Mechanistic Investigations of Ketosteroid Isomerase

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Enzymes are the macromolecules within cells that accelerate the specific chemical transformations needed for life. Much of biochemistry research in the mid-20th century focused on identifying the individual reactions that are found in biology and how these reactions come together to make metabolic pathways. As these reactions and pathways were discovered, focus shifted to the specific enzymes responsible for each reaction. Much of this work involved necessary basic characterization, and studies by many investigators were able to describe enzyme-catalyzed reactions in terms of chemical transformations that involve enzyme-bound cofactors (which are often vitamins or derived from vitamins) and specific amino acid side chains to facilitate certain types of chemical transformations. The majority of work in enzymology in the second half of the 20th century focused on determining these chemical pathways for the various transformations catalyzed by enzymes. This phase of research in biochemistry and enzymology has been highly successful.A second fundamental question is: What are the mechanisms that enzymes use to achieve their enormous rate enhancements and exquisite specificities? Speculations and models about the origin of the catalytic power of enzymes accompanied the above studies, but did not advance to attain the near-closure that the chemical mechanisms have. Important generalizations have been made, such as the use of binding energy from interactions within enzyme active sites to facilitate catalysis, thereby linking specificity and catalysis. Nevertheless, there are multiple, competing descriptions of enzymatic catalysis. One measure of the degree of closure in an academic area is the descriptions of that area in textbooks. For the mechanisms of enzyme specificity and rate enhancements, the treatments vary greatly from text to text and often provide present anecdotal information about particular enzymes or the names of models without clear description.A goal of this project is to provide in-depth and fundamental understanding of how enzymes achieve their rate enhancements. This field is ripe for advances because of the powerful tools that have been developed over the past decades. The project will utilize traditional approaches but also combine them with additional chemical, physical, and computational tools. Such a broad-based approach will be required to attain an integrated understanding of enzyme energetics and physical properties and this in turn will require intense focus on a system that is amenable to such an assault. Prior research has shown that the enzyme ketosteroid isomerase (KSI) is ideal; it is highly tractable and amenable to plethora of approaches. Two modes of catalysis that are used by KSI and common to many enzymes will be explored: general acid/base catalysis (GABC) and transition state complemetarity (via a so-called oxyanion hole).An important component of this project is introducing high school students to careers in science to increase the broadening participation of underrepresented groups. A workshop will be initiated for high school students that will include interactions and discussions with graduate students. These workshops are intended to inform students with a clear interest in science how to train for a scientific career. The students will receive advice and mentorship through this program to cultivate the students interest in science. The project will continue to promote the training of students at all levels through experimental research and curriculum development.