Information on EC 3.4.23.B11 - spumapepsin

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.23.B11
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
spumapepsin
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Good cleavage at the peptide bonds: Asn-Thr, Asn-Gln, Asn-Cys and Asn-Ala
show the reaction diagram
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-
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CAS REGISTRY NUMBER
COMMENTARY hide
9001-92-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
prototype foamy virus
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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the RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses.The RNase H domain might be involved in the stabilization of the protease dimer, while the reverse transcriptase domain is essential for RNA dependent protease activation
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Gag protein + H2O
?
show the reaction diagram
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recombinant His6-tagged spumaretroviral Gag proteins expressed in Escherichia coli strain BL21, determination of cleavage/processing sites by N-terminal sequence determination and mass spectrometry, overview
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-
?
Gag protein precursor + H2O
?
show the reaction diagram
Gag protein-derived peptides + H2O
?
show the reaction diagram
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recombinant peptide substrate derived from spumaretroviral Gag protein
-
-
?
GB1-GFP + H2O
?
show the reaction diagram
GSYVVNCNTKK + H2O
GSYVVN + CNTKK
show the reaction diagram
-
-
-
?
HSRV pr71Gag + H2O
?
show the reaction diagram
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efficiently but not completely cleaved at the p68gag/p3 cleavage site
-
-
?
IHLIAAVKAV + H2O
IHLIA + AVKAV
show the reaction diagram
-
-
-
?
PDGVFPVTTPDLR + H2O
PDGVF + PVTTPDLR
show the reaction diagram
-
-
-
?
PEGVFYTDGSR + H2O
PEGVF + YTDGSR
show the reaction diagram
-
-
-
?
PGHWVNQVGHR + H2O
PGHWVN + QVGHR
show the reaction diagram
-
-
-
?
PIQHIRSVTGE + H2O
PIQHIR + SVTGE
show the reaction diagram
Pol protein precursor + H2O
?
show the reaction diagram
PQHWVNQVGHR + H2O
PQHWVN + QVGHR
show the reaction diagram
-
-
-
?
pr74 + H2O
?
show the reaction diagram
PRAVNTVTOR + H2O
PRAVN + TVTOR
show the reaction diagram
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-
-
?
PRAVNTVTQR + H2O
PRAVN + TVTQR
show the reaction diagram
PRCIINAILGG + H2O
PRCIIN + AILGG
show the reaction diagram
-
-
-
?
PRCRIINAILGG + H2O
PRCRIIN + AILGG
show the reaction diagram
-
-
-
?
PYVVNCNTK + H2O
PYVVN + CNTK
show the reaction diagram
-
-
-
?
RAVNTVTQ + H2O
RAVN + TVTQ
show the reaction diagram
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-
-
?
SRAVNTVTOS + H2O
SRAVN + TVTOS
show the reaction diagram
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oligopeptide representing the Gag(nucleocapsi)-cleavage site in human foamy virus
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?
SRAVNTVTQS + H2O
SRAVN + TVTQS
show the reaction diagram
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molecular model of Human foamy virus protease PR indicated, role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of Human foamy virus protease PR in comparison with Human immunodeficiency virus type 1 protease PR analyzed
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-
?
SRAVYTITPE + H2O
SRAVY + TITPE
show the reaction diagram
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-
-
?
VPTINNVHTS + H2O
VPTIN + NVHTS
show the reaction diagram
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-
-
?
VSQAYPIVG + H2O
VSQAY + PIVG
show the reaction diagram
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X: Cys and Ala are preferred at P2 position
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?
VSQCYPIVG + H2O
VSQCY + PIVG
show the reaction diagram
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X: Cys and Ala are preferred at P2 position
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Gag protein precursor + H2O
?
show the reaction diagram
Pol protein precursor + H2O
?
show the reaction diagram
-
-
-
-
?
pr74 + H2O
?
show the reaction diagram
additional information
?
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cholic acid
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inhibitory to both the separate protease domain and to protease-reverse transcriptase. Cholic acid binds in the proposed protease domain dimerization interface and appears to impair formation of the correct dimer
lithocholic acid
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additional information
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HIV-1 inhibitors darunavir, tipranavir, and indinavir, have no effect on foamy virus protease in vitro
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NaCl
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assay at 3 M, no activity at salt concentrations of 0.2-0.4 M Na. In vitro dimerization of the protease domain is inducible at high salt concentrations. This effect might be caused by a hydrophobic dimerization interface, which under high ionic strength disfavors the monomeric state
additional information
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identification of a specific protease-activating RNA motif PARM located in the pol region of viral RNA, which stimulates protease activity in vitro and in vivo. PARM spans the A and B regions of the central purine-rich sequences of the prototype foamy virus (pre)genomic RNA. PARM enables foamy virus protease-reverse transcriptase to form a proteolytically active dimer in vitro as well as in vivo. At least two foamy virus protease-reverse transcriptase molecules bind to the PARM and only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation.The PARM displays a distinct RNA folding, important for protease activation and thus virus maturation
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.91
GSYVVNCNTKK
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pH 6.6, 37C
1
IHLIAAVKAV
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pH 6.6, 37C
0.56
SRAVNTVTOS
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pH 6.6, 37C
0.17 - 1.3
SRAVNTVTQS
0.15
SRAVYTITPE
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pH 6.6, 37C
0.03
VPTINNVHTS
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pH 6.6, 37C
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.003
GSYVVNCNTKK
Human spumaretrovirus
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pH 6.6, 37C
0.002
IHLIAAVKAV
Human spumaretrovirus
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pH 6.6, 37C
0.006
SRAVNTVTOS
Human spumaretrovirus
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pH 6.6, 37C
0.0017 - 0.0071
SRAVNTVTQS
0.001
SRAVYTITPE
Human spumaretrovirus
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pH 6.6, 37C
0.0002
VPTINNVHTS
Human spumaretrovirus
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pH 6.6, 37C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.6 - 0.75
cholic acid
1
lithocholic acid
Simian foamy virus
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protease-reverse transcriptase, pH 6.4, 20C, presence of 3 M NaCl
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.21
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Q8R mutant
6.24
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H22L mutant
6.3
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wild-type
6.5 - 9
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Q8R/T28D double mutant, increased pH optimum identified
6.5
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H22L/T28D double mutant, increased pH optimum identified
6.65
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S25T mutant, increased pH optimum identified
6.81
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T28D mutant, increased pH optimum identified
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 9
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pH range analyzed
5.8 - 7.6
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pH 5.8: about 65% of maximal activity, pH 7.6: about 45% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
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2 * 10000, SDS-PAGE
24500
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x * 24500, recombinant enzyme, SDS-PAGE
84500
prototype foamy virus
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gel filtration
86500
prototype foamy virus
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1 * 86500, calculated
87500
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x * 87500, HPLC followed by light scattering
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 24500, recombinant enzyme, SDS-PAGE
monomer
additional information
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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stability parameters for wild-type and mutant Human foamy virus protease PR forms determined using maltose binding protein fusion constructs
684018
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50% loss of activity at 0.75 M urea
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the dimeric enzyme is quite unstable compared to the enzyme of other species
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
fusion proteins, wild-type and mutant forms, gel filtration and SDS-PAGE
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wild-type and mutant forms, SDS-PAGE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli, wild-type and mutant enzymes, in fusion with maltose binding protein
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expression as His6-tagged enzyme in Escherichia coli
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expression in Escherichia coli
expression of a 141 residue long part of the HFV as a fusion protein with maltose binding protein in Escherichia coli
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the enzyme is cloned into a modified thioredoxin fusion vector that carries a His-tag in the centrally located surface loop of the Escherichia coli trxA protein, bacterially expressed as a soluble fusion protein
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Virion-associated RT assay is performed in BHK cells, ATCC CCL-10. Analysis of titers of wild-type and mutant proviruses transiently transfected into HEK-293T cells. Titers of Pol cleavage site mutants after infection measured in HEL cells.
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H22L
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single mutant created
H22L/T28D
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double mutant created
Q8R
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single mutant created
Q8R/T28D
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double mutant created
S25T
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single mutant created
T28D
-
single mutant created
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
increased stability for mutant forms identified
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