Making New Enzymes from Old

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9814455The goal of this research project is to develop and explore the applications of an experimental approach that will allow a systematic alteration of the properties of existing enzymes. Through a method called site specific modulation the investigators propose the alteration of selected amino acids in an enzyme by an approach that combines the strengths of oligonucleotide-directed mutagenesis (absolute site specificity) and chemical modification (introduction of a wide range of structural analogs). A range of amino acid analog reagents will be prepared to apply this method to several specific enzymes. The kinetics of carbonic anhydrase will be altered by modifications in both the zinc ligands and in the identity of the proton shuttle group. The substrate specificity and stereospecificity of malate dehydrogenase will be tuned by the selective introduction of unnatural amino acids into the active site. Finally, these investigators plan to use the knowledge that has been gained from alterations in the metal binding site of carbonic anhydrase to design and introduce a new metal ion binding site and commensurate new catalytic activity into malate dehydrogenase.Enzymes, natures' catalysts, control and accelerate all chemical reactions in living systems. Researchers have not yet succeeded in designing completely new catalysts that can mimic the remarkable efficiency of enzymes. These investigators will be exploring a method that has the promise of expanding the scope of existing enzymes, thus allowing them to catalyze new reactions. Designing altered enzymes that can specifically be tuned to accelerate targeted chemical reactions can have broad new commercial and basic applications.