Catalytic Antibody (catalytic + antibody)

Distribution by Scientific Domains
Chemistry100%

Selected Abstracts

Theoretical Study of Catalytic Efficiency of a Diels,Alderase Catalytic Antibody: An Indirect Effect Produced During the Maturation Process

CHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008
Sergio Martí Dr.
Abstract The Diels,Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39,A-11, that catalyze a Diels,Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate,protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate. [source]

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity

JOURNAL OF MOLECULAR RECOGNITION, Issue 5 2008
Rui Huo
Abstract In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t -butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH,+,VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni2+ -immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0,3.0,mg of proteins from 1,L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288,U/µmol. Copyright © 2008 John Wiley & Sons, Ltd. [source]

From cofactor to enzymes.

THE CHEMICAL RECORD, Issue 6 2001
-phosphate-dependent enzymes, The molecular evolution of pyridoxal-
Abstract The pyridoxal-5,-phosphate (vitamin B6)-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B6 enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B6 enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the , family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller , family (tryptophan synthase , as the prototype enzyme) is structurally and functionally more homogenous. Both the D -alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5,-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B6 enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5,-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B6 enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5,-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:436,447, 2001 [source]

On the Generation of Catalytic Antibodies by Transition State Analogues

CHEMBIOCHEM, Issue 4 2003
Montserrat Barbany
Abstract The effective design of catalytic antibodies represents a major conceptual and practical challenge. It is implicitly assumed that a proper transition state analogue (TSA) can elicit a catalytic antibody (CA) that will catalyze the given reaction in a similar way to an enzyme that would evolve (or was evolved) to catalyze this reaction. However, in most cases it was found that the TSA used produced CAs with relatively low rate enhancement as compared to the corresponding enzymes, when these exist. The present work explores the origin of this problem, by developing two approaches that examine the similarity of the TSA and the corresponding transition state (TS). These analyses are used to assess the proficiency of the CA generated by the given TSA. Both approaches focus on electrostatic effects that have been found to play a major role in enzymatic reactions. The first method uses molecular interaction potentials to look for the similarity between the TSA and the TS and, in principle, to help in designing new haptens by using 3D quantitative struture,activity relationships. The second and more quantitative approach generates a grid of Langevin dipoles, which are polarized by the TSA, and then uses the grid to bind the TS. Comparison of the resulting binding energy with the binding energy of the TS to the grid that was polarized by the TS provides an estimate of the proficiency of the given CA. Our methods are used in examining the origin of the difference between the catalytic power of the 1F7 CA and chorismate mutase. It is demonstrated that the relatively small changes in charge and structure between the TS and TSA are sufficient to account for the difference in proficiency between the CA and the enzyme. Apparently the environment that was preorganized to stabilize the TSA charge distribution does not provide a sufficient stabilization to the TS. The general implications of our findings and the difficulties in designing a perfect TSA are discussed. Finally, the possible use of our approach in screening for an optimal TSA is pointed out. [source]

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity

JOURNAL OF MOLECULAR RECOGNITION, Issue 5 2008
Rui Huo
Abstract In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t -butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH,+,VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni2+ -immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0,3.0,mg of proteins from 1,L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288,U/µmol. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Structure of a pseudomerohedrally twinned monoclinic crystal form of a pyridoxal phosphate-dependent catalytic antibody

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2005
Béatrice Golinelli-Pimpaneau
The purification, crystallization and structure determination at 2.3,Ĺ resolution of the complex of the pyridoxal-5,-phosphate (PLP) dependent catalytic antibody 15A9 with a phosphopyridoxyl- l -alanine (PPL- l -alanine) substrate analogue are described. The crystal belongs to space group P21, with two molecules in the asymmetric unit related by non-crystallographic symmetry. The unit-cell parameters are a = 63.5, b = 81.7, c = 79.3,Ĺ and , is fortuitously 90°. Refinement of the structure converged at unacceptably high R factors. Although the traditional analysis of intensity distribution did not indicate twinning, pseudomerohedral twinning was revealed by a newer test based on local intensity differences [Padilla & Yeates (2003), Acta Cryst. D59, 1124,1130]. When the potential twinning operator was included in SHELX, the structure could be satisfactorily refined with a twinning fraction of 0.46, indicating a nearly perfect hemihedrally twinned crystal. One of the active sites is occupied by the phosphopyridoxyl- l -alanine ligand, while one iodide ion mimics the cofactor phosphate group in the other. Four other iodide ions are present in the structure: two are involved in specific intermolecular contacts and two dictate the conformation of the CDRH3 loop in each molecule. [source]

On the Generation of Catalytic Antibodies by Transition State Analogues

CHEMBIOCHEM, Issue 4 2003
Montserrat Barbany
Abstract The effective design of catalytic antibodies represents a major conceptual and practical challenge. It is implicitly assumed that a proper transition state analogue (TSA) can elicit a catalytic antibody (CA) that will catalyze the given reaction in a similar way to an enzyme that would evolve (or was evolved) to catalyze this reaction. However, in most cases it was found that the TSA used produced CAs with relatively low rate enhancement as compared to the corresponding enzymes, when these exist. The present work explores the origin of this problem, by developing two approaches that examine the similarity of the TSA and the corresponding transition state (TS). These analyses are used to assess the proficiency of the CA generated by the given TSA. Both approaches focus on electrostatic effects that have been found to play a major role in enzymatic reactions. The first method uses molecular interaction potentials to look for the similarity between the TSA and the TS and, in principle, to help in designing new haptens by using 3D quantitative struture,activity relationships. The second and more quantitative approach generates a grid of Langevin dipoles, which are polarized by the TSA, and then uses the grid to bind the TS. Comparison of the resulting binding energy with the binding energy of the TS to the grid that was polarized by the TS provides an estimate of the proficiency of the given CA. Our methods are used in examining the origin of the difference between the catalytic power of the 1F7 CA and chorismate mutase. It is demonstrated that the relatively small changes in charge and structure between the TS and TSA are sufficient to account for the difference in proficiency between the CA and the enzyme. Apparently the environment that was preorganized to stabilize the TSA charge distribution does not provide a sufficient stabilization to the TS. The general implications of our findings and the difficulties in designing a perfect TSA are discussed. Finally, the possible use of our approach in screening for an optimal TSA is pointed out. [source]

Theoretical Study of Catalytic Efficiency of a Diels,Alderase Catalytic Antibody: An Indirect Effect Produced During the Maturation Process

CHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008
Sergio Martí Dr.
Abstract The Diels,Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39,A-11, that catalyze a Diels,Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate,protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate. [source]