutagenesis, the C112S mutant is monomeric, ubiquitin binding crystal structure analysis, overview | Severe acute respiratory syndrome-related coronavirus |
D165A | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
E168A | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
E168D | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
E168R | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
L163Q | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
Y265A | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
Y265F | site-directed mutagenesis, the mutant shows reduced activity in deubiquitination compared to the wild-type enzyme | Severe acute respiratory syndrome-related coronavirus |
Y274A | site-directed mutagenesis, inactive mutant | Severe acute respiratory syndrome-related coronavirus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
6-Mercaptopurine | competitive and reversible inhibitor | Severe acute respiratory syndrome-related coronavirus | |
6-thioguanine | competitive and reversible inhibitor | Severe acute respiratory syndrome-related coronavirus | |
N-cyclohexyl-2-aminethanesulfonic acid | the molecule binds near the catalytic triad | Severe acute respiratory syndrome-related coronavirus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten steady-state enzyme-kinetics of wild-type and mutant enzymes, overview | Severe acute respiratory syndrome-related coronavirus | |
0.73 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant wild-type enzyme, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.01 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168R, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.1 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168D, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.2 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant L163Q, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.26 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.35 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant Y265F, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
2.48 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant Y265A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
3.33 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant D165A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Severe acute respiratory syndrome-related coronavirus | P0C6U8 | polyprotein; SARS-CoV | - |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3), the secretion tag is removed and the purification continued by nickel affinity chromatography, gel filtration, and ultrafiltration | Severe acute respiratory syndrome-related coronavirus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
FRLKGG-4-nitroanilide + H2O | - |
Severe acute respiratory syndrome-related coronavirus | FRLKGG + 4-nitroaniline | - |
? | |
ubiquitin-7-amido-4-trifluoromethylcoumarin + H2O | the enzyme catalyzes proteolytic processing of the viral polyprotein and also shows significant in vitro deubiquitinating and de-ISGylating activities. The enzyme binds ubiquitin, the ubiquitin core makes mostly hydrophilic interactions with the enzyme, while the Leu-Arg-Gly-Gly C-terminus of ubiquitin is located in the catalytic cleft of the enzyme. The ubiquitin core binds to the palm, thumb and fingers domains of the enzyme, while its final four C-terminal residues bind into a narrow channel by a network of hydrogen bonds and reach towards the active site | Severe acute respiratory syndrome-related coronavirus | ubiquitin + 7-amino-4-trifluoromethylcoumarin | - |
? |
Synonyms | Comment | Organism |
---|---|---|
papain-like protease | - |
Severe acute respiratory syndrome-related coronavirus |
PLpro | - |
Severe acute respiratory syndrome-related coronavirus |
severe acute respiratory syndrome coronavirus papain-like protease | - |
Severe acute respiratory syndrome-related coronavirus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Severe acute respiratory syndrome-related coronavirus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant Y265A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.04 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant D165A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.09 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant L163Q, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.29 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.36 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168R, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.61 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168D, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.61 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant Y265F, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.79 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant wild-type enzyme, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Severe acute respiratory syndrome-related coronavirus |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.015 | - |
6-thioguanine | pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.015 | - |
6-Mercaptopurine | pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus |
General Information | Comment | Organism |
---|---|---|
malfunction | mutations of residues in the enzyme-ubiquitin interface lead to reduced catalytic activity | Severe acute respiratory syndrome-related coronavirus |
additional information | the enzyme comprises the viral polyprotein residues 1541-1858 | Severe acute respiratory syndrome-related coronavirus |
physiological function | the enzyme is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus. It also shows significant in vitro deubiquitinating and de-ISGylating activities | Severe acute respiratory syndrome-related coronavirus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant Y265A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.08 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant D165A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.23 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168A, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.36 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant L163Q, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.45 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant Y265F, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
0.55 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168D, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.01 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant mutant E168R, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus | |
1.08 | - |
ubiquitin-7-amido-4-trifluoromethylcoumarin | recombinant wild-type enzyme, pH 7.4, 30°C | Severe acute respiratory syndrome-related coronavirus |