Mechanisms of biosynthesis of long chain fatty acids in eukaryotic cells are the focus of our investigation. The fatty acid synthase is a key enzyme in palmitate synthesis and the overall energy metabolism of humans. Diet and hormones regulate the synthesis of the synthase and its mRNA. Elucidation, therefore, of the expression of this enzyme and its mechanism of action is essential to our knowledge of fatty acid and energy metabolism in humans in normal and disease conditions such as obesity and diabetes. Fatty acid synthases of animal tissues and yeast are complexes of multifunctional proteins. The structure-function relationships of component enzymes and the organization of protein subunits in native synthases will be studied in detail. The entry of the substrates acetyl and malonyl in the process of chain elongation and the specificity of the acetyl and malonyl transacylases will be established. The involvement of a common serine-OH in the formation of oxyesters and the order and kinetics of transfer of acetyl and malonyl moieties from CoA to serine-OH, pantetheine- SH, and cysteine-SH will be determined by using chromophoric substrates and native synthase as well as active-site labeled synthases. The peptides containing the active serine, cysteine and pantetheine will be isolated and their amino acid sequence will be established. These sequences will be vital in isolating the cDNA coding for the structural protein and in identifying these sites on the deduced amino acid sequence of the protein subunit. Because of the large size of synthase mRNA (12-16 kb), known cDNA fragments coding for the thioesterase component of the synthase, as well as oligonucleotides synthesized according to known protein sequences, will be used to synthesize, screen and identify cDNA segments from synthase mRNA. The availability of cDNA coding for the entire synthase should lead to a complete sequence of the synthase protein and to physical, kinetic and functional studies of the protein altered by site-directed mutagenesis. This will be an exceptionally powerful approach to understanding the physiological relevance and structure-function relationships of these complex multifunctional proteins. cDNA coding for component enzymes or combinations thereof will be cloned into E. coli or yeast expression vectors. The proteins produced will be isolated, their enzymatic and physical properties established, and attempts will be made to reconstitute the fatty acid synthesizing system. Methodologies to be employed include enzymology, protein chemistry, lipid chemistry, nucleic acid chemistry and molecular and cellular biology.
