The enzyme CTP:phosphocholine cytidylyltransferase (CT) is a critical enzyme for the biosynthesis of phosphatidylcholine (PC), a major lipid component of mammalian cellular membranes. In a cell, translocation of CT from a soluble pool to the membrane surface results in enzyme activation. Prior work has established that CT from rat is modular and comprises several functional domains, including a catalytic domain and a lipid-binding domain. Recent work, however, has localized the lipid-binding region of CT from the nematode C. elegans to a 21 amino acid stretch postulated to form an amphipathic alpha helix. The proposed research will investigate the mechanism of lipid activation of CT at a molecular level using C. elegans CT as a model enzyme. The specific aims are to 1) identify the hydrophobic amino acid side chains of the putative amphipathic alpha helix dictating interaction with cellular membranes, 2) correlate amino acid composition of the amphipathic helix to lipid specificity, and 3) determine the mechanism of inhibition of CT in the absence of activating lipids. This study will utilize site-directed mutagenesis to alter individual amino acids, facilitating identification of individual chemical functional groups that are critical for lipid activation of CT. Completion of the research project will enable development of a refined model of the mechanism of regulation of the members of the CT family. A detailed understanding of the natural mechanism of CT regulation and the molecular determinants for interaction of CT with activating lipid molecules is essential for the eventual development of synthetic inhibitors of CT and therapeutic agents that alter CT activity in vivo.