Information on EC 3.4.24.68 - tentoxilysin

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The expected taxonomic range for this enzyme is: Clostridium tetani

EC NUMBER
COMMENTARY hide
3.4.24.68
-
RECOMMENDED NAME
GeneOntology No.
tentoxilysin
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of -Gln76-/-Phe- bond in synaptobrevin (also known as neuronal vesicle-associated membrane protein, VAMP)
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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-
-
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CAS REGISTRY NUMBER
COMMENTARY hide
107231-12-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Harvard
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-
Manually annotated by BRENDA team
strain Y-IV-3
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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TeNT cleaves vesicle-associated membrane protein-2, thereby inhibiting neurotransmitter release in the central nervous system to elicit spastic paralysis
physiological function
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cleavage of synaptobrevin results in inhibition of release of neurotransmitters glycine and gamma-amino butyric acid from inhibitory interneurons causing spastic paralysis
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
rat synaptobrevin 2 + H2O
?
show the reaction diagram
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catalytic activity of all mutants
-
?
synaptobrevin + H2O
?
show the reaction diagram
Synaptobrevin + H2O
Hydrolyzed synaptobrevin
show the reaction diagram
synaptobrevin-2 + H2O
?
show the reaction diagram
vesicle-associated membrane protein VAMP + H2O
?
show the reaction diagram
vesicle-associated membrane protein-2 + H2O
?
show the reaction diagram
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neuronal SNARE protein, i.e. VAMP2
-
-
?
additional information
?
-
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
synaptobrevin + H2O
?
show the reaction diagram
synaptobrevin-2 + H2O
?
show the reaction diagram
-
i.e. vesicle associated membrane protein-2, VAMP-2
-
-
?
vesicle-associated membrane protein-2 + H2O
?
show the reaction diagram
-
neuronal SNARE protein, i.e. VAMP2
-
-
?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cobalt
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zinc-dependent endoproteinase, can replace zinc
Nickel
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zinc-dependent endoproteinase, can replace zinc
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ala-Ser-Gln-Phe-Glu-Thr-Ser
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synthetic peptide containing cleavage site of synaptobrevin, inhibits toxin action on buccal ganglion of Aplysia californica
Gln-Phe-Glu-Thr
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synthetic peptide containing cleavage site of synaptobrevin, inhibits toxin action on buccal ganglion of Aplysia californica
NaOCl
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inactivation
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Proteases
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
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assay at
7.4
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
-
effect of medium composition on the production of tetanus toxin. The highest final average yield of tetanus toxin is obtained at 9.7 mg/ml starting level of glucose and 43.5 g/l N-T Case TT as carbon and nitrogen source
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
150000
150700
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Clostridium tetani, calculated from amino acid sequence
additional information
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amino acid sequence homologies between tetanus toxin TeNT and botulinum toxins BoNT/A, B and E
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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1 * 100000, C-terminal heavy chain, + 1 * 50000, N-terminal light chain
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycolipoprotein
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gangliosides are bound to the C-terminal receptor-binding domain via two carbohydrate-binding sites, termed the lactose-binding site or the W pocket, and the sialic acid-binding site or the R pocket, GM1a bound to the W pocket, and GD3 bound to the R pocket
proteolytic modification
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TeNT is produced as a 150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain and a C-terminal heavy chain domain linked through a single disulfide bond
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals obtained with the hanging drop method
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purified C-terminal receptor binding domain with bound ganglioside GT2 in presence and absence of lactose, 10 mg/ml protein in 20 mM Tris-HCl buffer, pH 7.9, containing 100 mM NaCl are mixed with the carbohydrate moiety of GT2 at 1:8 molar ratio, vapor diffusion hanging drop method, 0.002 ml of protein-ligand solution are mixed with 0.002 ml of well solution containing 100 mM bis(trispropane) buffer, pH 7.0, 25% polyethylene glycol 2000 and 300 mM ammonium sulfate, equilibration against 0.5 ml well solution at 19C, X-ray diffraction structure determination and analysis at 2.0-2.1 A resolution, molecular replacement method
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sitting drop vapor diffusion method. The structure provides insight into the active site, the importance of the nucleophilic water and the role of the zinc ion
tetanus neurotoxin C fragment, vapor-diffusion method, hanging drops and sitting drops, thick rod-shaped crystals, space group P2(1)2(1)2(1), unit cell dimensions a : 67.4 A, b : 79.7 A, c : 91.1 A
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 60
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Tm-value 58.4C
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
extremely sensitive to oxidants
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31426
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, in 10 mM HEPES buffer, pH 7.2, 50 mM NaCl, after freezing in liquid N2, stable
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4C, both native and recombinant TeNT L-chains show significant decreases after 3-4 days of storage
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
His-tagged teTN-LC protein
single-chain, two-chain and L-chain form; very toxic! Booster injection of tetanus toxoid before starting research with tetanus toxin advisable, human anti-tetanus neurotoxin antibodies available
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TeNT Hc fragment mutants
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tetanus neurotoxin light chain
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wild-type and mutated recombinant Hc-fragments
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Clostridium tetani
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Clostridium tetani; expressed in Escherichia coli JM101 using three different plasmid vectors
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expressed in Escherichia coli
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tetanus neurotoxin light chain, expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D1222L
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lactose-binding site mutant, mutation generated by PCR
D1309A
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site-directed mutagenesis
D1309N
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site-directed mutagenesis
E1310A
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site-directed mutagenesis
E1310Q
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site-directed mutagenesis
F1305A
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site-directed mutagenesis
G1215F
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sialic acid binding site mutant, mutation generated by PCR
G1300F
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lactose-binding site mutant, mutation generated by PCR
H1271A
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lactose-binding site mutant, mutation generated by PCR
H1271W
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lactose-binding site mutant, mutation generated by PCR
H1293A
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lactose-binding site mutant, mutation generated by PCR
K1295A
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site-directed mutagenesis
K1297A
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site-directed mutagenesis
N1219I
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lactose-binding site mutant, mutation generated by PCR
N1220I
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lactose-binding site mutant, mutation generated by PCR
R1168A
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site-directed mutagenesis
R1168K
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site-directed mutagenesis
R1226F
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sialic acid binding site mutant, mutation generated by PCR
R1226L
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sialic acid binding site mutant, mutation generated by PCR
S1287A
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lactose-binding site mutant, mutation generated by PCR
W1289G
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lactose-binding site mutant, mutation generated by PCR
W1289L
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lactose-binding site mutant, mutation generated by PCR
W1303A
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site-directed mutagenesis
Y1170A
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site-directed mutagenesis
Y1290A
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lactose-binding site mutant, mutation generated by PCR
Y1290F
Y1290K
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site-directed mutagenesis
Y1290S
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site-directed mutagenesis
Y1292K
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site-directed mutagenesis
additional information
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construction of mutated forms of HCR/T that lack one or both carbohydrate-binding pocket, loss of gangliosides binding ability leads to loss of neuron binding ability of the toxin, both of the W and R pockets are necessary for high affinity binding to neuronal and non-neuronal cells, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine