9321371 Drueckhammer This project will use synthetic Coenzyme A (CoA) analogs to study fundamental mechanistic questions in enzyme catalyzed reactions of CoA esters. This work is based on methodology developed in the P.I.'s lab for the convenient synthesis of CoA/CoA ester analogs. The largest effort will be directed at probing the involvement of enolate and/or enol intermediates in enzyme catalyzed reactions of acetyl-CoA and other CoA esters. Stable analogs of the enolate and enol forms of acetyl-CoA will be prepared, and studied as inhibitors of citrate synthase. The protonation state of these analogs in isotopically enriched form bound to the enzyme will be studied by NMR and in some cases FTIR. Similar studies will be conducted with related enzymes, including malate synthase, crotonase, and acetyl-CoA dependent acetyltransferases. This project is expected to provide new insights into the enzyme catalyzed reactions of CoA esters and general principles of enzyme reaction mechanisms. %%% Dr. Drueckhammer will study the reactions of enzymes which require Coenzyme A for their function. Enzymes are catalysts which carry out chemical reactions in biological systems. Understanding how these enzymes function is at the heart of understanding biological processes at a fundamental chemical level. About 4% of all enzymes require coenzyme A (CoA) for their catalytic activity. CoA serves as a reactant or a reactant carrier in these enzymatic reactions. CoA is also synthesized in biological systems by a series of enzyme catalyzed reactions. In previous work in this lab, a method was developed for using the enzymes which make CoA to make altered forms (analogs) of natural CoA. This research will use this method to make a series of CoA analogs designed to serve as probes for studying the reactions of CoA utilizing enzymes. This will help reveal how enzymes form chemical bonds to make complex biological molecules from simple building blocks, and how enzymes break down com plex compounds in food to produce biological energy. ***
