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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM046278-01
Application #
3305680
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1991-08-01
Project End
1995-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Stoops, J K; Cheng, R H; Yazdi, M A et al. (1997) On the unique structural organization of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. J Biol Chem 272:5757-64
You, Y; Elmore, S; Colton, L L et al. (1996) Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum. J Bacteriol 178:3177-87
Kolodziej, S J; Penczek, P A; Schroeter, J P et al. (1996) Structure-function relationships of the Saccharomyces cerevisiae fatty acid synthase. Three-dimensional structure. J Biol Chem 271:28422-9
Zhu, H; Ownby, D W; Riggs, C K et al. (1996) Assembly of the gigantic hemoglobin of the earthworm Lumbricus terrestris. Roles of subunit equilibria, non-globin linker chains, and valence of the heme iron. J Biol Chem 271:30007-21
Stoops, J K; Schroeter, J P; Kolodziej, S J et al. (1994) Structure-function relationships of human alpha 2-macroglobulin. Three-dimensional structures of native alpha 2-macroglobulin and its methylamine and chymotrypsin derivatives. Ann N Y Acad Sci 737:212-28
Stoops, J K; Kolodziej, S J; Schroeter, J P et al. (1992) Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure. Proc Natl Acad Sci U S A 89:6585-9
Schroeter, J P; Kolodziej, S J; Wagenknecht, T et al. (1992) Three-dimensional structures of the human alpha 2-macroglobulin-methylamine and chymotrypsin complexes. J Struct Biol 109:235-47
Stoops, J K; Baker, T S; Schroeter, J P et al. (1992) Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images. J Biol Chem 267:24769-75