The overall goal is to determine the structural basis for catalysis by catalytic antibodies (abzymes). The understanding of the catalysis will be broadened by determining how structural differences between abzymes and natural enzymes influence specificity, kinetics, mechanism, and reaction profile. To facilitate such comparisons, this research will focus on the aldol reaction, which is critical for carbon-carbon bond formation in organic and bioorganic chemistry. This reaction was selected for study because of the development of a growing family of successful aldolase abzymes has been demonstrated in the synthesis of anti-cancer agents, and as potential pro-drug activators in cancer therapy. The goal is to investigate antibody and enzyme aldolase complexes with inhibitors or substrate analogs, and design structure-based mutants. The introduction of co-factors and redesign of inhibitors will expand our understanding of catalysis. Related to these objectives, it is intended to study the evolution of catalysis in abzymes, beginning with the progenitor germline antibodies. Structural comparisons with the germline will provide valuable insight on how the accumulation of random somatic mutations can lead to the increase in intrinsic binding energy that is necessary for catalysis. This work will provide a unique opportunity to compare mechanisms employed by nature and matured on an evolutionary timescale with antibody catalysts generated by immune response within a few months. The individual objectives are: Crystal structures of aldolase antibody 33F12 in complex with different inhibitors and substrates Crystal structures of new aldolase antibodies with their corresponding inhibitors or substrate analogs Crystal structures of engineered and mutant aldolase antibodies Ultra-high resolution aldolase enzyme structure Evolution of catalysis and specificity Crystal structures of antibodies generated by reactive immunization catalyzing other transformations Structure of catalytic antibodies generated and selected by phage display Structural information will be obtained by x-ray crystallography, and structures will be performed to determine mechanisms of catalysis and binding. The structural work can be harnessed to provide better catalysts for synthesis organic chemistry and synthesis of new drugs.
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