Information on EC 3.4.17.18 - carboxypeptidase T

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.17.18
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RECOMMENDED NAME
GeneOntology No.
carboxypeptidase T
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
releases a C-terminal residue, which may be hydrophobic or positively charged
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
89623-65-4
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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-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
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-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
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
additional information
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not: phenylmethylsulfonyl fluoride, carbobenzoxy-Ala-Ala-Phe-CH2Cl, Hg2+, p-hydroxymercuribenzoate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Subtilisin
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cleavage of the carboxypeptidase precursor
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.6 - 5.9
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
0.92
N-Carbobenzoxy-Ala-Ala-Arg
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-
0.026
N-Carbobenzoxy-Ala-Ala-Leu
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-
0.78
N-Carbobenzoxy-Ala-Ala-Lys
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-
0.013
N-carbobenzoxy-Ala-Ala-Phe
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-
0.01
N-Carbobenzoxy-Ala-Ala-Trp
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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 - 18
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
57
N-Carbobenzoxy-Ala-Ala-Arg
Thermoactinomyces vulgaris
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12.5
N-Carbobenzoxy-Ala-Ala-Leu
Thermoactinomyces vulgaris
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-
5
N-Carbobenzoxy-Ala-Ala-Lys
Thermoactinomyces vulgaris
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2.6
N-carbobenzoxy-Ala-Ala-Phe
Thermoactinomyces vulgaris
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0.06
N-Carbobenzoxy-Ala-Ala-Trp
Thermoactinomyces vulgaris
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.68 - 360
benzyloxycarbonyl-Ala-Ala-Arg
202291
11 - 155
benzyloxycarbonyl-Ala-Ala-Leu
170609
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.2
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0.2 ml 50 mM Tris-HCl pH 8.0, 5 mM CaCl2, 0.2 ml 2,4-dinitrophenyl-Ala-Ala-Arg, 37C
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60 - 70
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36928
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1 * 36928, deduced from sequence
48477
1 * 48477, deduced from sequence
48480
calculated from sequence
48697
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1 * 48697, deduced from sequence
48700
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calculated from sequence
SUBUNITS
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
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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
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X-ray diffraction at 2.35 A resolution
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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2 h, 50% loss of activity
37
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8 h, 1 mM Ca2+, 20% loss of activity
65
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10% denatured protein after 2 h at 65C, 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+
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resistant against trypsin and subtilisin
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by gel filtration on a Superdex TM 75 column and by using a CABS-Sepharose column
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by gel filtration on a Superdex-75 column and by using a CABS-Sepharose column
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native CpT isolated from inclusion bodies, purified by molecular exclusion chromatography and on cation-exchange resin
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purification from inclusion bodes
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two chromatography steps
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in stable protoplast type L-form of Proteus mirabilis
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into the vector pET23a for expression in Escherichia coli BL21DE3 pLysS cells
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wild-type and mutant pro-cpT genes cloned into pET23a vector and expressed in the Escherichia coli BL21(DE3)pLysS cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A243G
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successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
A243G/D253G/T255D
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successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
D253G/T255D
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successive substitution of residues in the CpT S1'-subsite by similar residues of CpB, retains substrate specificity of the wild-type
D260G
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mutant, the influence of residues at positions 260 and 262 on a broad substrate specifity is studied
D260G/T262I
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mutant, the influence of residues at positions 260 and 262 on a broad substrate specifity is studied
D260G/T262K
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mutant, the influence of residues at positions 260 and 262 on a broad substrate specifity is studied
D260G/T262R
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mutant, the influence of residues at positions 260 and 262 on a broad substrate specifity is studied
D260N
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decrease in activity with both substrates benzyloxycarbonyl-Ala-Ala-Leu and benzyloxycarbonyl-Ala-Ala-Arg
D263N
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mutant acquires carboxypeptidase A-like selectivity
G207S/A243G/D253G/T255D
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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
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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
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mutant CPT6
G215S/A251G/T257A/D260G/T262D
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mutant CPT5
H68N
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mutant, it is shown that the His68 residue is not a structural determinant of specifity
L211Q
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mutant acquires carboxypeptidase B-like properties
L254N
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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
L254S
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increase in activity with both substrates benzyloxycarbonyl-Ala-Ala-Leu and benzyloxycarbonyl-Ala-Ala-Arg
T257D
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decrease in activity with substrate benzyloxycarbonyl-Ala-Ala-Leu, increase in activity with substrate benzyloxycarbonyl-Ala-Ala-Arg
T262G
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increase in catalytic activity
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
best conditions: 30 mM Tris-HCl pH 9.0, 0.5 M NaCl, 30% glycerol, 2 mM dithiothreitol, 2 mM cysteine, 10 mM CaCl2, 40C for 12 h
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refolding after treatment with 8 M urea
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation
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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