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Catalytic antibodies

Catalytic antibodies. Classes of biological catalysts Enzymes, RNA and antibodies Antibodies are proteins (immunoglobulins) that circulate in the blood and function as defense against foreign molecules Antibodies form specific stable complexes with antigens ( anti body gen erators).

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Catalytic antibodies

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  1. Catalytic antibodies Classes of biological catalysts • Enzymes, RNA and antibodies • Antibodies are proteins (immunoglobulins) that circulate in the blood and function as defense against foreign molecules • Antibodies form specific stable complexeswith antigens (antibody generators)

  2. Catalytic antibodies Jencks 1969 • If complementarity between the active site and the transition state contribute significantly to enzyme catalysis, it should be possible to synthesise an enzyme by constructing such an active site • Synthesis of a hapten which resembles the transition state of a given reaction

  3. Catalytic antibodies Structure of immunoglobulins • IgG (Figure 8.1) Y shaped • Antigen binding site located on tip of the Y arms • Fab arms connected to Fc stem domain via a flexible hinge • Two identical H chains and L chains • Each chain has a N-terminal VHand VL domain that together form the antigen binding site • Covalent disulfide bridges between H and H-L

  4. Catalytic antibodies Structure and function of IgG • CH1, CH2, CH3 responsible for effector functions • Loops of VH and VL involved in antigen binding • Three hypervariable stretches of amino sequence present in both chains • Complementary-determining regions (Fig. 8.2)

  5. Catalytic antibodies Antigen binding • Very tight binding (Kd = 10-6 - 10-12 M) Binding energy provided by: • Hydrophobic and electrostatic interactions • Large contact area (600 Å2) • Interaction with about 7 AA residues of antigen

  6. Catalytic antibodies Antibody technology • Affinity maturation: IgM  polyclonal IgG • Need for identical monoclonal antibodies • Fusion of B-cells from the spleen with cancer (myeloma) cells, resulting in hybridoma cells • Köhler and Milstein 1976

  7. Catalytic antibodies Generation of catalytic MABs Fig. 8.3 • Antigen development Design of transition state analog Hapten synthesis Hapten-carrier protein conjugate • Production of hybridoma cells • Preparation of MABs • Selection for catalysis

  8. Catalytic antibodies Hybridomatissue culture • Azaserine and hypoxanthine: alternative route for purine biosynthesis by hybridoma cells • Cell line selection by limiting dilution • Propagation in mice (ascites) or tissue culture • Time-consuming and expensive • Not easily accessible to genetic engineering • Human MABs: ethical aspects

  9. Catalytic antibodies Phage display libraries Fig. 8.4 • Display of antibody Fv fragments on surface of bacteriophage • Construction of libraries by amplification of antibody genes encoding for VH and VL fragments • Gene fusion with phage coat protein gene • Multiplication of fusion product in bacteria

  10. Catalytic antibodies Phage selection Fig. 8.5 • Hapten coated to surface • Phage binding • Elution by competition with free hapten • Multiplication and purification • Panning procedure (repeated four times) • Cloning of selected antibody DNA • Easy accessible to genetic manipulation

  11. Catalytic antibodies Antibody catalysis • Design of stable transition state analog • Ester hydrolysis: tetrahedral intermediate • Scheme 8.1 Phosphonate anologs • MM kinetics, inhibition by TS-analog, narrow substrate specificity, product inhibition

  12. Catalytic antibodies Antibody catalysis • To date more than 100 reactions reported • Ester bond hydrolysis, -elimination, C-C bond formation, cyclization, decarboxylation, transesterification, redox reactions • Diels-Alder reactions, oxy-Cope rearrangement • Scheme 8.2 • Potential for asymmetric synthesis

  13. Catalytic antibodies Transition state theory • 1 / KTS = (kcat / kuncat) / Km • 1 / KTS : association constant for transition state to the catalytic antibody • Perfect TS-analog: KTS = Ki hapten • kcat / kuncat = Km / Ki

  14. Catalytic antibodies Structure-function relationship • Scheme 8.3 • Cloning of variable domains and expression as single chain antigen binding protein (scFv) • AA sequence: molecular modelling of CDR’s antibody binding site • Role of Arg and His in catalysis and formation of covalent acyl-antibody intermediate

  15. Catalytic antibodies Catalytic potential of antibodies • Low kcat values, rate enhancement 103 fold • Stabilisation of TS with 30-40 kJ / mol • Enzymes: 60 - 130 kJ / mol • The best catalytic antibodies approach the worst enzymes in catalytic power (Fig. 8.7) • Electronic constraints, passive binding • Enzyme dynamics: increase of catalytic efficiency

  16. Catalytic antibodies Application of antibodies • Industrial catalyst, analytical tool, drug • Requires specific development • Expensive • Fine chemicals, resolution of racemates • Non natural cofactors, biosensors etc. • Economical, technical and fundamental restrictions

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