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Information on EC 3.4.17.18 - carboxypeptidase T and Organism(s) Thermoactinomyces vulgaris and UniProt Accession P29068

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     3 Hydrolases
         3.4 Acting on peptide bonds (peptidases)
             3.4.17 Metallocarboxypeptidases
                3.4.17.18 carboxypeptidase T
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This record set is specific for:
Thermoactinomyces vulgaris
UNIPROT: P29068 not found.
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The taxonomic range for the selected organisms is: Thermoactinomyces vulgaris
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
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releases a C-terminal residue, which may be hydrophobic or positively charged
Synonyms
cpt, carboxypeptidase t, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metallocarboxypeptidase T
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
89623-65-4
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,4-dinitrophenyl-alanyl-alanyl-arginine + H2O
2,4-dinitrophenyl-Ala-Ala + Arg
show the reaction diagram
-
-
-
?
benzyloxycarbonyl-Ala-Ala-Arg + H2O
benzyloxycarbonyl-Ala-Ala + L-Arg
show the reaction diagram
-
-
-
?
benzyloxycarbonyl-Ala-Ala-Leu + H2O
benzyloxycarbonyl-Ala-Ala + L-Leu
show the reaction diagram
-
-
-
?
benzyloxycarbonyl-alanyl-alanyl-leucine + H2O
benzyloxycarbonyl-Ala-Ala + Leu
show the reaction diagram
-
-
-
?
N-2,4-dinitrophenyl-Ala-Ala-Arg + H2O
N-2,4-dinitrophenyl-Ala-Ala-Arg + L-arginine
show the reaction diagram
-
-
-
?
N-benzyloxycarbonyl-Ala-Ala-Glu + H2O
N-benzyloxycarbonyl-Ala-Ala + L-glutamate
show the reaction diagram
-
-
-
?
N-benzyloxycarbonyl-Ala-Ala-Leu + H2O
N-benzyloxycarbonyl-Ala-Ala + L-leucine
show the reaction diagram
-
-
-
?
N-carbobenzoxy-Ala-Ala-Leu + H2O
N-carbobenzoxy-Ala-Ala + Leu
show the reaction diagram
-
-
-
?
2,4-dinitrophenyl-Ala-Ala-Arg + H2O
2,4-dinitrophenyl-Ala-Ala + Arg
show the reaction diagram
2,4-dinitrophenyl-Ala-Ala-Leu-Arg + H2O
2,4-dinitrophenyl-Ala-Ala-Leu + Arg
show the reaction diagram
N-2,4-dinitrophenyl-Ala-Ala-Arg + H2O
N-2,4-dinitrophenyl-Ala-Ala-Arg + L-arginine
show the reaction diagram
-
-
-
-
?
N-benzyloxycarbonyl-Ala-Ala-Glu + H2O
N-benzyloxycarbonyl-Ala-Ala + L-glutamate
show the reaction diagram
-
-
-
-
?
N-benzyloxycarbonyl-Ala-Ala-Leu + H2O
N-benzyloxycarbonyl-Ala-Ala + L-leucine
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-Ala-Ala-Arg + H2O
N-carbobenzoxy-Ala-Ala + Arg
show the reaction diagram
-
-
-
?
N-carbobenzoxy-Ala-Ala-Leu + H2O
N-carbobenzoxy-Ala-Ala + Leu
show the reaction diagram
-
-
-
?
N-carbobenzoxy-Ala-Ala-Lys + H2O
N-carbobenzoxy-Ala-Ala + Lys
show the reaction diagram
-
-
-
?
N-carbobenzoxy-Ala-Ala-Phe-OH + H2O
N-carbobenzoxy-Ala-Ala + Phe
show the reaction diagram
-
-
-
?
N-carbobenzoxy-Ala-Ala-Trp + H2O
N-carbobenzoxy-Ala-Ala + Trp
show the reaction diagram
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
zinc-dependent enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
N-sulfamoyl-L-phenylalanine
-
1,10-phenanthroline
-
-
additional information
-
not: phenylmethylsulfonyl fluoride, carbobenzoxy-Ala-Ala-Phe-CH2Cl, Hg2+, p-hydroxymercuribenzoate
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Subtilisin
-
cleavage of the carboxypeptidase precursor
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.6
2,4-dinitrophenyl-alanyl-alanyl-arginine
0.27 - 1.35
benzyloxycarbonyl-Ala-Ala-Arg
0.04 - 0.43
benzyloxycarbonyl-Ala-Ala-Leu
0.02 - 0.15
benzyloxycarbonyl-alanyl-alanyl-leucine
2.8 - 4.4
N-2,4-dinitrophenyl-Ala-Ala-Arg
0.18 - 0.8
N-benzyloxycarbonyl-Ala-Ala-Glu
0.016 - 0.75
N-benzyloxycarbonyl-Ala-Ala-Leu
0.027
N-Carbobenzoxy-Ala-Ala-Leu
wild-type enzyme, pH and temperature not specified in the publication
4 - 5.9
2,4-dinitrophenyl-alanyl-alanyl-arginine
0.02 - 0.063
N-benzyloxycarbonyl-Ala-Ala-Leu
0.92
N-Carbobenzoxy-Ala-Ala-Arg
-
-
0.026
N-Carbobenzoxy-Ala-Ala-Leu
-
-
0.78
N-Carbobenzoxy-Ala-Ala-Lys
-
-
0.013
N-carbobenzoxy-Ala-Ala-Phe
-
-
0.01
N-Carbobenzoxy-Ala-Ala-Trp
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 4.3
2,4-dinitrophenyl-alanyl-alanyl-arginine
0.6 - 19
benzyloxycarbonyl-Ala-Ala-Arg
1.1 - 9.9
benzyloxycarbonyl-Ala-Ala-Leu
2 - 22.8
benzyloxycarbonyl-alanyl-alanyl-leucine
0.04 - 14.6
N-2,4-dinitrophenyl-Ala-Ala-Arg
7.2 - 23.6
N-benzyloxycarbonyl-Ala-Ala-Glu
1.6 - 22.8
N-benzyloxycarbonyl-Ala-Ala-Leu
6
N-Carbobenzoxy-Ala-Ala-Leu
wild-type enzyme, pH and temperature not specified in the publication
14 - 18
N-2,4-dinitrophenyl-Ala-Ala-Arg
5.6 - 10
N-benzyloxycarbonyl-Ala-Ala-Leu
57
N-Carbobenzoxy-Ala-Ala-Arg
-
-
12.5
N-Carbobenzoxy-Ala-Ala-Leu
-
-
5
N-Carbobenzoxy-Ala-Ala-Lys
-
-
2.6
N-carbobenzoxy-Ala-Ala-Phe
-
-
0.06
N-Carbobenzoxy-Ala-Ala-Trp
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.68 - 360
benzyloxycarbonyl-Ala-Ala-Arg
11 - 155
benzyloxycarbonyl-Ala-Ala-Leu
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11
N-sulfamoyl-L-phenylalanine
mutant enzyme A243G, pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.2
-
0.2 ml 50 mM Tris-HCl pH 8.0, 5 mM CaCl2, 0.2 ml 2,4-dinitrophenyl-Ala-Ala-Arg, 37°C
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
activity assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
hydrolysis of N-2,4-dinitrophenyl-Ala-Ala-Arg
25
-
activity assay, N-benzyloxycarbonyl-Ala-Ala-Leu and N-benzyloxycarbonyl-Ala-Ala-Glu
37
-
activity assay, N-2,4-dinitrophenyl-Ala-Ala-Arg
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
CBPT_THEVU
424
1
47476
Swiss-Prot
-
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36928
-
1 * 36928, deduced from sequence
36930
38000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
carboxypeptidase T complexes with substrate analogs benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid by molecular replacement at resolutions of 1.57 A and 1.62 A. The conservative Leu211 and Leu254 residues are structural determinants for recognition of hydrophobic substrates, whereas Asp263 is for recognition of positively charged substrates. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 is responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S10 subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis
crystals of carboxypeptidase T complexes with the transition-state analogs N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine are grown in microgravity in a capillary using the counter-diffusion technique. Conformation of the transition state analogue of the hydrophobic substrate in the complex with carboxypeptidase T is similar to that of the ground state analogue
crystals of the mutant enzyme A243G complexed with N-sulfamoyl-L-phenylalanine are formed under the microgravity conditions by the method of counterx02diffusion through a gel layer in a capillary. The structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine is solved at a resolution of 1.35 A and compared it with the structure of similar complex formed by carboxypeptidase T
in complex with N-benzyloxycarbonyl-L-leucine, at 1.38 A resolution. The structure of the complex is almost identical to that of the free carboxypeptidase T molecule, and a SO42- ion is also localized in the active site. The S1 subsite of carboxypeptidase T is a very conservative structure and negligibly differs from corresponding sites of carboxypeptidase A and carboxypeptidase B in the composition and the 3D structure. The S1 subsite is close to the catalytic zinc ion and to the residues Arg71, Arg147, Arg129, and Glu277 important for catalysis
mutant enzyme G215S/A251G/T257A/D260G/T262D is crystallized and its three-dimensional structure is determined at 1.29 A resolution by X-ray crystallography. The S1' subsite of carboxypeptidase T has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite
X-ray diffraction at 2.35 A resolution
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A243G
successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
A243G/D253G/T255D
successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
D253G/T255D
successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
D260G
mutant, the influence of residues at positions 260 and 262 on a broad substrate specificity is studied
D260G/T262I
mutant, the influence of residues at positions 260 and 262 on a broad substrate specificity is studied
D260G/T262K
mutant, the influence of residues at positions 260 and 262 on a broad substrate specificity is studied
D260G/T262R
mutant, the influence of residues at positions 260 and 262 on a broad substrate specificity is studied
D260N
decrease in activity with both substrates benzyloxycarbonyl-Ala-Ala-Leu and benzyloxycarbonyl-Ala-Ala-Arg
D263N
mutant acquires carboxypeptidase A-like selectivity
G207S/A243G/D253G/T255D
successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
G207S/A243G/T250A/D253G/T255D
successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
G215S/A251G/D253_L254insT/T257A/D260G/T262D
mutant CPT6
G215S/A251G/T257A/D260G/T262D
L211Q
mutant acquires carboxypeptidase B-like properties
L254S
increase in activity with both substrates benzyloxycarbonyl-Ala-Ala-Leu and benzyloxycarbonyl-Ala-Ala-Arg
T257D
decrease in activity with substrate benzyloxycarbonyl-Ala-Ala-Leu, increase in activity with substrate benzyloxycarbonyl-Ala-Ala-Arg
T262G
increase in catalytic activity
H68N
-
mutant, it is shown that the His68 residue is not a structural determinant of specificity
L254N
-
mutant, hydrolysis efficiency of substrates with C-terminal Leu and Arg not changed, 28-fold decrease in activity towards the substrate with C-terminal Glu
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
2 h, 50% loss of activity
37
-
8 h, 1 mM Ca2+, 20% loss of activity
65
-
10% denatured protein after 2 h at 65°C, 30 mM Tris-HCl pH 9.0, 0.5 M NaCl, 30% glycerol, 2 mM dithiothreitol, 2 mM cysteine, 10 mM CaCl2, rapid denaturing occurs without CaCl2 under the same conditions
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Ca2+ important for thermostability
-
Ca2+ stabilizes against thermal denaturation, contains four binding sites for Ca2+
-
resistant against trypsin and subtilisin
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by gel filtration on a Superdex TM 75 column and by using a CABS-Sepharose column
native CpT isolated from inclusion bodies, purified by molecular exclusion chromatography and on cation-exchange resin
by gel filtration on a Superdex-75 column and by using a CABS-Sepharose column
-
purification from inclusion bodes
-
two chromatography steps
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in the Escherichia coli BL21(DE3)pLysS cells
expression of mutant enzyme A243G is carried out Escherichia coli BL21(DE3) pLysS cells
into the vector pET23a for expression in Escherichia coli BL21DE3 pLysS cells
wild-type and mutant pro-cpT genes cloned into pET23a vector and expressed in the Escherichia coli BL21(DE3)pLysS cells
expression in Escherichia coli
-
expression in stable protoplast type L-form of Proteus mirabilis
-
into the vector pET23a for expression in Escherichia coli BL21DE3 pLysS cells
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
refolding after treatment with 8 M urea
best conditions: 30 mM Tris-HCl pH 9.0, 0.5 M NaCl, 30% glycerol, 2 mM dithiothreitol, 2 mM cysteine, 10 mM CaCl2, 40°C for 12 h
-
refolding after treatment with 8 M urea
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation
reconstruction of the primary specificity pocket of CpT to make it like CpB neither enhances the CpT5 activity with a substrate possessing C-terminal Arg, nor lowers the activity with a substrate carrying C-terminal Leu. Notwithstanding the considerable structural similarity of CpT and CpB, the mechanisms underlying their substrate specificities are different
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Smulevitch, S.V.; Osterman, A.L.; Galperina, O.V.; Matz, M.V.; Zagnitko, O.P.; Kadyrov, R.M.; Tsaplina, I.A.; Grishin, N.V.; Chestukhina, G.G.; Stepanov, V.M.
Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase T. A metalloenzyme endowed with dual substrate specificity
FEBS Lett.
291
75-78
1991
Thermoactinomyces vulgaris (P29068), Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Teplyakov, A.; Polyakov, K.; Obmolova, G.; Strokopytov, B.; Kuranova, I.; Osterman, A.; Grishin, N.; Smulevitch, S.; Zagnitko, O.; Galperina, O.; Matz, M.; Stepanov, V.
Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris
Eur. J. Biochem.
208
281-288
1992
Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Stepanov, V.M.
Carboxypeptidase T
Methods Enzymol.
248
675-683
1995
Thermoactinomyces vulgaris, Thermoactinomyces vulgaris INMI
Manually annotated by BRENDA team
Bushueva, A.M.; Shevelev, A.B.; Gumpert, J.; Chestukhina, G.G.; Serkina, A.V.; Hoischen, C.; Matz, M.V.; Kuryatova, M.V.; Stepanov, V.M.
Expression of the carboxypeptidase T gene from Thermoactinomyces vulgaris in stable protoplast type L-forms of Proteus mirabilis
FEMS Microbiol. Lett.
159
145-150
1998
Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Stepanov, V.M.
Carboxypeptidase T
Handbook of Proteolytic Enzymes (Barrett,A. J. ;Rawlings,N. D. ,Woessner)
1
835-837
2004
Streptomyces griseus, Thermoactinomyces vulgaris, Saccharothrix mutabilis subsp. capreolus (P39041)
-
Manually annotated by BRENDA team
Trachuk, L.; Letarov, A.; Kudelina, I.A.; Yusupova, M.P.; Chestukhina, G.G.
In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies
Protein Expr. Purif.
40
51-59
2005
Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Akparov, V.K.h.; Grishin, A.M.; Yusupova, M.P.; Ivanova, N.M.; Chestukhina, G.G.
Structural principles of the wide substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T. Reconstruction of the carboxypeptidase B primary specificity pocket
Biochemistry (Moscow)
72
416-423
2007
Thermoactinomyces vulgaris (P29068), Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Grishin, A.M.; Akparov, V.K.h.; Chestukhina, G.G.
Leu254 residue and calcium ions as new structural determinants of carboxypeptidase T substrate specificity
Biochemistry (Moscow)
73
1140-1145
2008
Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Grishin, A.M.; Akparov, V.K.h.; Chestukhina, G.G.
Structural principles of the broad substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T--role of amino acid residues at positions 260 and 262
Protein Eng. Des. Sel.
21
545-551
2008
Thermoactinomyces vulgaris (P29068), Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Timofeev, V.I.; Kuznetsov, S.A.; Akparov, V.K.h.; Chestukhina, G.G.; Kuranova, I.P.
Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine
Biochemistry (Moscow)
78
252-259
2013
Thermoactinomyces vulgaris (P29068), Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Akparov, V.K.; Timofeev, V.I.; Khaliullin, I.G.; ?vedas, V.; Chestukhina, G.G.; Kuranova, I.P.
Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris
FEBS J.
282
1214-1224
2015
Thermoactinomyces vulgaris (P29068)
Manually annotated by BRENDA team
Akparov, V.K.; Timofeev, V.I.; Kuranova, I.P.; Rakitina, T.V.
Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1 subsite from pancreatic carboxypeptidase B
Acta Crystallogr. Sect. F
74
638-643
2018
Thermoactinomyces vulgaris (P29068), Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Akparov, V.K.; Timofeev, V.I.; Khaliullin, I.G.; Konstantinova, G.E.; Kuranova, I.P.; Rakitina, T.V.; Svedas, V.K.
Mobile loop in the active site of metallocarboxypeptidases as an underestimated determinant of substrate specificity
Biochemistry (Moscow)
83
1594-1602
2018
Thermoactinomyces vulgaris (P29068), Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Akparov, V.K.; Timofeev, V.I.; Khaliullin, I.G.; Svedas, V.; Kuranova, I.P.; Rakitina, T.V.
Crystal structures of carboxypeptidase T complexes with transition-state analogs
J. Biomol. Struct. Dyn.
36
3958-3966
2018
Thermoactinomyces vulgaris (P29068)
Manually annotated by BRENDA team