Information on EC 3.4.21.114 - equine arterivirus serine peptidase

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The expected taxonomic range for this enzyme is: Arterivirus

EC NUMBER
COMMENTARY
3.4.21.114
-
RECOMMENDED NAME
GeneOntology No.
equine arterivirus serine peptidase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cleavage of (Glu/Gln)-/-(Gly/Ser/Ala) in arterivirus replicase translation products ORF1a and ORF1ab
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
cleavage of C-N-linkage
-
;
-
hydrolysis of peptide bond
-
;
-
CAS REGISTRY NUMBER
COMMENTARY
97162-88-4
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
porcine reproductive and respiratory syndrome virus nonstructural protein 4 is a critical apoptosis inducer dependent on its serine protease activity, the full-length of nsp4 structure is required for its apoptosis-inducing activity, the enzyme can induce apoptosis in diverse cell lines, e.g. in HeLa cells, overview
malfunction
the ability of nsp4 to induce apoptosis is significantly impaired by His39, Asp64, and Ser118 mutations
additional information
residues His39, Asp64, and Ser118 are essential for the enzyme to trigger apoptosis
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
arterivirus replicase ORF1a + H2O
?
show the reaction diagram
-
cleavage sites are Glu1430/Gly and Glu1452/Ser, ORF1a can be cleaved following two alternative pathways. In the majority nsp3-8 precursors are cleaved at the nsp4/5 site yielding nsp3-4 and nsp5-8. The latter product is then cleaved at nsp7/8 only. In the alternative proteolytic cascade the nsp4/5 site remains uncleaved while the nsp5/6 and nsp6/7 sites are processed. nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, when nsp2 cofactor is absent the alternative pathway is used
-
-
?
arterivirus replicase ORF1a protein + H2O
?
show the reaction diagram
-
-
-
?
arterivirus replicase ORF1a protein + H2O
?
show the reaction diagram
nsp4 and an unidentified protease is responsible for the cleavage sites nsp2/3, nsp3/4, nsp4/5 and nsp5/6 in the ORF1a protein
-
-
?
arterivirus replicase ORF1a protein + H2O
?
show the reaction diagram
-
-
-
?
arterivirus replicase ORF1ab + H2O
?
show the reaction diagram
-
cleavage sites are the junctions at nsp3/4, Nsp4/5, 2 internal nsp5 sites, nsp5/6, p80/p50, p50/p26, p26/p12, enzyme cleaves between Glu and Gly, Glu and Ser or Glu and Lys
-
-
?
arterivirus replicase ORF1b + H2O
?
show the reaction diagram
-
protein is processed by nsp4 serine protease
-
-
?
arterivirus replicase ORF1b + H2O
?
show the reaction diagram
-
protein is processed by nsp4 serine protease at the following sites: nsp9/10 i.e. p80/p50, cleavage occurs between Glu2370 and Ser, nsp10/11 i.e. p50/p26, cleavage occurs between Gln2837 and Ser, nsp11/12 i.e. nsp26/p12, cleavage between Glu3056 and Gly
-
-
?
arterivirus replicase ORF1b + H2O
?
show the reaction diagram
-
protein is processed by nsp4 serine protease
-
-
?
Dabsyl-Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu-Gly-Arg-His-Phe-Glu-EDANS + H2O
Dabsyl-Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu + Gly-Arg-His-Phe-Glu-EDANS
show the reaction diagram
-
-
?
Lys-Asp-Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu-Gly-Arg-His-Phe-Thr-Trp + H2O
Lys-Asp-Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu + Gly-Arg-His-Phe-Thr-Trp
show the reaction diagram
-
-
?
NSP3'4 protein + H2O
?
show the reaction diagram
recombinantly expressed substrate NSP3'4 covering the last 8 residues of nonstructural protein 3 and the whole amino acid sequence of nonstructural protein 4 mutant S118A
-
-
?
proform caspase-3 + H2O
mature caspase-3 + ?
show the reaction diagram
activation
-
?
proform caspase-8 + H2O
mature caspase-8 + ?
show the reaction diagram
activation
-
?
proform caspase-9 + H2O
mature caspase-9 + ?
show the reaction diagram
activation
-
?
Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu-Gly-Arg-His-Phe-Thr-Trp-Tyr-Gln + H2O
Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu + Gly-Arg-His-Phe-Thr-Trp-Tyr-Gln
show the reaction diagram
-
-
?
additional information
?
-
nsp4 cleaves multiple GluX(Gly/Ser/Ala) sites, X: cleavage site
-
-
?
additional information
?
-
-
protease has a specificity for Glu/Gly or Glu/Ser substrates
-
-
?
additional information
?
-
-
enzyme is the main viral protease
-
-
?
additional information
?
-
-
the enzyme controls the maturation of the replicase complex. The C-terminal domain has a modulating role in nsp4-mediated autoprocessing of the arterivirus replicase polyproteins
-
-
-
additional information
?
-
-
the enzyme cleaves the cognate nsp6/7- and nsp7/8 site in in vitro synthesized substrates
-
-
-
additional information
?
-
the enzyme shows a 3C-like serine protease activity
-
-
-
additional information
?
-
nsp4 cleaves multiple GluX(Gly/Ser/Ala) sites, X: cleavage site
-
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
proform caspase-3 + H2O
mature caspase-3 + ?
show the reaction diagram
Q91F54
activation
-
?
proform caspase-8 + H2O
mature caspase-8 + ?
show the reaction diagram
Q91F54
activation
-
?
proform caspase-9 + H2O
mature caspase-9 + ?
show the reaction diagram
Q91F54
activation
-
?
arterivirus replicase ORF1a + H2O
?
show the reaction diagram
-
ORF1a can be cleaved following two alternative pathways. In the majority nsp3-8 precursors are cleaved at the nsp4/5 site yielding nsp3-4 and nsp5-8. The latter product is then cleaved at nsp7/8 only. In the alternative proteolytic cascade the nsp4/5 site remains uncleaved while the nsp5/6 and nsp6/7 sites are processed. nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, when nsp2 cofactor is absent the alternative pathway is used
-
-
?
additional information
?
-
-
enzyme is the main viral protease
-
-
?
additional information
?
-
-
the enzyme controls the maturation of the replicase complex. The C-terminal domain has a modulating role in nsp4-mediated autoprocessing of the arterivirus replicase polyproteins
-
-
-
additional information
?
-
Q91F54
the enzyme shows a 3C-like serine protease activity
-
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.24
dabsyl-Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu-Gly-Arg-His-Phe-Glu-EDANS
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0073
dabsyl-Lys-Thr-Ala-Tyr-Phe-Gln-Leu-Glu-Gly-Arg-His-Phe-Glu-EDANS
porcine reproductive and respiratory syndrome virus
A1E8J1
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
21000
calculated from amino acid sequence
651859
31000
SDS-PAGE
651859
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
proteolytic modification
-
ORF1a is subject to at least 5 proteolytic cleavages, resulting in five non-structural protein 1-6
proteolytic modification
replicase of EAV processed by at least 3 viral proteases, resulting in the generation of non-structural proteins Nsp1-6
proteolytic modification
-
replicase of EAV processed by at least 3 viral proteases, resulting in the generation of non-structural proteins Nsp1-6
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
glutathione-Sepharose affinity chromatography, gel filtration
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3).
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
fusion protein with maltose-binding protein
-
large scale production of recombinant nsp4 in Escherichia coli. The nsp4 proteinase is expressed either fused to maltose binding protein or carrying a C-terminal hexahistidine tag
-
ORF1a, fusion protein with glutathione S-transferase
-
GST fusion protein expressed in Escherichia coli BL21(DE3)
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)., recombinant expression in HeLa cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D1129E
mutant is able to cleave the Nsp4/5 site with wild-type efficiently but does not process the Nsp3/4 and Nsp5/6 junctions
D1129K
no Nsp3456 cleavage
D1129V
no Nsp3456 cleavage
H1103G
complete inhibition of Nsp3456 processing
H1103R
complete inhibition of Nsp3456 processing
H1198L
completely abolished processing of Nsp4/5 and Nsp5/6
H1198R
completely abolished processing of Nsp4/5 and Nsp5/6
H1198Y
completely abolished processing of Nsp4/5 and Nsp5/6
S1184C
mutation has no effect on Nsp3456 processing
S1184F
mutation has no effect on Nsp3456 processing
S1184I
mutation has no effect on Nsp3456 processing
S1184I
-
no cleavage between nsp9 and nsp10
S1184Y
mutation has no effect on Nsp3456 processing
T1179D
completely abolished processing of Nsp4/5 and Nsp5/6
T1179G
reduced cleavage of Nsp4/5, no cleavage of Nsp5/6 site
T1179N
retains wild-type activity towards Nsp4/5, cleaves Nsp5/6 with increased efficiency
T1179S
reduced cleavage of Nsp4/5, no cleavage of Nsp5/6 site
H1103G
-
complete inhibition of Nsp3456 processing
-
H1103R
-
complete inhibition of Nsp3456 processing
-
S1184C
-
mutation has no effect on Nsp3456 processing
-
S1184F
-
mutation has no effect on Nsp3456 processing
-
S1184I
-
mutation has no effect on Nsp3456 processing
-
D64A
site-directed mutagenesis, the mutant shows significantly reduced ability to induce apoptosis compared to the wild-type enzyme
H39A
site-directed mutagenesis, the mutant shows significantly reduced ability to induce apoptosis compared to the wild-type enzyme
S118A
catalytic residue, mutant protein inactive
S118A
site-directed mutagenesis, the mutant shows significantly reduced ability to induce apoptosis compared to the wild-type enzyme