The de novo synthesis of fatty acids from acetyl- and malonyl-CoA in animals is catalyzed by an enzyme comprised of two identical, multifunctional polypeptide subunits. The thioester substrates are loaded on a common 4'-phosphopantetheine site and the incorrectly loaded substrate is removed by reaction with coenzyme A through a kinetic self-editing mechanism. The covalently-attached acyl-intermediates are then transfered to sites for condensation, Beta-ketoacyl reduction, dehydration, enoyl reduction, and finally deacylation following completion of chain elongation. In the proposed study, a variety of techniques such as transient and steady-state kinetic analyses, equilibrium substrate binding, reactivity of thiol groups, hybridization and crosslinking will be employed to elucidate a detailed catalytic mechanism of this enzyme and its functional conformation states. Chemical modification with specific reagents will be carried out to detect and characterize amino-acid residues essential for activity. Experiments are designed to test for the apparent """"""""half-of-the-sites"""""""" reactivity of 5,5 dithiobis (2,-nitrobenzoic acid), and for a requirement of specific subunit interactions for condensation and acyl-transfer between hydroxyl, pantetheine, and cysteine sites implicated by the recently postulated """"""""head-to-tail"""""""" model. A """"""""half-of-the-sites"""""""" model has been proposed by us to account for the unusual behavior of malic enzyme which supplies a portion of NADPH equivalents for fatty acid synthesis. The validity of this model will be examined by end group analysis, peptide mapping, and by determining the stoichiometry of catalytic sites and activity of matrix-bound monomeric units of the enzyme. Ultimately, results of this study will enhance our knowledge of lipid metabolism and provide a basis for the prevention, diagnosis, and treatment of disorders related to lipid metabolism such as artereosclerosis and obesity.
| Hsu, R Y; Glynias, M J; Satterlee, J et al. (1992) Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants. Biochem J 284 ( Pt 3):869-76 |
| Satterlee, J; Hsu, R Y (1991) Duck liver malic enzyme: sequence of a tryptic peptide containing the cysteine residue labeled by the substrate analog bromopyruvate. Biochim Biophys Acta 1079:247-52 |
| Tian, W X; Wang, Y S; Hsu, R Y (1989) Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate. Biochim Biophys Acta 998:310-6 |
