Literature summary for 3.4.22.B50 extracted from

  • Chou, C.Y.; Lai, H.Y.; Chen, H.Y.; Cheng, S.C.; Cheng, K.W.; Chou, Y.W.
    Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease (2014), Acta Crystallogr. Sect. D, 70, 572-581.
    View publication on PubMed

Cloned(Commentary)

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 (Commentary)

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

Protein Variants Comment Organism
D165A 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
C112S site-directed mutagenesis, the C112S mutant is monomeric, ubiquitin binding crystal structure analysis, overview 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
Y265F 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
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
Y274A site-directed mutagenesis, inactive mutant Severe acute respiratory syndrome-related coronavirus

Inhibitors

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 [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
1.2
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant L163Q, 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
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
3.33
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant D165A, 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
1.26
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant E168A, 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

Organism

Organism UniProt Comment Textmining
Severe acute respiratory syndrome-related coronavirus P0C6U8 polyprotein; SARS-CoV
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
PLpro
-
Severe acute respiratory syndrome-related coronavirus
papain-like protease
-
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 [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Severe acute respiratory syndrome-related coronavirus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.04
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant D165A, pH 7.4, 30°C Severe acute respiratory syndrome-related coronavirus
0.01
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant Y265A, 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
0.36
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant E168R, 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
0.09
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant L163Q, 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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Severe acute respiratory syndrome-related coronavirus

Ki Value [mM]

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

General Information Comment Organism
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
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

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.01
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant E168R, pH 7.4, 30°C Severe acute respiratory syndrome-related coronavirus
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
0.45
-
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
1.08
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant wild-type enzyme, 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
0.23
-
ubiquitin-7-amido-4-trifluoromethylcoumarin recombinant mutant E168A, pH 7.4, 30°C Severe acute respiratory syndrome-related coronavirus