Literature summary for 3.4.11.15 extracted from

Dura, M.A.; Rosenbaum, E.; Larabi, A.; Gabel, F.; Vellieux, F.M.; Franzetti, B.
The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea (2009), Mol. Microbiol., 72, 26-40.

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Crystallization (Commentary)

Crystallization (Comment)	Organism
the 12-subunit PhTET3 complex is crystallized and its 3D structure is solved at a resolution of 1.9 A by molecular replacement	Pyrococcus horikoshii

Inhibitors

Inhibitors	Comment	Organism	Structure
amastatin	0.002 mM, 77% inhibition	Pyrococcus horikoshii
bestatin	0.5 mM, 34% inhibition	Pyrococcus horikoshii
Ca2+	1 mM,% inhibition	Pyrococcus horikoshii
Cd2+	1 mM,% inhibition	Pyrococcus horikoshii
CHAPS buffer	-	Pyrococcus horikoshii
chymostatin	1 mM, 22% inhibition	Pyrococcus horikoshii
Cu2+	1 mM,% inhibition	Pyrococcus horikoshii
EDTA	0.02 mM, 85% inhibition	Pyrococcus horikoshii
Mg2+	1 mM,% inhibition	Pyrococcus horikoshii
Mn2+	1 mM,% inhibition	Pyrococcus horikoshii
additional information	the enzyme is insensitive to the papain and trypsin inhibitor antipain, to the cysteine proteases inhibitor E-64, to the serine and cysteine proteases inhibitor leupeptin, to the aspartate proteases inhibitor pepstatin, and to the metalloendopeptidases inhibitor phosphoramidon. Puromycin, an inhibitor of some exopeptidases, has practically no effect on the activity of PhTET3	Pyrococcus horikoshii
pefabloc	5 mM, 20% inhibition	Pyrococcus horikoshii
Zn2+	1 mM,% inhibition	Pyrococcus horikoshii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Co2+	1 mM, 2.4fold activation	Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
40000	-	12 * 40000, SDS-PAGE, the active sites of the PhTET complexes are located inside the particles, in the catalytic chambers that are enclosed by the tetrahedron apices. The walls of these chambers are positively charged, and each one contains three active sites arranged in a circular way. Adjacent to the active sites, the specificity pockets appear as small cavities. The geometry of these pockets is different in each PhTET complex, as well as their location in the particles relative to the active sites. In PhTET3, the specificity pocket is formed by the side-chains of D262, T240, T295 and T297, which leads to a negative charge	Pyrococcus horikoshii
500000	-	dodecameric complex, gel filtration	Pyrococcus horikoshii

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O59485	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-alanyl-L-alanine 4-nitroanilide + H2O	L-alanine 4-nitroanilide accumulates very fast in the reaction mixture, and the L-alanine 4-nitroanilide/4-nitroaniline ratio diminishes from 123 at 5 min to 69 at 25 min, therefore indicating that the enzyme is mostly cleaving the substrate to L-alanine 4-nitroanilide, and then also this last product to give 4-nitroaniline	Pyrococcus horikoshii	L-alanine + L-alanine 4-nitroanilide	-	?
L-alanyl-L-alanyl-L-alanine 4-nitroanilide + H2O	L-alanyl-L-alanine 4-nitroanilide is produced, but also consumed by the enzyme while 4-nitroaniline is generated after a lag phase. At 26 min the ratios L-alanyl-L-alanine 4-nitroaniline/4-nitroaniline and L-alanyl 4-nitroanilide/4-nitroaniline are 8 and 33 respectively	Pyrococcus horikoshii	L-alanine + L-alanyl-L-alanine 4-nitroanilide	-	?
L-arginine 4-nitroanilide + H2O	66.6% of the activity compared to L-lysine 4-nitroanilide	Pyrococcus horikoshii	L-arginine + 4-nitroaniline	-	?
L-glutamate 4-nitroanilide + H2O	20.1% of the activity compared to L-lysine 4-nitroanilide	Pyrococcus horikoshii	L-glutamate + 4-nitroaniline	-	?
L-leucine 4-nitroanilide + H2O	11.4% of the activity compared to L-lysine 4-nitroanilide	Pyrococcus horikoshii	L-leucine + 4-nitroaniline	-	?
L-lysine 4-nitroanilide + H2O	-	Pyrococcus horikoshii	L-lysine + 4-nitroaniline	-	?
additional information	poor activity is detected against L-ornithinyl- and L-glutaminyl-7-amido-4-methylcoumarin, and L-aspartyl-, L-methionyl- and L-alanyl 4-nitroanilide, while the enzyme shows very poor or no activity against the other aminoacyl-4-nitroanilides or aminoacyl-7-amido-4-methylcoumarines assayed. No activity can be measured against any of the N-terminal blocked derivatives	Pyrococcus horikoshii	?	-	?

Subunits

Subunits	Comment	Organism
dodecamer	12 * 40000, SDS-PAGE, the active sites of the PhTET complexes are located inside the particles, in the catalytic chambers that are enclosed by the tetrahedron apices. The walls of these chambers are positively charged, and each one contains three active sites arranged in a circular way. Adjacent to the active sites, the specificity pockets appear as small cavities. The geometry of these pockets is different in each PhTET complex, as well as their location in the particles relative to the active sites. In PhTET3, the specificity pocket is formed by the side-chains of D262, T240, T295 and T297, which leads to a negative charge	Pyrococcus horikoshii

Synonyms

Synonyms	Comment	Organism
PH1821	-	Pyrococcus horikoshii
PhTET3	-	Pyrococcus horikoshii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
80	-	half-life: 17.83 min	Pyrococcus horikoshii
90	-	half-life: 3.44 min	Pyrococcus horikoshii
95	-	half-life: 55.1 min	Pyrococcus horikoshii
100	-	half-life: 27 min	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	-	Pyrococcus horikoshii

pH Range

pH Minimum	pH Maximum	Comment	Organism
6	8.5	pH 6: about 50% of maximal activity, pH 8.0: about 90% of maximal activity	Pyrococcus horikoshii

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Release 2023.1 (January 2023)