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Francesc X Aviles - One of the best experts on this subject based on the ideXlab platform.
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amyloid formation by human carboxypeptidase d transthyretin like domain under physiological conditions
Journal of Biological Chemistry, 2014Co-Authors: Javier Garciapardo, Ricardo Granamontes, Marc Fernandezmendez, Angels Ruyra, Nerea Roher, Julia Lorenzo, Francesc X Aviles, Salvador VenturaAbstract:Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily Carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.
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crystal structure of novel metallocarboxypeptidase inhibitor from marine mollusk nerita versicolor in complex with human carboxypeptidase a4
Journal of Biological Chemistry, 2012Co-Authors: Giovanni Covaleda, Francesc X Aviles, Maday Alonso Del Rivero, Maria A Chavez, David ReverterAbstract:NvCI is a novel exogenous proteinaceous inhibitor of metalloCarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 A resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of Carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.
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the nmr structure and dynamics of the two domain tick carboxypeptidase inhibitor reveal flexibility in its free form and stiffness upon binding to human carboxypeptidase b
Biochemistry, 2008Co-Authors: David Pantojauceda, Joan L. Arolas, Francesc X Aviles, Pascal Garcia, Eva Lopezhernandez, Daniel Padro, Francisco J BlancoAbstract:The structure of the tick carboxypeptidase inhibitor (TCI) and its backbone dynamics, free and in complex with human carboxypeptidase B, have been determined by NMR spectroscopy. Although free TCI has the same overall fold as that observed in the crystal structures of its complexes with metallocarboxypeptidase types A and B, there are structural differences at the linker between the two domains. The linker residues have greater flexibility than the globular domains, and the C-terminal residues are highly flexible in free TCI. Upon formation of a complex with carboxypeptidase B, TCI becomes more rigid, especially at the level of the linker and at the C-terminus, which is inserted into the active site groove of the carboxypeptidase. Solvent exchange rates of the backbone amide protons also show a strong reduction of the local TCI dynamics and a stabilization of its structure upon complex formation. The findings are consistent with a recognition mechanism that primarily involves the C-terminal domain, which adjusts its conformation and that of the linker, thus facilitating complex stabilization by further interactions between the N-terminal domain and an exosite of the carboxypeptidase. This adaptability enables TCI to tune its global conformation for proper interaction with distinct types of Carboxypeptidases by a mechanism of induced fit. Our results provide new information about the structure-function relationship and stability of a molecule with potential biomedical applications in thrombolytic therapy. Furthermore, the plasticity of TCI makes it an ideal scaffold for developing stronger and/or more specific inhibitors directed toward modulating the activity of metalloCarboxypeptidases.
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Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B.
Insect Biochemistry and Molecular Biology, 2006Co-Authors: Àlex Bayés, Josep Vendrell, Monica Rodriguez De La Vega, Francesc X Aviles, Maarten Anthonie Jongsma, Jules BeekwilderAbstract:Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different Carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of Carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.
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The three-dimensional structures of tick carboxypeptidase inhibitor in complex with A/B Carboxypeptidases reveal a novel double-headed binding mode
Journal of Molecular Biology, 2005Co-Authors: Joan L. Arolas, Julia Lorenzo, Grzegorz M Popowicz, Francesc X Aviles, Christian Petro Sommerhoff, Robert Huber, Tad A. HolakAbstract:The tick carboxypeptidase inhibitor (TCI) is a proteinaceous inhibitor of metallo-Carboxypeptidases present in the blood-sucking tick Rhipicephalus bursa. The three-dimensional crystal structures of recombinant TCI bound to bovine carboxypeptidase A and to human carboxypeptidase B have been determined and refined at 1.7 A and at 2.0 A resolution, respectively. TCI consists of two domains that are structurally similar despite the low degree of sequence homology. The domains, each consisting of a short α-helix followed by a small twisted antiparallel β-sheet, show a high level of structural homology to proteins of the β-defensin-fold family. TCI anchors to the surface of mammalian Carboxypeptidases in a double-headed manner not previously seen for carboxypeptidase inhibitors: the last three carboxy-terminal amino acid residues interact with the active site of the enzyme in a way that mimics substrate binding, and the N-terminal domain binds to an exosite distinct from the active-site groove. The structures of these complexes should prove valuable in the applications of TCI as a thrombolytic drug and as a basis for the design of novel bivalent carboxypeptidase inhibitors.
Christian Petro Sommerhoff - One of the best experts on this subject based on the ideXlab platform.
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The three-dimensional structures of tick carboxypeptidase inhibitor in complex with A/B Carboxypeptidases reveal a novel double-headed binding mode
Journal of Molecular Biology, 2005Co-Authors: Joan L. Arolas, Julia Lorenzo, Grzegorz M Popowicz, Francesc X Aviles, Christian Petro Sommerhoff, Robert Huber, Tad A. HolakAbstract:The tick carboxypeptidase inhibitor (TCI) is a proteinaceous inhibitor of metallo-Carboxypeptidases present in the blood-sucking tick Rhipicephalus bursa. The three-dimensional crystal structures of recombinant TCI bound to bovine carboxypeptidase A and to human carboxypeptidase B have been determined and refined at 1.7 A and at 2.0 A resolution, respectively. TCI consists of two domains that are structurally similar despite the low degree of sequence homology. The domains, each consisting of a short α-helix followed by a small twisted antiparallel β-sheet, show a high level of structural homology to proteins of the β-defensin-fold family. TCI anchors to the surface of mammalian Carboxypeptidases in a double-headed manner not previously seen for carboxypeptidase inhibitors: the last three carboxy-terminal amino acid residues interact with the active site of the enzyme in a way that mimics substrate binding, and the N-terminal domain binds to an exosite distinct from the active-site groove. The structures of these complexes should prove valuable in the applications of TCI as a thrombolytic drug and as a basis for the design of novel bivalent carboxypeptidase inhibitors.
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a carboxypeptidase inhibitor from the tick rhipicephalus bursa isolation cdna cloning recombinant expression and characterization
Journal of Biological Chemistry, 2005Co-Authors: Joan L. Arolas, Julia Lorenzo, Francesc X Aviles, A Rovira, J Castella, Christian Petro SommerhoffAbstract:A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits Carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.
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a carboxypeptidase inhibitor from the medical leech hirudo medicinalis isolation sequence analysis cdna cloning recombinant expression and characterization
Journal of Biological Chemistry, 1998Co-Authors: David Reverter, Josep Vendrell, Francesc X Aviles, Francesc Canals, Jeanny Horstmann, Hans Fritz, Christian Petro SommerhoffAbstract:A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metalloCarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like Carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
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a carboxypeptidase inhibitor from the medical leech hirudo medicinalis
1998Co-Authors: David Reverter, Josep Vendrell, Francesc X Aviles, Francesc Canals, Jeanny Horstmann, Hans Fritz, Christian Petro SommerhoffAbstract:A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptasepolymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metalloCarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like Carboxypeptidases, with equilibrium dissociation constants Ki of 0.2‐ 0.4 3 10 29 M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
Josep Vendrell - One of the best experts on this subject based on the ideXlab platform.
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Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B.
Insect Biochemistry and Molecular Biology, 2006Co-Authors: Àlex Bayés, Josep Vendrell, Monica Rodriguez De La Vega, Francesc X Aviles, Maarten Anthonie Jongsma, Jules BeekwilderAbstract:Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different Carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of Carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.
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The Crystal Structure of the Inhibitor-complexed Carboxypeptidase D Domain II and the Modeling of Regulatory Carboxypeptidases
Journal of Biological Chemistry, 2001Co-Authors: Patrick Aloy, Josep Vendrell, Verònica Companys, Miquel Coll, Lloyd D Fricker, Francesc X Aviles, F. Xavier Gomis-rüthAbstract:Abstract The three-dimensional crystal structure of duck carboxypeptidase D domain II has been solved in a complex with the peptidomimetic inhibitor, guanidinoethylmercaptosuccinic acid, occupying the specificity pocket. This structure allows a clear definition of the substrate binding sites and the substrate funnel-like access. The structure of domain II is the only one available from the regulatory carboxypeptidase family and can be used as a general template for its members. Here, it has been used to model the structures of domains I and III from the former protein and of human carboxypeptidase E. The models obtained show that the overall topology is similar in all cases, the main differences being local and because of insertions in non-regular loops. In both carboxypeptidase D domain I and carboxypeptidase E slightly different shapes of the access to the active site are predicted, implying some kind of structural selection of protein or peptide substrates. Furthermore, emplacement of the inhibitor structure in the active site of the constructed models showed that the inhibitor fits very well in all of them and that the relevant interactions observed with domain II are conserved in domain I and carboxypeptidase E but not in the non-active domain III because of the absence of catalytically indispensable residues in the latter protein. However, in domain III some of the residues potentially involved in substrate binding are well preserved, together with others of unknown roles, which also are highly conserved among all Carboxypeptidases. These observations, taken together with others, suggest that domain III might play a role in the binding and presentation of proteins or peptide substrates, such as the pre-S domain of the large envelope protein of duck hepatitis B virus.
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crystal structure of avian carboxypeptidase d domain ii a prototype for the regulatory metallocarboxypeptidase subfamily
The EMBO Journal, 1999Co-Authors: F X Gomisruth, Josep Vendrell, Verònica Companys, Lloyd D Fricker, Francesc X Aviles, Y Qian, Miquel CollAbstract:The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N‐terminal α/β‐hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel‐like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This α/β‐hydrolase subdomain is followed by a C‐terminal 80 residue β‐sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar‐binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory Carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.
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a carboxypeptidase inhibitor from the medical leech hirudo medicinalis isolation sequence analysis cdna cloning recombinant expression and characterization
Journal of Biological Chemistry, 1998Co-Authors: David Reverter, Josep Vendrell, Francesc X Aviles, Francesc Canals, Jeanny Horstmann, Hans Fritz, Christian Petro SommerhoffAbstract:A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metalloCarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like Carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
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a carboxypeptidase inhibitor from the medical leech hirudo medicinalis
1998Co-Authors: David Reverter, Josep Vendrell, Francesc X Aviles, Francesc Canals, Jeanny Horstmann, Hans Fritz, Christian Petro SommerhoffAbstract:A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptasepolymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metalloCarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like Carboxypeptidases, with equilibrium dissociation constants Ki of 0.2‐ 0.4 3 10 29 M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
David Reverter - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of novel metallocarboxypeptidase inhibitor from marine mollusk nerita versicolor in complex with human carboxypeptidase a4
Journal of Biological Chemistry, 2012Co-Authors: Giovanni Covaleda, Francesc X Aviles, Maday Alonso Del Rivero, Maria A Chavez, David ReverterAbstract:NvCI is a novel exogenous proteinaceous inhibitor of metalloCarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 A resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of Carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.
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a carboxypeptidase inhibitor from the medical leech hirudo medicinalis isolation sequence analysis cdna cloning recombinant expression and characterization
Journal of Biological Chemistry, 1998Co-Authors: David Reverter, Josep Vendrell, Francesc X Aviles, Francesc Canals, Jeanny Horstmann, Hans Fritz, Christian Petro SommerhoffAbstract:A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metalloCarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like Carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
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a carboxypeptidase inhibitor from the medical leech hirudo medicinalis
1998Co-Authors: David Reverter, Josep Vendrell, Francesc X Aviles, Francesc Canals, Jeanny Horstmann, Hans Fritz, Christian Petro SommerhoffAbstract:A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptasepolymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metalloCarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like Carboxypeptidases, with equilibrium dissociation constants Ki of 0.2‐ 0.4 3 10 29 M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
Ib Svendsen - One of the best experts on this subject based on the ideXlab platform.
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the primary structure of carboxypeptidase s3 from penicillium janthinellum ibt 3991
FEBS Letters, 1995Co-Authors: Ib Svendsen, Estera Szwajcer DayAbstract:The complete amino acid sequence of penicillopeptidase S3, a serine carboxypeptidase isolated from Penicillium janthinellum IBT 3991, has been determined. The enzyme consists of 481 amino acids arranged in a single polypeptide chain. Six glycosylation sites were established in positions 41, 218, 256, 326, 384 and 392. The molecule contains six cysteinyl residues among which disulfide bridges was established between Cys-71-Cys-333 and Cys-233-Cys-289. Carboxypeptidase S3 is homologous to carboxypeptidase PEPF (or carboxypeptidase I) from Aspergillus niger (67% identical positions). It is proposed that these enzymes form a separate sub-family among the serine Carboxypeptidases.
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the primary structure of carboxypeptidase s1 from penicillium janthinellum
FEBS Letters, 1993Co-Authors: Ib Svendsen, Theo Hofmann, James A Endrizzi, James S Remington, Klaus BreddamAbstract:The complete amino acid sequence of carboxypeptidase Sl from Penicillium janthinellum has been determined by N-terminal sequencing of the reduced and vinylpyridinated protein and of peptides obtained by cleavage with cyanogen bromide, iodosobenzoic acid, hydroxylamine, endoproteinase LysC, endoproteinase AspN and Glu-specific proteinase from B. licheniformis. The enzyme consists of a single peptide chain of 433 amino acid residues and contains 9 half-cystine residues and one glycosylated asparagine residue. A comparison to other Carboxypeptidases shows that the enzyme is homologous to carboxypeptidase-Y and carboxypeptidase-MIII from malt. Specificity and binding of substrates are discussed from a three-dimensional model based on the known structure of carboxypeptidase-Y from Saccharomyces cereviciae and carboxypeptidase II from wheat.