Fatty acids are essential components of biological membranes in eukaryotes and in higher organisms. In these organisms, the derivatives of fatty acids serve as highly concentrated energy stores, hormones, and intracellular messengers. Moreover, in humans, fatty acids play a major role in arteriosclerosis, which can lead to heart attack and stroke. To understand fully the etiology of arteriosclerosis we must first understand fatty acid synthesis on the molecular level. In this study, the yeast fatty acid synthase will be used as a model system for fatty acid synthesis because this enzyme is well suited to a variety of physicochemical and molecular biological approaches. The yeast fatty acid synthase is a macromolecular complex (M-r = 2.4 x 10[6]) comprised of six copies of two different subunits called alpha and beta (alpha6beta6). The structural organization of the complex will be studied using different electron microscopic techniques (negative stain, cryo, and immuno) together with image processing to enhance structural details and to achieve a three-dimensional reconstruction of the complex. The six sites of fatty acid synthesis will be located using an active site-directed undecagold label by scanning electron microscopy. How the six alpha and six beta subunits (M-r equals approximately 220,000 each) self-assemble to form the active enzyme and how the functional domains are organized within the subunits will be studied by electron microscopy and analytical ultracentrifugation and by immunoelectron microscopy, respectively. An enzyme containing a truncated alpha subunit lacking the N-terminal acyl carrier protein domain will be examined to assess its role in the structure and function of the complex and to determine the role of the C-terminal portion of the alpha subunit in the total structure and overall function of the enzyme. Site-directed mutagenesis will be employed to obtain yeast synthase lacking the primer function (acetyl transacylase) and the 4'-phosphopantetheine prosthetic group to investigate the mechanism of the transacetylation pathway by enzyme kinetics.
