Information on EC 3.4.17.B1 - Sulfolobus solfataricus carboxypeptidase

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The expected taxonomic range for this enzyme is: Sulfolobus solfataricus

EC NUMBER
COMMENTARY hide
3.4.17.B1
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
Sulfolobus solfataricus carboxypeptidase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Release of basic, acidic and aromatic amino acids from the respective benzoylglycated and benzyloxycarbonylated amino acids. Slow hydrolysis of aliphatic amino acids. No activity with benzyloxycarbonyl-Pro and benzyloxycarbonyl-Trp
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
benzoyl-glycyl-arginine + H2O
?
show the reaction diagram
benzoyl-glycyl-lysine + H2O
?
show the reaction diagram
-
-
?
benzoyl-L-arginine + H2O
benzoic acid + L-arginine
show the reaction diagram
benzyloxycarbonyl-Ala + H2O
L-alanine + benzyloxycarbonate
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Ala-Ser-methyl ester + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Ala-Val-methyl ester + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Arg + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Asp + H2O
?
show the reaction diagram
benzyloxycarbonyl-Asp + H2O
L-aspartate + benzyloxycarbonate
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Glu-methyl ester + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Gly-Gly-Phe + H2O
?
show the reaction diagram
benzyloxycarbonyl-Leu-Ala-methyl ester + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Leu-Gly-methyl ester + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Phe + H2O
?
show the reaction diagram
-
-
?
benzyloxycarbonyl-Phe + H2O
L-phenylalanine + benzyloxycarbonate
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Phe-Leu + H2O
benzyloxycarbonyl-Phe + Leu
show the reaction diagram
-
-
-
-
benzyloxycarbonyl-Tyr-ethyl ester + H2O
?
show the reaction diagram
-
-
?
Bz-Gly + H2O
benzoic acid + glycine
show the reaction diagram
-
-
-
-
?
Fur-Gly + H2O
furoic acid + glycine
show the reaction diagram
-
-
-
-
?
furylacryloyl-L-phenylalanine + H2O
furylacrylic acid + L-phenylalanine
show the reaction diagram
L-Bz-Ala + H2O
benzoic acid + L-alanine
show the reaction diagram
-
-
-
-
?
L-Bz-Arg + H2O
benzoic acid + L-arginine
show the reaction diagram
-
-
-
-
?
L-Bz-Glu + H2O
benzoic acid + L-glutamate
show the reaction diagram
-
-
-
-
?
L-Bz-His + H2O
benzoic acid + L-histidine
show the reaction diagram
-
-
-
-
?
L-Bz-Leu + H2O
benzoic acid + L-leucine
show the reaction diagram
-
-
-
-
?
L-Bz-Met + H2O
benzoic acid + L-methionine
show the reaction diagram
-
-
-
-
?
L-Bz-Phe + H2O
benzoic acid + L-phenylalanine
show the reaction diagram
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-
-
-
?
L-Fur-Leu + H2O
furoic acid + L-leucine
show the reaction diagram
-
-
-
-
?
L-Fur-Phe + H2O
furoic acid + L-phenylalanine
show the reaction diagram
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-
-
-
?
N-[3-(2-Furylacryloyl)]-L-Ala-L-Lys + H2O
N-[3-(2-Furylacryloyl)]-L-Ala + L-Lys
show the reaction diagram
peptide + H2O
?
show the reaction diagram
protein + H2O
peptides
show the reaction diagram
additional information
?
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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
peptide + H2O
?
show the reaction diagram
protein + H2O
peptides
show the reaction diagram
additional information
?
-
P80092
CPSso, unlike most known carboxypeptidases, removes any amino acid from the C-terminus of short peptides, with the sole exception of proline, and also hydrolyzes N-blocked amino acids, thus acting as an aminoacylase, overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
can partially substitute for Zn2+, activates
additional information
Ni2+, Mn2+, Ca2+, and Mg2+ are ineffective
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
o-phenanthroline
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p-(chloromercuri)benzene sulfonate
-
-
additional information
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not affected by EDTA, pepstatin, and bestatin
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
leupeptin
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activates
phosphoramidon
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activates
PMSF
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activates
tosyl-lysine chloromethyl ketone
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activates
additional information
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not affected by EDTA, pepstatin, and bestatin, enzyme activity is increased 3 to 4fold when yeast extract instead of glucose is used in the cell culture medium
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.17
benzoyl-glycyl-arginine
pH 6.5, 60°C
0.23
benzoyl-glycyl-lysine
pH 6.5, 60°C
0.061
Benzyloxycarbonyl-Arg
pH 6.5, 60°C
0.22
Benzyloxycarbonyl-Asp
pH 6.5, 60°C
0.69
benzyloxycarbonyl-Gly-Gly-Phe
pH 6.5, 60°C
0.31
Benzyloxycarbonyl-Phe
pH 6.5, 60°C
0.061 - 0.149
Bz-Gly
0.028
Fur-Gly
-
at pH 6.5 and 35°C
0.014
L-Bz-Ala
-
at pH 6.5 and 35°C
0.014
L-Bz-Arg
-
at pH 6.5 and 35°C
0.019
L-Bz-Glu
-
at pH 6.5 and 35°C
0.005 - 0.181
L-Bz-His
0.501
L-Bz-Leu
-
at pH 6.5 and 35°C
0.028
L-Bz-Met
-
at pH 6.5 and 35°C
0.034 - 0.144
L-Bz-Phe
0.103
L-Fur-Leu
-
at pH 6.5 and 35°C
0.013
L-Fur-Phe
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at pH 6.5 and 35°C
0.38
N-[3-(2-Furylacryloyl)]-L-Ala-L-Lys
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pH 6.5, 50°C
additional information
additional information
kinetics and thermodynamics, recombinant His-tagged CPSso, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.97 - 16.6
benzoyl-glycyl-arginine
21.2
benzoyl-glycyl-lysine
Sulfolobus solfataricus
P80092
pH 6.5, 60°C
10.4
Benzyloxycarbonyl-Arg
Sulfolobus solfataricus
P80092
pH 6.5, 60°C
6
Benzyloxycarbonyl-Asp
Sulfolobus solfataricus
P80092
pH 6.5, 60°C
36.4
benzyloxycarbonyl-Gly-Gly-Phe
Sulfolobus solfataricus
P80092
pH 6.5, 60°C
3.4
Benzyloxycarbonyl-Phe
Sulfolobus solfataricus
P80092
pH 6.5, 60°C
1.03 - 4.15
Bz-Gly
2.3
Fur-Gly
Sulfolobus solfataricus
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at pH 6.5 and 35°C
2.8
L-Bz-Ala
Sulfolobus solfataricus
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at pH 6.5 and 35°C
3.63
L-Bz-Arg
Sulfolobus solfataricus
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at pH 6.5 and 35°C
4.3
L-Bz-Glu
Sulfolobus solfataricus
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at pH 6.5 and 35°C
2.05 - 8.83
L-Bz-His
8.35
L-Bz-Leu
Sulfolobus solfataricus
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at pH 6.5 and 35°C
2.95
L-Bz-Met
Sulfolobus solfataricus
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at pH 6.5 and 35°C
4.3 - 4.87
L-Bz-Phe
3.58
L-Fur-Leu
Sulfolobus solfataricus
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at pH 6.5 and 35°C
3.25
L-Fur-Phe
Sulfolobus solfataricus
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at pH 6.5 and 35°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.022
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crude cell extract, cells grown on glucose, exponential phase
0.029
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crude cell extract, cells grown on glucose, stationary phase
0.079
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crude cell extract, cells grown on yeast extract, stationary phase
0.095
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crude cell extract, cells grown on yeast extract, exponential phase
4.1
purified enzyme, substrate benzyloxycarbonyl-Phe
8.6
purified enzyme, substrate benzyloxycarbonyl-Asp
16.3
purified enzyme, substrate benzyloxycarbonyl-Arg
21.9
purified enzyme, substrate benzoyl-glycyl-arginine
27.2
purified enzyme, substrate benzoyl-glycyl-lysine
38.7
purified enzyme, substrate benzyloxycarbonyl-Gly-Gly-Phe
additional information
substrate specificity, esterase activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9
broad optimum, assay at pH 6.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
CPSso is maximally active at room temperature
additional information
-
calculation of activation energies, thermodynamics
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 85
linear increase, still active at room temperature
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.9
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42000
4 * 42000, SDS-PAGE
160000
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gel filtration
170000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
additional information
disulfide bridge formed by Cys286 and Cys293 and protease-resistant N- and C-terminal stretches, 3D-structure model, mass spectrometry, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
3D structure is developed by a molecular modeling approach. The Monte Carlo method is used to compare the crystallographic coordinates of the possible molecular structures for the enzyme with the small angle X-ray scattering profiles
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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at the extremes of the pH-stability curve, NaCl does not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein
649646
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 25
instable at, glycerol stabilizes
20
-
stable for several days at room temperature in buffer containing 10 mM imidazole-HCl, 10 mM potassium acetate and 23 mM Tris-HCl, pH 7.2, in the presence of 2 mM 2-mercaptoethanol and 50% (v/v) glycerol
25
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the enzyme gradually loses its activity resulting in a complete inactivation after 96 h. The nanobioconjugate of the enzyme immobilized on silica-coated magnetic nanoparticles leads to a substantial increase in stability, up to 85% of initial activity being retained after 96 h
40
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in the presence of ethanol at 40°C and various concentrations the inactivation profiles shows that the enzyme has a residual activity of 50% after 6 h, which decreases to 20% after 24 h incubation. The nanobioconjugate of the enzyme immobilized on silica-coated magnetic nanoparticles reveales a significantly improved stability in ethanol at the different tested concentrations compared with free enzyme, up to 80-90% of residual activity after 6 h, and 70% after 24 h incubation in 80% ethanol being retained
50
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after 1 h of incubation at 50°C and 1 MPa in the absence of substrate, the activity is decreased by 30%. This effect is abolished when the enzyme is incubated at 200 MPa. Activity loss at atmospheric pressure is about 7% in 10 min, the activity can be stabilized completely at 300 MPa; in the absence of glycerol and 2-mercaptoethanol, at 50°C, the enzyme undergoes a slow thermal inactivation upon dilution in an aqueous buffer at pH 6.5. This loss of activity can be inhibited when the enzyme is maintained at high pressure. At higher temperatures, higher pressures (up to 400 MPa) are required to maintain the enzyme in its active state
70
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inactivation rate constants of both holo- and apo-enzyme is determined at 70°C over a broad pH range. At pH values below 5.7, the metal-depleted enzyme is substantially more stable than the native form, a probable consequence of a reduction in electrostatic repulsion. In contrast, at any pH value above 5.7 loss of Zn2+ severely impairs enzyme stability. Below pH 5 the apoenzyme is also significantly destabilized
80
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first-order irreversible thermal inactivation of the metal-depleted enzyme shows an activation energy value of 205.6 kJ/mol, which is considerably lower than that of the holoenzyme (494.4 kJ/mol). The values of activation free energies, enthalpies and entropies also dropp with metal removal. Thermal inactivation of the apoenzyme is very quick at 80°C, whereas the holoenzyme is stable at the same temperature. The bivalent cation exhibits a major stabilizing role. Chaotropic salts strongly destabilize the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. The inactivation rate is also increased by sodium sulfate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme; holoenzyme is stable at 80°C, while the apoenzyme is rapidly inactivated
85
stable for 15 min
95
17% remaining activity
additional information
hydrophobic interactions between Leu7 and Leu376 increase protein thermostability
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
10% glycerol stabilizes
13% remaining activity in 0.1% SDS at 70°C after 180 min, stable at 40°C
40% remaining activity in 8 M urea after 55 min at 40°C
after dilution of the carboxypeptidase in MES buffer, pH 6.5, in the absence of glycerol and 2-mercaptoethanol, the enzyme undergoes a slow loss of activity
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immobilization of the enzyme on magnetic nanoparticles improves long-term stability at room temperature compared to the free native enzyme and also results in a significantly higher stability in organic solvents at 40°C
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in the absence of glycerol and beta-mercaptoethanol, at 50°C, the enzyme undergoes a slow thermal inactivation upon dilution in an aqueous buffer at pH 6.5. This loss of activity can be inhibited when the enzyme is maintained at high pressure. At higher temperatures, higher pressures (up to 400 MPa) are required to maintain the enzyme in its active state.
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inactivation is strongly temperature and pressure dependent
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetonitril
dimethylformamide
Ethanol
Methanol
tetrahydrofuran
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, buffer containing 10 mM imidazole-HCl, 10 mM potassium acetate and 23 mM Tris-HCl, pH 7.2, in the presence of 2 mM beta-mercaptoethanol and 50% (v/v) glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity purification of histidine-tagged enzyme
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by affinity chromatography
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recombinant HIs6-tagged wild-type and mutant CPSso from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
to electrophoretic homogeneity, 76fold
Wild-type and mutated histidine-tagged enzyme are purified by Ni-chelate affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as an N-terminus His-tagged protein in Escherichia coli
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expression in Escherichia coli
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expression of His6-tagged wild-type and mutant CPSso in Escherichia coli strain BL21(DE3)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A380S
site-directed mutagenesis, the mutation does not affect enzyme activity
L376D
site-directed mutagenesis, the mutant displays higher values of thermodynamic activation parameters with respect to those of wild type and shows a dramatic drop in thermostability
L376N
site-directed mutagenesis, the mutant displays lower values of thermodynamic activation parameters with respect to those of wild type and shows a dramatic drop in thermostability
L7D
site-directed mutagenesis, the mutant displays higher values of thermodynamic activation parameters with respect to those of wild type and shows a dramatic drop in thermostability
L7N
site-directed mutagenesis, the mutant displays lower values of thermodynamic activation parameters with respect to those of wild type and shows a dramatic drop in thermostability
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
additional information
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effect of temperature and pressure on conformational modifications and enzyme-substrate interactions are generally dissimilar and barely predictable