Information on EC 3.4.14.10 - tripeptidyl-peptidase II

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.4.14.10
-
RECOMMENDED NAME
GeneOntology No.
tripeptidyl-peptidase II
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Release of an N-terminal tripeptide from a polypeptide
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
101149-94-4
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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
Ala-Ala-Ala-4-nitroanilide + H2O
Ala-Ala-Ala + 4-nitroaniline
show the reaction diagram
Ala-Ala-Ala-p-nitroanilide + H2O
Ala-Ala-Ala + p-nitroaniline
show the reaction diagram
-
24% of the activity with Ala-Ala-Phe-p-nitroanilide
-
-
?
Ala-Ala-Phe 2-naphthylamide + H2O
Ala-Ala-Phe + 2-naphthylamine
show the reaction diagram
-
at 41% the rate of Ala-Ala-Phe 7-amido-4-methylcoumarin
-
?
Ala-Ala-Phe 7-amido-4-methylcoumarin + H2O
Ala-Ala-Phe + 7-amino-4-methylcoumarin
show the reaction diagram
Ala-Ala-Phe-4-nitroanilide + H2O
Ala-Ala-Phe + 4-nitroaniline
show the reaction diagram
Ala-Ala-Phe-7-amido-4-methylcoumarin + H2O
Ala-Ala-Phe + 7-amino-4-methylcoumarin
show the reaction diagram
Ala-Ala-Phe-p-nitroanilide + H2O
Ala-Ala-Phe + p-nitroaniline
show the reaction diagram
angiotensin III + H2O
?
show the reaction diagram
-
-
-
-
?
Arg-Ala-(dehydro)Ala-Val-Ala + H2O
?
show the reaction diagram
-
inhibitor, at 0.05% the rate of Arg-Arg-Ala-(phospho)Ser-Val-Ala hydrolysis
-
-
?
Arg-Ala-Ser-Val-Ala + H2O
Arg-Ala-Ser + Val-Ala
show the reaction diagram
-
-
-
-
?
Arg-Arg-Ala-(phospho)Ser-Val + H2O
?
show the reaction diagram
-
the Ala-(phospho)Ser bond is cleaved at the same rate as that of Arg-Arg-Ala-(phospho)Ser-Val-Ala
-
-
?
Arg-Arg-Ala-(phospho)Ser-Val-Ala + H2O
Arg-Arg-Ala + (phospho)Ser-Val-Ala
show the reaction diagram
Arg-Arg-Ala-Ser-Val + H2O
Arg-Arg-Ala + Ser-Val
show the reaction diagram
-
-
-
-
?
Arg-Arg-Ala-Ser-Val-Ala + H2O
Arg-Arg-Ala + Ser-Val-Ala
show the reaction diagram
-
better substrate than the phosphorylated peptide
-
-
?
Cholecystokinin + H2O
?
show the reaction diagram
-
-
-
-
?
cholecystokinin octapeptide fragment + H2O
?
show the reaction diagram
-
-
-
-
?
cholecystokinin-8-sulfate + H2O
Asp-Tyr(SO3H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH2
show the reaction diagram
-
-
-
?
Gln-7-amido-4-methylcoumarin + H2O
Gln + 7-amino-4-methylcoumarin
show the reaction diagram
-
-
-
-
?
Gly-Val-Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala + H2O
Gly-Val-Leu + Arg-Arg-Ala-(phospho)Ser-Val-Ala
show the reaction diagram
-
major cleavage site: L-Arg, followed by cleavage at Ala-(phospho)Ser
-
-
Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala + H2O
Gly-Val-Leu + Arg-Arg-Ala-Ser-Val-Ala
show the reaction diagram
-
-
-
-
?
His-Leu-His 2-naphthylamide + H2O
His-Leu-His + 2-naphthylamine
show the reaction diagram
-
at 12% the rate of Ala-Ala-Phe 4-methylcoumarin 7-amide
-
?
KKE-5-[(2-aminoethyl) amino]-naphthalene-1-sulfonic acid-Q9K-4'-dimethylaminoazobenzene-4'-sulfonyl + H2O
?
show the reaction diagram
-
-
-
-
?
L-Ala-L-Ala-L-Ala 4-nitroanilide + H2O
L-Ala-L-Ala-L-Ala + 4-nitroaniline
show the reaction diagram
L-Ala-L-Ala-L-Phe 4-nitroanilide + H2O
L-Ala-L-Ala-L-Phe + 4-nitroaniline
show the reaction diagram
L-Ala-L-Ala-L-Phe-p-nitroanilide + H2O
L-Ala-L-Ala-L-Phe + p-nitroaniline
show the reaction diagram
-
-
-
?
L-Ala-L-Phe-L-Pro-p-nitroanilide + H2O
L-Ala-L-Phe-L-Pro + p-nitroaniline
show the reaction diagram
Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala + H2O
Leu-Arg-Arg + Ala-(phospho)Ser-Val-Ala
show the reaction diagram
-
poor substrate
-
-
?
Lys-Arg-Ala-Ser-Val + H2O
Lys-Arg-Ala + Ser-Val
show the reaction diagram
-
-
-
-
?
N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin + H2O
N-succinyl-Leu-Leu-Val-Tyr + 7-amino-4-methylcoumarin
show the reaction diagram
-
-
-
-
?
neurokinin + H2O
?
show the reaction diagram
-
-
-
-
?
Phe-Pro-Ala 2-naphthylamide + H2O
Phe-Pro-Ala + 2-naphthylamine
show the reaction diagram
-
at 6% the rate of Ala-Ala-Phe 4-methylcoumarin 7-amide
-
?
Pro-Pro-Ala-p-nitroanilide + H2O
Pro-Pro-Ala-p-nitroaniline
show the reaction diagram
-
12% of the activity with Ala-Ala-Phe-p-nitroanilide
-
-
?
Q20RRGRR + H2O
Q17RRGRR + Q14RRGRR + QQQ
show the reaction diagram
-
little activity
-
-
?
Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg + H2O
Val-Gly-Ala + His-Ala-Gly + Glu-Tyr-Gly + Ala-Glu-Ala + Leu-Glu-Arg
show the reaction diagram
-
peptide derived from human hemoglobin alpha-chain, residues 17-31, sequential release of tripeptides from free N-terminus, cleaved into 5 tripeptides by human enzyme, cleavage of Gly25-Ala bond occurs at a lower rate than Ala19-His and Gly22-Glu
-
?
Val-Leu-Arg-Arg-Ala-Ser-Val-Ala + H2O
Val-Leu-Arg + Arg-Ala-Ser-Val-Ala
show the reaction diagram
-
-
the latter product is cleaved at a higher rate than the substrate
?
Val-Tyr-Ser 2-naphthylamide + H2O
Val-Tyr-Ser + 2-naphthylamine
show the reaction diagram
-
at 3% the rate of Ala-Ala-Phe 4-methylcoumarin 7-amide
-
?
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
additional information
?
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S)-1-oxo-1-[(2S)-2-(5-propyl-1H-imidazol-2-yl)-2,3-dihydro-1H-indol-1-yl]butan-2-amine
-
IC50: 4 nM
(2S)-1-oxo-1-[(2S)-2-(5-propyl-1H-imidazol-2-yl)-2,3-dihydro-1H-indol-1-yl]propan-2-amine
-
IC50: 6 nM
(2S)-1-[(2S)-2-(5-ethyl-1H-imidazol-2-yl)-2,3-dihydro-1H-indol-1-yl]-1-oxopropan-2-amine
-
IC50: 23 nM
(2S)-2-[[(2S)-2-aminobutanoyl]amino]butanoic acid
-
-
(2S)-2-[[(2S)-2-aminobutanoyl]amino]pentanoic acid
-
-
(2S)-aminobutyryl-(4S)-fluoro-L-proline n-butylamide
-
-
(2S)-aminobutyryl-4-cis-benzyl-L-proline n-butylamide
-
-
(S)-valinyl-(S)-phenylalaninol amide
-
-
1,10-phenanthroline
-
-
2,2'-Dithiopyridine
-
strong
2-(2-amino-3-methyl-butyrylamino)-4-phenyl-butyric acid
-
-
2-amino-1-[2-(4-propyl-1H-imidazol-2-yl)-2,3-dihydroindol-1-yl]ethanone
-
IC50: 36 nM
2-amino-3-methyl-N-(3-phenyl-propyl)-butyramide
-
-
2-amino-3-methyl-N-phenethyl-butyramide
-
-
2-amino-N-benzyl-3-methyl-butyramide
-
-
3,4-dichloroisocoumarin
-
-
Acetyl-Val-Leu-Leu-Arg-Ala-Ser-Val-Ala
-
weak
Ala-Ala-Ala-OH
-
-
Ala-Ala-OH
-
-
Ala-Ala-Phe-chloromethyl ketone
Ala-Ala-Phe-chloromethylketone
Ala-Ala-Phe-CMK
-
-
Ala-Ala-Pro-Ala-OH
-
-
Ala-Phe-OH
-
-
Ala-Pro-Ala-OH
-
-
Ala-Pro-OH
-
-
aminobutyryl-L-Pro-n-butylamide
-
-
aminobutyryl-Phe-n-butylamide
-
-
aminobutyryl-Pro-n-butylamide
-
-
Arg-Ala-(dehydro)Ala-Val-Ala
-
-
Arg-Ala-(phospho)Ser-Val-Ala
-
-
Arg-Ala-DELTAAla-Val-Ala
-
competitive
-
Arg-Arg-Ala
-
-
Arg-Arg-Ala-(dehydro)Ala-Val-Ala
-
less effective than Arg-Ala-(dehydro)Ala-Val-Ala
Arg-Arg-Ala-(phospho)Ser-Val-Ala
-
inhibits cleavage of Leu-Arg bond, Gly-Val-Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala as substrate
Asp-Tyr-Met-OH
-
-
azetepane
-
-
azetidine
-
-
butabindide
butabindide oxalate
-
specific TPPII inhibitor
butyloxycarbonyl-Val-Nle-OH
-
-
CH3-Nle-Nle-NHCH3
-
-
CH3CO-Val-Nvl-OH
-
-
CH3OCO-Val-Nvl-OH
-
-
Cu2+
-
strong
Diazoacetyl norleucine methyl ester
-
weak
diisopropyl fluorophosphate
Gly-Gly-Phe-OH
-
-
Gly-Phe-OH
-
-
Gly-Trp-Met-OH
-
-
Gly-Trp-OH
-
-
Gly-Tyr-OH
-
-
Gly-Val-Phe-OH
-
-
Guanidinovaleric acid-Arg-Ala-(phospho)Ser-Val
-
-
Hg2+
-
strong, reversible by dialysis against 2 mM DTT
Ile-Pro-Ile-OH
-
-
iodoacetate
-
-
isovaleryl-L-Leu-L-Arg-L-Arg-L-Ala-L-Ser-L-Val-L-Ala
-
-
L-Ala-L-Ala-L-Phe-chloromethane
-
-
L-Ala-L-Ala-L-Phe-chloromethylketone
-
-
L-Val-L-Leu-L-Arg-L-Arg-L-Ala-L-Ser-L-Val-L-Ala
-
-
Leu-Arg-Arg-Ala-(phospho)Ser-Val
-
weak, Gly-Val-Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala as substrate
Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala
-
weak, Gly-Val-Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala as substrate
Met-Met-OH
-
-
Met-Tyr-OH
-
-
N-[[(L-arginyl-L-alanyl)amino](oxo)acetyl]-L-valyl-L-alanine
-
potent inhibitor
Nle-Nle-NHCH3
-
-
Nle-Nle-OH
-
-
Pepstatin
-
weak
PhCH2-Val-Nlv-OH
-
-
-
PhCH2OCO-Val-Nle-O(CH2)3CH3
-
-
-
PhCH2OCO-Val-Val-OH
-
-
-
pipecolinic acid
-
-
piperidine
-
-
Ser-Val-Ala
-
-
Soybean trypsin inhibitor
-
weak
-
trans-epoxysuccinyl-L-leucinamido(4-guanidino)butane
-
i.e. E-64, weak
Val-(4-PhCH2O)Phe-OH
-
-
Val-(beta-naphthyl)Ala-OH
-
-
Val-(S)-cyclohexylalanyl-NH(CH2)3CH3
-
-
Val-(S)-cyclohexylalanyl-OH
-
-
Val-Ala-OH
-
-
Val-Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala
-
hydrolysis of Gly-Val-Leu-Arg-Arg-Ala-(phospho)Ser-Val-Ala
Val-Met-OH
-
-
Val-Nle-NH(CH2)3CH3
-
-
Val-Nle-NH(CH2)3Ph
-
-
Val-Nle-NHCH3
-
-
Val-Nle-O(CH2)3CH3
-
-
Val-Nle-OH
-
-
Val-Nvl-NH(CH2)3CH3
-
-
Val-Nvl-NHCH3
-
-
Val-Nvl-OH
-
-
Val-Phe-NH(CH2)3CH3
-
-
Val-Phe-NH(CH2)CH3
-
-
Val-Phe-NHCH3
-
-
Val-Phe-O(CH2)3CH3
-
-
Val-Phe-O(CH2)CH3
-
-
Val-Phe-OCH3
-
-
Val-Phe-OH
-
-
Val-phenylglycine-NHCH3
-
-
Val-phenylglycine-OH
-
-
Val-Tyr-NHCH3
-
-
Val-Tyr-OH
-
-
Val-Val-NHCH3
-
-
Val-Val-OH
-
-
Z-Gly-Leu-Ala-OH
-
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
the subunits form an active complexwith a MW of 1000000 Da, the complex can sponateously dissociate in vitro into dimers which retain 1/10th of the original specific activity. The dissociated enzyme can reassociate at elevated temperatures, provided the protein concentration is sufficiently high. This reassiciation is accompanied by a reactivation. At 30°C more than 7fold activation after 2 h. Arg-Arg-Ala and Ser-Val-Ala at 0.01 mM enhance reactivation
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0036 - 0.13
Ala-Ala-Ala-4-nitroanilide
0.016
Ala-Ala-Phe 4-methylcoumarin 7-amide
-
-
0.012 - 0.33
Ala-Ala-Phe-4-nitroanilide
0.11 - 0.47
Ala-Ala-Phe-7-amido-4-methylcoumarin
0.02
Ala-Ala-Phe-p-nitroanilide
-
pH 7.5, 37°C
0.008 - 0.015
Arg-Arg-Ala-(phospho)Ser-Val-Ala
0.008 - 20
L-Ala-L-Ala-L-Ala 4-nitroanilide
0.017 - 30
L-Ala-L-Ala-L-Phe 4-nitroanilide
0.013 - 1
L-Ala-L-Ala-L-Phe-p-nitroanilide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25 - 33
Ala-Ala-Ala-4-nitroanilide
0.38 - 114
Ala-Ala-Phe-4-nitroanilide
0.25 - 100
L-Ala-L-Ala-L-Ala 4-nitroanilide
0.38 - 500
L-Ala-L-Ala-L-Phe 4-nitroanilide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000005 - 0.0039
Ala-Ala-Ala-4-nitroanilide
52375
0.0000038 - 0.0035
Ala-Ala-Phe-4-nitroanilide
115188
3.4 - 3900
L-Ala-L-Ala-L-Ala 4-nitroanilide
202268
3.82 - 3500
L-Ala-L-Ala-L-Phe 4-nitroanilide
202267
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.67
(2S)-2-[[(2S)-2-aminobutanoyl]amino]butanoic acid
-
-
1
(2S)-2-[[(2S)-2-aminobutanoyl]amino]pentanoic acid
-
-
0.00032
(2S)-aminobutyryl-(4S)-fluoro-L-proline n-butylamide
-
-
0.00014
(2S)-aminobutyryl-4-cis-benzyl-L-proline n-butylamide
-
-
2.1
(S)-valinyl-(S)-phenylalaninol amide
-
-
0.57
2-(2-amino-3-methyl-butyrylamino)-4-phenyl-butyric acid
-
-
0.033 - 0.086
2-amino-3-methyl-N-(3-phenyl-propyl)-butyramide
-
-
0.114
2-amino-3-methyl-N-phenethyl-butyramide
-
-
0.08
2-amino-N-benzyl-3-methyl-butyramide
-
-
0.014
Ala-Ala-Ala-OH
-
-
5
Ala-Ala-OH
-
-
0.114
Ala-Ala-Pro-Ala-OH
-
-
1.14
Ala-Phe-OH
-
-
0.003
Ala-Pro-Ala-OH
-
-
1.4
Ala-Pro-OH
-
-
0.0094
aminobutyryl-Phe-n-butylamide
-
-
0.00008
aminobutyryl-Pro-n-butylamide
-
-
0.01
Arg-Arg-Ala
-
pH 7.5, 37°C
0.05
Asp-Tyr-Met-OH
-
-
0.006
azetepane
-
-
0.02
azetidine
-
-
0.000007 - 0.14
butabindide
0.11
Gly-Gly-Phe-OH
-
-
0.02
Gly-Trp-Met-OH
-
-
0.47 - 0.65
Gly-Trp-OH
0.029
Gly-Val-Phe-OH
-
-
0.001
Ile-Pro-Ile-OH
-
-
0.001 - 2
L-Val-L-Leu-L-Arg-L-Arg-L-Ala-L-Ser-L-Val-L-Ala
2
Met-Tyr-OH
-
-
0.009
Nle-Nle-NHCH3
-
-
1.43
Nle-Nle-OH
-
-
0.035
pipecolinic acid
-
-
0.012
piperidine
-
-
0.01
Ser-Val-Ala
-
pH 7.5, 37°C
0.21
Val-(4-PhCH2O)Phe-OH
-
-
0.06
Val-(beta-naphthyl)Ala-OH
-
-
0.057
Val-(S)-cyclohexylalanyl-NH(CH2)3CH3
-
-
3
Val-(S)-cyclohexylalanyl-OH
-
-
9
Val-Ala-OH
-
-
2
Val-Met-OH
-
-
0.014 - 0.029
Val-Nle-NH(CH2)3Ph
0.066
Val-Nle-NHCH3
-
-
0.29
Val-Nle-O(CH2)3CH3
-
-
0.86
Val-Nle-OH
-
-
0.003 - 0.028
Val-Nvl-NH(CH2)3CH3
0.43
Val-Nvl-OH
-
-
0.02
Val-Phe-NH(CH2)3CH3
-
-
0.022
Val-Phe-NHCH3
-
-
0.23
Val-Phe-O(CH2)3CH3
-
-
0.26
Val-Phe-OCH3
-
-
0.35
Val-Phe-OH
-
-
0.029 - 0.6
Val-phenylglycine-OH
0.857
Val-Tyr-OH
-
-
0.011
Val-TyrNHCH3
-
-
0.046
Val-Val-NHCH3
-
-
1.66 - 1.67
Val-Val-OH
additional information
additional information
-
Ki-value above 5 mM: Met-Met-OH, Asp-Tyr-OH, PhCH2-Val-Nvl-OH, PhCH2OCO-Val-Nvl-OH, CH3-Nl2-Nle-OCH3. Ki-value above 3.0 mM: CH3-Nle-Nle-NHCH3, PhCH2-Nle-Nle-NHCH3, PhCH2OCO-Val-Nle-O(CH2)3CH3. Ki-value above 0.5 mM: CH3CO-Val-Nvl-OH
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000004
(2S)-1-oxo-1-[(2S)-2-(5-propyl-1H-imidazol-2-yl)-2,3-dihydro-1H-indol-1-yl]butan-2-amine
Rattus norvegicus
-
IC50: 4 nM
0.000006
(2S)-1-oxo-1-[(2S)-2-(5-propyl-1H-imidazol-2-yl)-2,3-dihydro-1H-indol-1-yl]propan-2-amine
Rattus norvegicus
-
IC50: 6 nM
0.000023
(2S)-1-[(2S)-2-(5-ethyl-1H-imidazol-2-yl)-2,3-dihydro-1H-indol-1-yl]-1-oxopropan-2-amine
Rattus norvegicus
-
IC50: 23 nM
0.000036
2-amino-1-[2-(4-propyl-1H-imidazol-2-yl)-2,3-dihydroindol-1-yl]ethanone
Rattus norvegicus
-
IC50: 36 nM
0.000007
butabindide
Rattus norvegicus
-
IC50: 7 nM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.068
-
recombinant enzyme from cell lysate, at 37°C
4.245
-
-
12
-
after 8fold purification, at 37°C
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 7.8
-
reaction with Ala-Ala-Phe-p-nitroanilide
7.6
optimal pH, substrate: Ala-Ala-Phe-4-nitroanilide
7.8
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
gastrocnemius muscle bearing MAC16 tumour
Manually annotated by BRENDA team
-
from newborn Hooded Lister rats
Manually annotated by BRENDA team
-
TPPII is located on the sperm acrosomal region
Manually annotated by BRENDA team
additional information
-
tissue distribution
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
radiation does not lead to nuclear translocation of TPPII
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
76000
-
SDS-PAGE
80000
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mature enzyme, estimated from amino acid sequence
135000
-
x * 135000, SDS-PAGE under reducing conditions
138000
145000
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His-tagged TPP II, SDS-PAGE
150000
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gel filtration
1000000
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oligomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cryoelectron microscopy and single-particle analysis show that the complex is built from two strands forming a quasihelical structure harboring a complex system of inner cavities. The dimensions of the TPP2 spindle are 55 x 28 nm and the strands are built of nine stacked dimers. The interior of each strand is permeated by a cavity system that features a suite of chambers at each dimer-dimer interface
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis against 2 mM Tris-HCl dissociation buffer, 0.5 mM 2-mercaptoethanol, 3% w/v glycerol, pH 8, gradually decreases activity, 30% glycerol stabilizes
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dithiothreitol and glycerol stabilize
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purified enzyme undergos a spontaneous dissociation upon storage, the dissociated enzyme has a specific activity which is 1/10th of that of the normal high MW complex
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spindle formation (at TPP II concentrations above 0.03 mg/ml) by strand paring causes both significant thermodynamic and kinetic stabilization
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, 90-95 loss of activity after storage of more than 3 years
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-70°C, frozen in liquid nitrogen, stable
-70°C, in microsomal extract, 3 months
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-70°C, stable for 4 years
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4°C, in microsomal extract, at least 3 days, less stable in phosphate buffer diluted extracts
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE column chromatography and Resource RPC hydrophobic column chromatography
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DEAE Sephacel column chromatography and hydroxyapatite column chromatography
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Ni Sepharose 6 column chromatography
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recombinant protein with His-tag
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using polyethyleneimine precipitation, (NH4)2SO4 precipitation, dialysis, anion-exchange chromatography, hydrophobic interaction chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
an 8 kb NcoI fragment covering the 5'-flanking region of the TPP2 gene, including the initiation codon, is cloned into a luciferase-containing reporter vector. HEK-293 cells and NIH3T3 cells are transiently transfected with the construct. Through sequential deletions and analysis of short PCR-fragments, the promoter can be localized to a 215 bp fragment upstream of the initiation codon
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expressed in Escherichia coli
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expressed in Escherichia coli JM109(pTD-lac-NdeI-PTPA-L2R1) cells
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expressed in Escherichia coli; expression in Escherichia coli
expressed in Escherichia coli; expression of murine TPP2 gene optimized for high-level expression in Escherichia coli
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expressed in Pichia pastoris
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expression in Escherichia coli
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normal and alternatively spliced cDNA variant which contains an additional 39 bp, encoding 13 amino acids in the C-terminal end of the protein, expression in human kidney 293 cells
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overexpression in 293-cells, enzyme forms active oligomers
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overexpression in 293-cells, enzyme i mainly in a nonassociated, inactive state
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
highly up-regulated in oral squamous cell carcinoma-derived cells; TPP2 mRNA and protein are significantly up-regulated in oral squamous cell carcinoma-derived cells compared with human normal oral keratinocytes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D387G
mutant shows an increase in pKa for kcat; the pKa values of mutant D387G for kcat (app) increase when Asp-387 is changed to glycine. Mutant shows a bell-shaped pH-dependence of kcat(app), possibly due to an impaired protonation of the leaving group. Mutant shows a decresed kcat for all substrates and a highly decreased kcat/Km compared to wild-type. In contrast to wild-type at higher pH the kcat (app) value decreases
D44A
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catalytic activity of the mutant enzyme is at least one order of magnitude lower than that of the wild-type enzyme
H264A
-
catalytic activity of the mutant enzyme is at least one order of magnitude lower than that of the wild-type enzyme
N362A
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catalytic activity of the mutant enzyme is at least one order of magnitude lower than that of the wild-type enzyme, mutation effects the quarternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of more than 10000 Da
S449A
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inactive mutant enzyme, mutation effects the quarternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of more than 10000 Da
D387G
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mutant shows bell-shaped pH-dependence of kcat, possibly due to an impaired protonation of the leaving group
E305K
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the mutant shows extremely low activity
E305Q
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the mutant shows extremely low activity
E331K
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the catalytic efficiency is reduced 20000fold for the E331K variant compared to the wild type enzyme
G375D
-
less than 0.1% of wild-type activity; mutant has only 0.1% activity of wild-type at pH 7.5. Mutant shows a marked decrease in pH optimum compared to wild-type
H267A
-
mutant shows bell-shaped pH-dependence of kcat, possibly due to an impaired protonation of the leaving group; mutant shows mostly a decresed kcat for all substrates and a highly decreased kcat/Km between 2 to fold orders of magnitude compared to wild-type. kcat shows a higher rise with pH in mutant H267A than in wild-type with substrate Ala-Ala-Phe-4-nitroanilide but at pH more than 7.6 kcat (app) decreases for substrate Ala-Ala-Phe-4-nitroanilide, resulting in a significantly better fit for a bell-shaped curve than for a sigmoidal
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine