Any feedback?
Information on EC 3.4.17.B1 - Sulfolobus solfataricus carboxypeptidase and Organism(s) Saccharolobus solfataricus and UniProt Accession P80092
for references in articles please use BRENDA:EC3.4.17.B1preliminary BRENDA-supplied EC number
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
3.4.17.B1 Sulfolobus solfataricus carboxypeptidaseSpecify your search results
Word Map
- 3.4.17.B1
-
hyperthermophilic
-
zinc-metalloenzyme
-
n-blocked
- asparagine
- archaeon
- synthesis
The taxonomic range for the selected organisms is: Saccharolobus solfataricus
The expected taxonomic range for this enzyme is: Saccharolobus solfataricus
The expected taxonomic range for this enzyme is: Saccharolobus solfataricus
Reaction Schemes
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
Synonyms
cpsso, sulfolobus solfataricus carboxypeptidase, carboxypeptidase s1, thermostable carboxypeptidase 1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
benzoyl-glycyl-L-lysine + H2O
benzoyl-glycine + L-lysine
-
-
-
?
benzyloxycarbonyl-Ala + H2O
L-alanine + benzyloxycarbonate
-
-
-
?
benzyloxycarbonyl-Ala-Ser-methyl ester + H2O
?
-
-
?
benzyloxycarbonyl-Ala-Val-methyl ester + H2O
?
-
-
?
benzyloxycarbonyl-Asp + H2O
L-aspartate + benzyloxycarbonate
-
-
-
?
benzyloxycarbonyl-L-Arg + H2O
benzyloxycarbonate + L-Arg
-
-
-
?
benzyloxycarbonyl-Leu-Ala-methyl ester + H2O
?
-
-
?
benzyloxycarbonyl-Leu-Gly-methyl ester + H2O
?
-
-
?
benzyloxycarbonyl-Phe + H2O
L-phenylalanine + benzyloxycarbonate
-
-
-
?
benzyloxycarbonyl-Phe-Leu + H2O
benzyloxycarbonyl-Phe + Leu
-
-
-
?
furylacryloyl-L-phenylalanine + H2O
furylacrylic acid + L-phenylalanine
-
-
-
?
N-[3-(2-Furylacryloyl)]-L-Ala-L-Lys + H2O
N-[3-(2-Furylacryloyl)]-L-Ala + L-Lys
-
-
-
?
benzyloxycarbonyl-Gly-Gly-Phe + H2O
?
best substrate
-
?
peptide + H2O
?
broad substrate specificity, overview
-
?
protein + H2O
peptides
broad substrate specificity, overview
-
?
additional information
?
-
enzyme also shows esterase activity, no activity with 4-nitroanilide substrates
-
?
additional information
?
-
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
-
-
?
additional information
?
-
-
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
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
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
-
-
?
additional information
?
-
-
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
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Zn2+
Zn2+
additional information
Ni2+, Mn2+, Ca2+, and Mg2+ are ineffective
Zn2+
removal of Zn2+ by dialysis leads to reversible activity loss, which is promptly restored by addition of 0.08 mM ZnCl2 to the assay mixture
Zn2+
metalloprotease, dependent on, reversible inactivation upon metal depletion
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
EDTA
complete inhibition, can be reversed by Zn2+ or to a lesser extent by Co2+
additional information
-
not affected by EDTA, pepstatin, and bestatin
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
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
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
kinetics and thermodynamics, recombinant His-tagged CPSso, overview
-
additional information
additional information
-
kinetics and thermodynamics, recombinant His-tagged CPSso, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.022
-
crude cell extract, cells grown on glucose, exponential phase
0.029
-
crude cell extract, cells grown on glucose, stationary phase
0.079
-
crude cell extract, cells grown on yeast extract, stationary phase
0.095
-
crude cell extract, cells grown on yeast extract, exponential phase
38.7
purified enzyme, substrate benzyloxycarbonyl-Gly-Gly-Phe
additional information
substrate specificity, esterase activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
60
additional information
additional information
-
calculation of activation energies, thermodynamics
additional information
calculation of activation energies, thermodynamics
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
disulfide bridge formed by Cys286 and Cys293 and protease-resistant N- and C-terminal stretches, 3D-structure model, mass spectrometry, overview
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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
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
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
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
additional information
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
LITERATURE
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
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
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
70
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
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
90
80
holoenzyme is stable at 80°C, while the apoenzyme is rapidly inactivated
additional information
50
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
50
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
90
enzyme looses activity at the rate of 25% in 10 min, a pressure raise of 400 MPa results in a 4fold decrease of the inactivation rate
additional information
hydrophobic interactions between Leu7 and Leu376 increase protein thermostability
additional information
-
hydrophobic interactions between Leu7 and Leu376 increase protein thermostability
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
13% remaining activity in 0.1% SDS at 70°C after 180 min, stable 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
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
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.
inactivation is strongly temperature and pressure dependent
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
acetonitril
93% remaining activity in 50% acetonitril at 40°C after 101 min, inactivation at 70°C
dimethylformamide
the enzyme gradually loses its activity by increasing the dimethylformamide in the solvent mixture, while the nanobioconiugate retains 80% of residual activity even in the presence of 80% dimethylformamide
Ethanol
Methanol
56% remaining activity in pure methanol at 40°C after 98 min, inactivation over this time period at 70°C, 11% remaining activity in 50% methanol at 70°C
tetrahydrofuran
19% remaining activity in 50% tetrahydrofuran after 42 min at 40°C, nactivation at 70°C
Ethanol
90% remaining activity in pure ethanol at 40°C after 98 min, inactivation over this time period at 70°C, 2% remaining activity in 50% ethanol at 70°C
Ethanol
nanobioconjugate of the enzyme immobilized on silica-coated magnetic nanoparticles exhibits enhanced stability in aqueous media at room temperature as well as in different organic solvents. The improved stability in ethanol paves the way to possible applications of immobilized enzyme, in particular as a biocatalyst for the synthesis of N-blocked amino acids
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant HIs6-tagged wild-type and mutant CPSso from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
Wild-type and mutated histidine-tagged enzyme are purified by Ni-chelate affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of His6-tagged wild-type and mutant CPSso in Escherichia coli strain BL21(DE3)
expressed as an N-terminus His-tagged protein in Escherichia coli
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
nanobioconjugate of the enzyme immobilized on silica-coated magnetic nanoparticles exhibits enhanced stability in aqueous media at room temperature as well as in different organic solvents. The improved stability in ethanol paves the way to possible applications of the immobilized enzyme, in particular as a biocatalyst for the synthesis of N-blocked amino acids. Another potential application might be amino acid racemate resolution, a critical and expensive step in chemical synthesis
additional information
-
effect of temperature and pressure on conformational modifications and enzyme-substrate interactions are generally dissimilar and barely predictable
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Villa, A.; Zecca, L.; Fusi, P.; Colombo, S.; Tedeschi, G.; Tortora, P.
Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus
Biochem. J.
295
827-831
1993
Saccharolobus solfataricus, Saccharolobus solfataricus (P80092), Saccharolobus solfataricus MT-4 / DSM 5833, Saccharolobus solfataricus P2 (P80092)
-
Colombo, S.; D'Auria, S.; Fusi, P.; Zecca, L.; Raia, C.A.; Tortora, P.
Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus
Eur. J. Biochem.
206
349-357
1992
Saccharolobus solfataricus (P80092), Saccharolobus solfataricus P2 (P80092)
Fusi, P.; Villa, M.; Burlini, N.; Tortora, P.; Guerritore, A.
Intracellular proteases from the extremely thermophilic archaebacterium Sulfolobus solfataricus
Experientia
47
1057-1060
1991
Saccharolobus solfataricus, Saccharolobus solfataricus MT-4 / DSM 5833
-
Occhipinti, E.; Bec, N.; Gambirasio, B.; Baietta, G.; Martelli, P.L.; Casadio, R.; Balny, C.; Lange, R.; Tortora, P.
Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions Sulfolobus solfataricus carboxypeptidase as a model enzyme
Biochim. Biophys. Acta
1764
563-572
2006
Saccharolobus solfataricus
Sommaruga, S.; De Palma, A.; Mauri, P.L.; Trisciani, M.; Basilico, F.; Martelli, P.L.; Casadio, R.; Tortora, P.; Occhipinti, E.
A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase
Proteins
71
1843-1852
2008
Saccharolobus solfataricus (P80092), Saccharolobus solfataricus
Colombo, S.; Toietta, G.; Zecca, L.; Vanoni, M.; Tortora, P.
Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus
J. Bacteriol.
177
:5561-5566
1995
Saccharolobus solfataricus (P80092)
Bec, N.; Villa, A.; Tortora, P.; Mozhaev, V.V.; Balny, C.; Lange, R.
Enhanced stability of carboxypeptidase from Sulfolobus solfataricus at high pressure
Biotechnol. Lett.
18
483-488
1996
Saccharolobus solfataricus (P80092), Saccharolobus solfataricus P2 (P80092)
-
Occhipinti, E.; Martelli, P.L.; Spinozzi, F.; Corsi, F.; Formantici, C.; Molteni, L.; Amenitsch, H.; Mariani, P.; Tortora, P.; Casadio, R.
3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering
Biophys. J.
85
1165-1175
2003
Saccharolobus solfataricus
Sommaruga, S.; Galbiati, E.; Penaranda-Avila, J.; Brambilla, C.; Tortora, P.; Colombo, M.; Prosperi, D.
Immobilization of carboxypeptidase from Sulfolobus solfataricus on magnetic nanoparticles improves enzyme stability and functionality in organic media
BMC Biotechnol.
14
82
2014
Saccharolobus solfataricus (P80092), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (P80092)
EXTERNAL LINKS (specific for EC-Number 3.4.17.B1)
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
