This project is aimed at further developmental of the synthesis of analogs of Co-enzyme A (CoA) and of CoA esters using the enzymes which catalyze the final steps in CoA biosynthesis. About 4% of all enzymes use CoA or a CoA ester as substrate, including many enzymes of biomedical and pharmaceutical importance. The availability of new and novel analogs of these compounds will facilitate studies of mechanism and inhibition of CoA utilizing. A major effort of this project will be directed at substantially increasing the scale of CoA analog synthesis by improving the source and thus quantity of dephospho-CoA kinase, which catalyzes the final enzymatic step in the synthetic method. One approach will be the use of the enzyme APS kinase, for which an over-expression system is available, as a dephospho-CoA kinase. APS kinase has been previously reported to exhibit dephospho-CoA kinase activity. Another approach will be the purification of dephospho-CoA kinase from Corynebacterium ammoniagenes for obtaining N-terminal amino acid sequence information to facilitate cloning of the dephospho-CoA kinase gene. This should provide the optimal solution for over-expression of dephospho-CoA kinase in the longer term. Another focus will be the improvement of the final aminolysis step in the synthesis of CoA analogs from a recently developed second generation CoA analog synthon. In one approach, a carboxylate group will be incorporate into the amine nucleophile to catalyze the aminolysis reaction. In a second approach, a more reactive N-alkylhydroxylamine nucleophile will be used, followed by reductive removal of the N-hydroxyl group from the hydroxamic acid product. The third major focus of this project will be the extension of synthetic methodology to analogs of CoA esters for which analogs have not been previously prepared. Special attention will be given to synthesis of analogs of myristoyl-CoA and palmitoyl-CoA which will designed to inhibit protein fatty acylation reactions. These analogs will be used in studies of the mechanism and enzymology of protein acylation. This work is expected to contribute to the understanding of control mechanisms of cell growth and differentiation related to cancer due to the vital role of protein fatty acylation in signal transduction mechanisms.
