Recent experiments have demonstrated the feasibility of using transition state analogue inhibitors to generate monoclonal antibodies that catalyze chemical transformations. This strategy provides us with unique tools for investigating chemical catalysis in proteins, as well as the means to create entirely new enzymatic activities for use in research, medicine and industry. Here, a combined chemical and genetic approach is proposed to study and improve the properties of one such antibody, a catalyst for the stereospecific conversion of chorismate into prephenate. Specifically, we plan to clone and sequence the genes for the chorismate mutase antibody 1F7 and engineer them for intracellular expression in a strain of the yeast Saccharomyces cerevisiae that lacks the normal gene for chorismate mutase (essential for tyrosine and phenylalanine biosynthesis). We will optimize the levels of expression and assembly of 1F7 to reconstitute the defective biosynthetic pathway and thereby allow the host cells to grow in the absence of exogenously added tyrosine and phenylalanine. We will also exploit this system to perform classical genetic selection on the antibody in order to improve its chemical efficiency. Thus, the host cells containing first-generation 1F7 will be grown under conditions such that only those cells survive that produce an improved version of the catalyst by random mutation. In these experiments we will be monitoring, in essence, the evolution of a protein catalyst not previously optimized by natural selection. Detailed characterization of the resulting 1F7 variants will enhance our understanding not only of how enzymes work but how they come to be the way they are. This combination of chemistry, immunology and molecular genetics provides a general strategy for developing highly efficient protein catalysts for biomedical and industrial applications. It is being funded by the Division of Chemistry and Molecular Biosciences through the Chemistry of Life Processes Initiative.
