The long term objective of this program is to elucidate the structure, mechanism of action, basis for specificity and evolutionary origins of animal lipogenic enzymes. In animals, the seven enzymes involved in de novo fatty acid synthesis from malonyl-CoA are integrated into two identical multifunctional polypeptides, which constitute the fatty acid synthetase (FAS). The growing fatty-acyl chain is translocated by a mobile 4'-phosphopantetheine arm sequentially and repeatedly through condensation, ketoreductase, dehydrase and enoyl reductase domains, ultimately to the site of chain termination. Termination of acyl chain growth can be catalyzed either at long chain-lengths by the resident thioesterase I domain of the FAS or at medium chain-lengths by a separate enzyme, thioesterase II. Thioesterase II is not a component of the FAS and is found only in certain specialized tissues such as mammary and uropygial glands. It is the substrate specificity of these thioesterases which controls the product specificity of the de novo lipogenic pathway in animal tissues. A major aim is to complete elucidation of the primary structure of rat FAS and to assign specific sequences to the various functional domains, DNA cloning and sequencing, active-site labeling, peptide sequencing and domain tagging with site-specific antibodies. Recombinant DNA vectors will be developed for expression of individual FAS domains as catalytically active monofunctional proteins and used to verify sequences assigned to specific functions of the FAS. A novel system for immobilizing catalytically competent hybrid FAS dimers will be used to evaluate the importance of intersubunit communications in the interdomain transfer of FAS reaction intermediates. Low resolution X-ray crystallography will be used in an attempt to provide a three-dimensional domain map for the FAS whcih accomodates structural and functional information available. The hypothesis that the multifunctional FAS evolved by fusion of genes coding for seven monofunctional proteins will be evaluated by compariosn of FAS sequences with sequences of monofunctional enzymes of related function and by appraisal of the enzymatic activity of individually expressed domains. The structure, mechanism and specificity of the mammalian chain-terminating thioesterases will be studied in detail by X-ray crystallography and mutagenesis. Structural relationships and evolutionary origins of these enzymes will be evaluated by sequence comparisons with long and medium-chain-specific bacterial thioesterases.
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