9722936 Drueckhammer The objective of this project is to study the mechanisms of catalysis by Coenzyme A (CoA) ester-utilizing enzymes using synthetic CoA analogs as probes. Further studies will be directed at investigation of the mechanism and the nature of the intermediate in the reaction of citrate synthase. Additional acetyl-CoA enol and enolate analogs will be prepared and studied as probes of citrate synthase, including analogs designed to permit direct observation of a stable enzyme-enol or enzyme-enolate complex. Further studies will be conducted on the mechanism of acetyl-CoA dependent acetyltransferases using newly prepared electrophilic ketone analogs of acetyl-CoA. These studies may provide new insights into the structure of the enzyme-intermediate and enzyme-transition state complexes and the means by which the acetyltransferases stabilize these species. Analogs of CoA will be prepared having the amide bond nearest the thiol group altered. These analogs will be used in initial investigations of the role of functional groups remote from the thiol group of CoA in stabilization and destabilization of ground state and transition state complexes in enzymatic reactions. Enzymes catalyze almost all of the biochemical reactions that occur in living organisms and it is estimated that about 4% of all enzymes require coenzyme A (CoA) or a derivative of CoA in their reactions. These include enzymes of industrial, biotechnological, and biomedical importance. This project is directed at understanding the underlying chemistry involved in the function of these enzymes. This work will provide new insights into how enzymes work, the basis for their malfunction, and the means by which their function may be modulated or altered.
