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
-
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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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
-
-
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
-
-
12.5
N-Carbobenzoxy-Ala-Ala-Leu
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5
N-Carbobenzoxy-Ala-Ala-Lys
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-
2.6
N-carbobenzoxy-Ala-Ala-Phe
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-
0.06
N-Carbobenzoxy-Ala-Ala-Trp
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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
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, 37°C
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
PDB
SCOP
CATH
UNIPROT
ORGANISM
Thermoactinomyces vulgaris;
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 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+
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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, 40°C 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
Show AA Sequence (145 entries)
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