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Chem*3560 Lecture 20: Fatty acid biosynthesis Fatty acid biosynthesis reverses the β -oxidation sequence β -Oxidation consists of a cycle of four reactions that include two dehydrogenase steps. Three steps are energetically favourable, and one complete cycle sums to ∆Go ' = –12 kJ/mol, which makes the overall process proceed in the direction of oxidation (Lehninger p.604-606). Fatty acid synthesis follows the same sequence in reverse, and uses two strategies to drive reactions in the opposite direction (Lehninger p. 770-771). 1) NADPH is used as reductant to reduce the ketoacyl and enoyl intermediates. Since the ratio [NADPH] / [NADP +] is high, this drives the reactions in the direction of reduction 2) Decarboxylation drives the elongation step that reverses the thiolase reaction. In order to add two carbons to elongate the chain, the three-carbon dicarboxylate malonyl-CoA – O2 C-CH2 -CO-SCoA condenses with the growing acyl chain, and loses its free carboxylate as CO2 . The energy made available by decarboxylation provides the driving force to maintain the synthesis direction. Acetyl CoA Carboxylase makes the malonyl CoA for fatty acid biosynthesis Acetyl CoA carboxylase is organized in three components, which like pyruvate carboxylase, includes a central biotin carrier protein (BCP) which has a biotin molecule covalently bonded to a lysine side chain. On one side is the ATP driven biotin carboxylase, which generates the active carboxylate donor carboxybiotin from bicarbonate ion at the cost of ATP hydrolysis: ATP + biotin + HCO3 – → ADP + Pi + biotin-CO2 – On the other side is the substrate specific transcarboxylase (or carboxyltransferase), which uses acetyl CoA as the carboxylate acceptor (Lehninger p.771). biotin-CO2 – + acetyl-CoA → biotin + malonyl-CoA. Unlike pyruvate carboxylase, where the three components are domains on a single polypeptide, acetyl-CoA carboxylase consists of three separate proteins held together in a single complex. Regulation of fatty acid biosynthesis Since fatty acid biosynthesis takes the opposite direction to β-oxidation, it must be regulated to prevent the two processes from operating as a futile cycle, in particular in animals where significant amounts of energy are stored as fats and fatty acids. Bacteria make fatty acids for membrane lipids, but do not use fatty acids and β-oxidation as a primary energy source, so regulation less strict and is mainly correlated with cell growth. In plants, fatty acid biosynthesis occurs in the chloroplasts, and is dependent on photosynthesis to provide substrate and NADPH (Lehninger p. 778-780).. In animals, the bulk of stored fat is found in adipose tissue . Fatty acids are released from triacylglycerol by hormone sensitive lipase, which is actually activated by protein kinase A in response to cyclic AMP induced by the hormones epinephrine and glucagon. Fatty acid is released from adipose tissue, and circulated in blood to those muscle tissues (e.g. cardiac) which use β-oxidation of fatty acid as an energy source. In contrast, fatty acid biosynthesis occurs primarily in the liver, and involves the following modes of regulation. 1) Compartmentation β -oxidation occurs in mitochondria or in a specialized organelle called the peroxisome, whereas fatty acid biosynthesis is cytoplasmic. 2) Substrate availability Fatty acid biosynthesis requires a cytoplasmic source of acetyl CoA. Acetyl CoA produced by β-oxidation or by oxidation of amino acids or pyruvate is located in mitochondria, and is directed towards the TCA cycle. Cytoplasmic acetyl CoA is produced when more citrate is produced than is needed for the TCA cycle to generate ATP (Lehninger Fig 21-11 p.779). The TCA cycle enzyme isocitrate dehydrogenase is under positive allosteric regulation by ADP levels in mitochondria. If ATP is not being consumed quickly enough, ADP levels drop, and isocitrate dehydrogenase loses activity. This causes unused isocitrate to accumulate, and because the aconitase equilibrium is unfavourable (K eq = 0.075), citrate builds up to very high levels. A transporter allows excess citrate to enter the cytoplasm, where it is broken down by citrate lyase. citrate + HSCoA + ATP → ADP + Pi + oxaloacetate + acetyl-CoA Oxaloacetate is reduced to malate by cytoplasmic NADH. Malate returns the mitochondria to allow the TCA cycle to continue, and enters by an exchange process via the same transporter that exports citrate. Strict segregation of cytoplasmic and mitochondrial compartments prevents citrate lyase from developing a futile cycle with the TCA cycle enzyme citrate synthase. 3) Enzyme regulation Fatty acid biosynthesis is subject to hormonal and allosteric regulation. Insulin (secreted when blood glucose levels are high) stimulates citrate lyase. Glucagon (secreted when blood glucose levels are low) and epinephrine (secreted when demand for ATP is anticipated) induce cyclic AMP, which stimulates protein kinase A to phosphorylate and inactivate acetyl CoA carboxylase (Lehninger p. 780). Cytoplasmic citrate is a positive allosteric effector of acetyl CoA carboxylase.