Investigation of the role of pre-organized binding pockets in protein catalysis

Catalytic antibody systems have been applied for investigating the role of pre-organized binding pockets in protein catalysis. Specifically, two families of catalytic antibodies have been explored in detail.; A family of decarboxylase antibodies that catalyze the decarboxylation of 5-nitro-3-carboxy-1,2-benzisoxazole have provided insight into the role of medium effects in enzymatic catalysis. Antibodies raised against hapten with a large, hydrophobic group exhibited modest activities (Chapter 2). Much more efficient decarboxylase antibodies were raised against a charged naphthalene disulfonate derivative. The crystal structure of one such antibody, 21D8, revealed that a melange of polar and nonpolar sites are more effectively exploited to achieve both substrate binding and acceleration of the reaction (Chapter 3). This structural information was also used to design site-directed mutants for detailed mechanistic characterization of 21D8, which provided insight into ligand binding modes (Chapter 4). In addition, structural analysis also revealed a striking similarity between the decarboxylase and a series of unrelated hydrolytic antibodies, suggesting an inherent limitation in the structural diversity provided by immune response against a variety of haptens. This structural similarity then led to the discovery of weak hydrolytic activity in 21D8 (Chapter 5), underscoring the relatively subtle changes that are involved in fine-tuning the immunoglobulin pocket for recognition of different ligands and for catalysis of different reactions.; Another antibody family that catalyzes a beta-elimination of a variety of benzisoxazole derivatives has provided a model for studying the effects of the precise positioning of catalytic groups within the binding pocket on the efficiency of the chemical catalysis achieved by enzymes. Successful cloning and sequencing of the two most active antibody catalysts, 34E4 and 35F 10, resulted in the construction of a plausible structural model (Chapter 6). Along with a number of residues that may line the binding pocket, the model identifies a single residue (GluH50) as the likely catalytic base, thereby providing specific targets for site-directed mutagenesis and detailed mechanistic characterization. Efficient production of 34E4 as a chimeric Fab has been achieved, which should greatly facilitate future structural studies.