The bacterial fatty acid biosynthesis (FASII) pathway is a promising target for antibacterial drug discovery and current research is focused on elucidating the substrate specificity of the b-ketoacyl-ACP synthase (KAS) enzymes in this pathway, identify the interactions between the components of the FASII pathway and discovering the unknown dehydratase, and studying the interaction of current enzyme inhibitors with FASII components in whole cells. Substrates are shuttled between the FASII enzymes by acyl carrier protein, a phosphopantetheinylated protein with a MW of 13 kDa in Mycobacterium tuberculosis and 7 kDa in Escherichia coli. While the KAS enzyme(s) involved in fatty acid elongation (KASI and KASII) utilize ACP-based substrates, the priming KAS enzyme (KASIII) is initially acylated by an acyl-CoA substrate. In order to compare and contrast the specificity of KASI/II and KASIII for the substrate carrier, we have synthesized substrates based on phosphopantetheine, CoA, ACP and ACP peptide mimics, and explored their interactions with the KASI enzyme from M. tuberculosis (KasA) as well as with the KASI and KASII enzymes from E. coli (ecFabB and ecFabF). Highlights of this work include the observation that a 14 residue malonyl-phosphopantetheine peptide can replace malonyl-ACP as the acceptor substrate in the ecFabF reaction. In addition, it was observed that the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the donor substrate, and that, provided this requirement was met, variation in the acceptor carrier had only had a small effect on kcat/Km. For the KASI enzymes it is proposed that the binding of ACP results in a conformational change toward a more accessible open form, thus facilitating the binding of the second substrate. Studies involving the components of the FASII system also involve the utilization of techniques to identify protein-protein interactions. Several data in the literature suggest the existence of protein-protein interactions within the FASII pathway. Efforts we employed toward characterizing this noncovalent complex include the development of a photoprobe by attaching a benzophenone group to the prosthetic PPant arm of AcpM (B4M-AcpM), the development of a bioorthogonal w-azido fatty acid probe to determine fatty acylated proteins in the cell and lastly, the incorporation of fluorescent tags into InhA in order to quantitate an interaction to KasA. Preliminary results indicated a covalent interaction between B4M-AcpM and proteins in M. smegmatis cell lysate. However, future optimizations of this method along with the incorporation of w-azido fatty acids into cells and the appropriate position to insert fluorescent probes into InhA are required to definitely identify physiologically relevant protein-protein interactions. In separate studies a novel drug-target identification method has been developed that relies on the incorporation of short-lived isotopes such as carbon-11 into the drug of interest. Radiolabeling is accomplished without altering the structure of the drug and size exclusion chromatography (SEC) is used to fractionate proteins following exposure to the radiotracer. Proof of concept experiments have so demonstrated interactions between the FabI enoyl-ACP reductase and the front-line tuberculosis drug 11C-isoniazid as well as with a carbon-11 labeled alkyl diaryl ether FabI inhibitor.