Engineering Enzymes and Antibodies
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Engineering Enzymes and Antibodies Donald Hilvert Phenomenal rate accelerations, exacting selectivities, and mild reaction conditions characterize biocatalysis and have generated considerable interest in nonbiological applications of enzymes. As a result of greater availability and purity, these molecules are now being used increasingly in organic synthesis to prepare complex natural products and novel materials.1 They are also being exploited industrially in the production of food additives, pharmaceuticals, and fine chemicals.2 Unfortunately, for many commercial applications, natural enzymes may be unsuitable. They may be unstable or difficult to isolate; they may function poorly at the temperatures, pH's, and substrate concentrations needed for reaction; or they may lack appropriate specificity. For many chemical reactions, a natural enzyme may not even exist. For example, natural biocatalysts for the synthetically valuable Diels-Alder cycloaddition have yet to be discovered. Protein engineering has the potential to significantly expand the range and utility of biocatalysts. The ability to design and synthesize a protein with tailored catalytic activity and specificity on demand would replace the long and often tedious search for natural enzymes having properties appropriate for a given application. This capability would also provide the means for creating catalysts that lack physiological counterparts. To devise effective and general strategies for generating new protein catalysts, a fundamental understanding of the processes of molecular recognition and catalysis is essential. The basic principles of enzymic catalysis are well known.3 Each enzyme has a distinct three-dimensional structure that binds substrate molecules in a highly specific fashion, thereby facilitating their transformation into products. Large rate accelerations result from selective stabilization of the rate-limiting transition state(s) along the reaction coordinate. This stabilization
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is achieved in part by having an active site that is complementary in shape and charge to the structure of the high-energy transition state. More specifically, enzymes reduce reaction barriers by exploiting binding energy to destabilize substrate molecules through strain or to compensate for unfavorable losses of rotational and translational entropy. In addition, many enzymes have arrays of functional groups to effect acid-base, nucleophilic-electrophilic, oxidation-reduction, or other mechanisms of catalysis. A binding site with high affinity for potential substrates is thus essential to any strategy to develop new enzymes. One day it may be possible to synthesize desired enzymes directly by predicting and then synthesizing their correct amino acid sequence; but this approach is still in its infancy. Today, well-defined binding sites in existing enzymes or antibodies provide an alternative and convenient starting point for building tailored active sites. It is the purpose of this article to discuss the use of site-directed mutagenesis and haptenic transit
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