The long term objective of this program is to elucidate the structure, mechanism of action and basis for specificity of enzymes involved in de novo fatty acid synthesis. In animals, the enzymes required for biosynthesis of fatty acids from malonyl-CoA are integrated into two identical multifunctional polypeptides of 272 kDa which constitute the fatty acids synthase (FAS). Based on previous studies, a model for FAS is proposed in which two centers for initiation, growth and termination of acyl chains are formed by the transferase, ketoacyl synthase and dehydrase of one subunit juxtaposed in head-to-tail orientation. Translocation of the growing acyl chain sequentially through the active centers of the constituent enzymes is mediated partly through covalent attachment of the intermediates to a mobile phosphopantetheine swinging arm and partly through dynamic interactions between component domains of the multifunctional complex. The nature of the final product is determined in part by the specificities of the transferase, ketoacyl synthase and thioesterase enzymes. We propose to test the model as follows. Recombinant FAS subunits each deficient in one of the seven catalytic functions will be expressed and recombined as heterodimers. By determining which mutants exhibit active-site complementation it will be possible to establish which domains of each subunit cooperate to form a center for acyl chain synthesis. By measuring the activities of heterodimers formed between normal and mutated subunits it will be possible to determine whether the two centers for acyl chain synthesis function independently. The importance of interdomain linkers in facilitating dynamic interactions between domains will be assessed by investigating the functional constraints associated with linker length and flexibility. Support for the refined model emerging from the functional studies will be sought at the structural level using immuno- electron-microscopy and ultimately by X-ray crystallography. Detailed characterization of individual FAS domains will be achieved by expression as independent enzymes. Proposed models for the catalytic mechanisms of the ketoacyl synthase, transferase and thioesterase will be evaluated by mutagenesis of putative active-site residues. The structural basis for the specificity of key enzymes will be explored by mapping the topography of the acyl-chain binding sites using substrate analogs and photoreactive reagents. Continuing collaborations with X-ray crystallographers will be pursued to obtain detailed three-dimensional models for the component enzymes of the FAS.
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