Bioc 460 - Dr. Miesfeld Spring 2008 1 of 9 pages Fatty Acid Synthase Lipid Metabolism 2 Key Concepts - Overview of fatty acid synthesis - Synthesis of palmitate from malonyl CoA - The Citrate Shuttle - Regulation of fatty acid synthesis KEY CONCEPT QUESTIONS IN THE LIPID METABOLISM: What properties of fatty acids make them a more suitable form of stored energy than glucose? What metabolic and hormonal signals control the activity of acetyl- CoA carboxylase? Fatty acid synthase is a multifunctional enzyme complex Fatty acid synthesis is mediated by a large enzyme complex called fatty acid synthase that consists of either seven different polypeptide chains (six enzymes and a carrier protein), as is the case in bacteria, or one large protein (~275 kD) encoding the same seven protein functions as found in animal cells. Importantly, while the chemistry of the four core reactions required for the removal or addition of C2 acetyl groups to the hydrocarbon chain are similar between fatty acid degradation and synthesis (however the direction of the reactions are reversed), the two pathways are in fact quite distinct in terms of the required enzymes, subcellular location and source of redox energy. For example, fatty acid degradation occurs in the mitochondrial matrix and utilizes FAD and NAD+ as the oxidants in two oxidation reactions, whereas, fatty acid synthesis occurs in the cytosol and is dependent on NADPH serving as the reductant in the two corresponding reduction reactions. Difference FA Synthesis FA Degradation subcellular location cytosol mitochondrial matrix carrier protein acyl carrier protein (ACP) Coenzyme A (CoA) enzymes all activities on a single polypeptide chain multiple enzymes required redox reductant is NADPH oxidants are NAD+ and FAD building block malonyl CoA (formed from Acetyl CoA) acetyl CoA The first step in fatty synthesis is the conversion of acetyl-CoA to malonyl-CoA through a reaction catalyzed by the enzyme acetyl CoA carboxylase . Malonyl-CoA serves as the donor of C2 acetyl groups during each round of the fatty acid synthesis reaction cycle. The E. coli acetyl CoA carboxylase enzyme consists of three subunits which encode a biotin carboxylase, a biotin carrier protein and a transcarboxylase as illustrated in figure 1 (acetyl CoA carboxylase in animals is a single large protein with the same three functional domains). This biotin-dependent reaction mechanism is similar to the pyruvate carboxylase reaction in the gluconeogenic pathway. In the first step of both reactions, the biotin carboxylase subunit of the enzyme uses ATP to form carboxyphosphate which is then dephosphorylated to drive the formation of carboxybiotin. The carboxybiotin arm then swings across the enzyme complex and positions the carboxyl group in a second active site where the transcarboxylase subunit transfers the carboxyl group from carboxybiotin to acetyl CoA to form the reaction product malonyl CoA. This same carboxyl group used to form malonyl CoA from acetyl CoA is removed by decarboxylation in step 4 of the fatty acid synthesis reaction cycle (decarboxylation is a highly exergonic reaction). Therefore, malonylBioc 460 - Dr. Miesfeld Spring 2008 2 of 9 pages Figure 1. CoA essentially serves as the "activated" carboxylated form of acetyl CoA. This is analogous to the role of oxaloacetate in the gluconeogenic pathway which represents the activated carboxylated form of pyruvate. Indeed, in both of these examples, one can think of ATP hydrolysis as the true driving force of the initial biosynthetic step because it is required for carboxylation of the first substrate in each of the pathways (acetyl CoA in the case of fatty acid synthesis and pyruvate in the gluconeogenic pathway). The fatty acid synthesis reaction cycle The four core reactions of fatty acid degradation and fatty acid synthesis are chemically similar although different enzymes are utilized and the two pathways are physically separated (degradation takes place in the mitochondrial matrix and fatty synthesis is a cytosolic pathway). Acetyl CoA enters the reaction cycle through malonyl CoA which is covalently linked to acyl carrier protein (ACP) through a thioester. Following decarboxylation of the malonyl group, and condensation with the enzyme-bound (Enz) fatty acyl (FA) group, the extended hydrocarbon chain is chemically modified and then translocated from ACP back to the fatty acid synthase enzyme. The reduced ACP thiol is then ready to accept another malonyl group and start the cycle over again. The role of this multifunctional enzyme complex in the fatty acid synthesis is illustrated in figure 2 where it can be seen that the growing hydrocarbon chain is covalently linked to either the ACP thiol group, or to a thiol group in the large fatty acid synthase enzyme complex. In this way the fatty acid synthase complex serves as the reaction hub for the entire fatty acid synthesis pathway. After each successive round of the cycle, the fatty acid chain grows by two carbons which represent the input acetyl-CoA in the acetyl-CoA carboxylase reaction. This is because ofBioc 460 - Dr. Miesfeld Spring 2008 3 of 9 pages Figure 2. Figure 3. the condensation step in the first reaction which results in decarboxylation of the incoming malonyl-CoA to produce a net increase of a single acetate unit (C2). The condensation reaction is catalyzed by the β-ketoacyl-ACP synthase (KS) subunit in which the acetyl group (or fatty acyl group in subsequent cycles) linked to the KS subunit at Cys163 is transferred to malonyl-ACP in a decarboxylation reaction leading to the formation of acetoacetyl-ACP. In the next reaction, acetoacetyl-ACP is then converted to D-3-hydroxybutyryl-ACP through a reduction reaction catalyzed by β-ketoacyl-ACP-reductase (KR) and NADPH oxidation. This is followed by a dehydration reaction catalyzed by β-hydroxyacyl-ACP-dehydratase (DH) to form α,β-trans-butenoyl-ACP, and a second NADPH-dependent reduction reaction catalyzed by the enzyme enoyl-ACP-reductase (ER) leading to the formation of butyryl-ACP. Lastly, the butyryl group is translocated to Cys163 of the KS subunit to regenerate ACP-SH which is then ready to accept another malonyl group in the next cycle. Let us take a closer look at these reaction steps to see just how cool this fat making protein machine really is. As shown in figure 3, step 1 involves the linkage of an acetyl group from acetyl CoA to a
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