Information on EC 3.4.22.46 - L-peptidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.22.46
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RECOMMENDED NAME
GeneOntology No.
L-peptidase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
autocatalytically cleaves itself from the polyprotein of the foot-and-mouth disease virus by hydrolysis of a Lys-/-Gly bond, but then cleaves host cell initiation factor eIF-4G at bonds -Gly-/-Arg- and -Lys-/-Arg-
show the reaction diagram
Best known from foot-and-mouth disease virus, but occurs in other aphthoviruses and cardioviruses. Destruction of initiation factor eIF-4G has the effect of shutting off host-cell protein synthesis while allowing synthesis of viral proteins to continue. The tertiary structure reveals a distant relationship to papain and, consistent with this, compound E-64 is inhibitory. Type example of peptidase family C28
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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CAS REGISTRY NUMBER
COMMENTARY hide
934238-57-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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significant conformational adaptation by the enzyme is important for substrate recognition, specificity differences between 3Cpro from different picornaviruses, overview
malfunction
metabolism
functions in the last step of nisin maturation as the leader-peptide peptidase
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Abz-KLKGAGQ-EDDnp + H2O
Abz-KLK + GAGQ-EDDnp
show the reaction diagram
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hydrolysis of FRET peptides
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-
?
Edans-EGAVSVRSQEIK-Dabcyl + H2O
?
show the reaction diagram
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-
-
?
eukaryotic initiation factor 4G + H2O
?
show the reaction diagram
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Lbpro cleaves two homologues of the host cell protein. Lbpro possesses specific binding sites at the non-prime side from S1 down to S7
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-
?
eukaryotic initiation factor eIF4G + H2O
?
show the reaction diagram
eukaryotic initiation factor eIF4GI + H2O
?
show the reaction diagram
eukaryotic initiation factor eIF4GII + H2O
?
show the reaction diagram
eukaryotic translation initiation factor eIF4GI + H2O
?
show the reaction diagram
foot-and-mouth disease leader protein + H2O
?
show the reaction diagram
human cyclin A + H2O
?
show the reaction diagram
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-
-
-
?
mengovirus polypeptide + H2O
?
show the reaction diagram
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-
-
-
?
nisin + H2O
?
show the reaction diagram
the final step in nisin maturation is the proteolytic cleavage of the first 23 amino-terminal residues by NisP
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-
?
nuclear factor-kappaB + H2O
?
show the reaction diagram
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-
-
-
?
poliovirus replicase-related polypeptide + H2O
?
show the reaction diagram
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-
-
-
?
SFANLGRTTL + H2O
SFANLG + RTTL
show the reaction diagram
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poor substrate for both isoforms Lbpro and sLbpro
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-
?
Val-Gln-Arg-Lys-Leu-Lys-4-methylcoumarin 7-amide + H2O
Val-Gln-Arg-Lys-Leu-Lys + 7-amino-4-methylcoumarin
show the reaction diagram
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the substrate corresponds to the six C-terminal amino acids of the leader protein
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?
VQRKLGAAGQ + H2O
VQRKLG + AAGQ
show the reaction diagram
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isoform sLbpro cleaves this substrate better than isoform Lbpro
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-
?
VQRKLGRAGQ + H2O
VQRKLG + RAGQ
show the reaction diagram
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isoform sLbpro cleaves this substrate better than isoform Lbpro
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-
?
VQRKLKGAGQ + H2O
VQRKLK + GAGQ
show the reaction diagram
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isoform sLbpro cleaves this substrate better than isoform Lbpro
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-
?
VQRKLKRAGQ + H2O
VQRKLK + RAGQ
show the reaction diagram
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isoform sLbpro cleaves this substrate better than isoform Lbpro
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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
eukaryotic initiation factor 4G + H2O
?
show the reaction diagram
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Lbpro cleaves two homologues of the host cell protein. Lbpro possesses specific binding sites at the non-prime side from S1 down to S7
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-
?
eukaryotic initiation factor eIF4G + H2O
?
show the reaction diagram
eukaryotic initiation factor eIF4GI + H2O
?
show the reaction diagram
eukaryotic initiation factor eIF4GII + H2O
?
show the reaction diagram
eukaryotic translation initiation factor eIF4GI + H2O
?
show the reaction diagram
foot-and-mouth disease leader protein + H2O
?
show the reaction diagram
nisin + H2O
?
show the reaction diagram
D9IXC0
the final step in nisin maturation is the proteolytic cleavage of the first 23 amino-terminal residues by NisP
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-
?
nuclear factor-kappaB + H2O
?
show the reaction diagram
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?
additional information
?
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S,3R,4R)-2-{[2-(2,3-dihydroxyphenyl)-5,8-dihydroxy-4-oxo-4H-chromen-7-yl]oxy}-4-hydroxy-4-(hydroxymethyl)tetrahydrofuran-3-yl alpha-D-galactopyranoside
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2-(3,4-dihydroxyphenyl)ethyl 4-O-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]-6-O-alpha-L-lyxopyranosyl-beta-D-glucopyranoside
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E64
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epoxide-based inhibitor of cysteine proteinases, two-step overall irreversible inhibition, with step one being reversible and step two irreversible. Extending E64 by addition of the dipeptide R-P to a compound termed E64-R-P-NH2, which irreversibly inhibits Lbpro with a Ki of 30 nM and k4 of 0.01/min, can serve as the basis for design of specific inhibitors of FMDV replication
E64-R-P-NH2
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0227
Val-Gln-Arg-Lys-Leu-Lys-4-methylcoumarin 7-amide
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0116
CA074
0.0034
E64
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pH 7.8, 37C
0.00003
E64-R-P-NH2
additional information
additional information
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kinetics mechanism of two-step overall irreversible inhibition by cysteinase inhibitors
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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self-processing activity of the enzyme on wild-type and mutant polyproteins, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 9.8
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less than 50% of maximal activity above and below, substrate Val-Gln-Arg-Lys-Leu-Lys-4-methylcoumarin 7-amide
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
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the L signal is mainly localized to the nuclei of infected cells, while VP1 is detected in the cytoplasm, thereafter the L signal is detected throughout the whole cell. The pattern of L sub-cellular localization of the L mutant viruses is similar to the WT virus, except for a delay in the cytoplasmic L protein signal
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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Lpro is localized to the nucleus of infected cells, and there is a correlation between the translocation of Lpro and the decrease in the amount of nuclear p65/RelA, a subunit of NF-kappa B
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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dimer in solution, determined by NMR-spectroscopy. Dimer can not be dissociated by increasing the ionic strength or by dilution
homodimer
additional information
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the peptide binding cleft, which contains the active site at its centre, is located at the interface between two beta-barrels. Unusually, picornaviral 3Cpro possess a Cys-His-Asp/Glu catalytic triad at the centre of this cleft, instead of the Ser-His-Asp arrangement of active-site residues
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures of LbproC51A, LbproC51A/C133S, sLbproC51A/C133S
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foot-and-mouth disease virus 3C protease mutant C95K/C142L/C163A complexed with a peptide substrates APAKQLLNFD and APAKELLNFD that spans P5-P5', 1:5.5 molar ratio of enzyme to peptide, 0.05 ml of of 3Cpro solution at 17 mg/ml protein in 100 mM HEPES, pH 7.0, 400 mM NaCl, 1 mM EDTA, 2 mM 2-mercaptoethanol, and 0.01% w/v sodium azide is mixed with 0.007 ml of 30 mM peptide dissolved in the same buffer, yielding a final protein concentration of 14.9 mg/ml, sitting-drop vapour diffusion, room temperature, mixing of 0.001 ml of protein complex solution with 0.003 ml reservoir composed of 40-43% PEG 400, 0.2 M LiSO4 and 0.1 M Tris, pH 8.0 X-ray diffraction structure determination and analysis
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isoform sLbpro in complex with inhibitor E-64-R-P-NH2, hanging drop vapor diffusion method, using 0.1 M sodium acetate pH 4.8, 0.9 M NaH2PO4 and 1.2 M K2HPO4
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hanging drop vapor diffusion method, using 0.1 M HEPES pH 7.5, 24-32% (v/v) 1,4-dioxane
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, phosphate buffer, 1 mM DTT, 50% glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitatio, Mono Q column chromatography, and Superdex 75 gel filtration
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ammonium sulfate precipitation, Mono Q column chromatography, and Superdex 75 gel filtration
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Ni-NTA column chromatography, Resource Q column chromatography, and Superdex 75 gel filtration
recombinant enzyme mutants from Escherichia coli by gel filtration
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recombinant GST-tagged enzyme from Escherichia coli strain JM101 by glutathione affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL-21
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expressed in Escherichia coli BL21(DE3)LysE cells
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expressed in Escherichia coli BL21(DE3)pLysS cells
expressed in Lactococcus lactis strain LAC71 and in Escherichia coli BL21 (DE3) cells
expression of enzyme mutants in Escherichia coli
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expression of FLAG- and HA-tagged FMDV A24-L1123 mutant enzyme in LF-BK cells. T7 RNA transcripts of SpeI-digested pA24-WT, pA24-L1123 and pA24-L tag mutants, as well as transcript of control plasmid pL, are translated in vitro in rabbit reticulocyte lysates. All synthesized L mutant proteins are able to cleave themselves from the precursor L-Vp4-Vp2', the Lbeta band is absent in all L mutant translation reactions. The tn insertion in these mutant L plasmids forces translation initiation in vitro from other than the second AUG codon
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expression of GST-tagged enzyme in Escherichia coli strain JM101
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expression of wild-type and mutant enzymes by in vitro transcription and translation using rabbit reticulocyte lysate
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PK-15 cells are transfected with a HA-tagged Labpro expression construct, together with a luciferase reporter plasmid with the porcine IFN-alpha1 promoter or the IFN-beta promoter and pRL-TK. At 24 h post-transfection, the cells were further transfected or mock-transfected with poly(I:C) followed by the dual-luciferase assay, quantitative real-time RT-PCR, overview
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
S484A
the mutation abrogates protease activity
C95K/C142L/C163A
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the C-terminally truncated, catalytically inactive mutant has wild-type binding activity but remains soluble at purified protein concentrations in excess of 10 mg/ml
C95K/C142S/C163A
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C-terminally truncated, catalytically inactive form of the type A10FMDV 3Cpro
D136N
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reduced activity
E48Q
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active mutant
H110L
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active mutant, nearly as active as wild-type
H120L
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no activity
H81L
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active mutant
L143A
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self-processes efficiently at the L(pro)/VP4 cleavage site containing P2 phenylalanine, self-processing at the eIF4GII sequence Asp-Phe-Gly-Arg-Gln-Thr is improved but shows more-extensive aberrant processing
L143M
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does not self-process efficiently at the L(pro)/VP4 cleavage site containing P2 phenylalanine
L200F
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the mutant is impaired in but still allows self-processing
R647P/S648P
the mutant has full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of the enzyme is no longer cleaved
L200F
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the mutant is impaired in activity but still allows self-processing
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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the enzym eis a target for inhibitor design for inhibition of viral replication