The majority of research in the field of catalytic antibodies has been directed toward the discovery of new chemical reactions that can be catalyzed by antibodies. While it is now clear from this work that a large number of reactions are amenable to antibody catalysis, the standard first-generation catalysts reported in these studies have poor activity compared to natural enzymes. We are interested in developing new methods that produce catalytic antibodies with higher activity, and in understanding structure/function relationships in antibody catalysis that will allow higher activity variants of first-generation catalysts to be engineered. We recently generated a hydrolytic antibody that catalyzes enantioselective amino acid ester and amide hydrolysis. This is one of the best catalytic antibodies generated to date for esterase activity (kcat=4 sec-1), and our preliminary mechanistic studies indicate that the antibody uses a mechanism of catalysis that is similar to serine proteases. In addition, we have solved the 3-dimensional crystal structure of this antibody with bound hapten at 2.5 Angstroms resolution (in collaboration with Robert Fletterick's group at UCSF). The active site structure is consistent with our kinetic data and further supports the mechanistic analogy with serine proteases. With this system, we have an unparalleled opportunity to delineate the important structure/function relations for hydrolytic antibodies and create more active variants of our first- generation catalyst. This proposal describes mechanistic studies, amidase kinetic analyses, mutagenesis studies, and an amidase-based functional selection in hybridomas that are designed around our unique catalytic antibody system. The mechanistic experiments are aimed at proving the existence of a covalent intermediate in catalysis and identifying whether intermediate formation or breakdown is rate-limiting. We propose to develop a more sensitive radioisotope assay to accurately measure the kinetics of antibody-catalyzed amide hydrolysis. Our mutagenesis studies are designed to identify catalytically important residues in the antibody active site, and construct more active antibody mutants that are improved in both kcat and KM. With our functional selection in hybridomas we intend to probe the immunoglobulin repertoire for antibodies that are better at catalyzing amide hydrolysis. The results of this research could significantly advance the field of catalytic antibodies by defining the limits of catalytic activity that can be incorporated into an immunoglobulin combining site and providing new selection-based methods for generating catalytic antibodies with higher levels of activity.
