Fatty Acid Synthesis Javed Khan Biotechnology Department PGCSTR

Fatty Acid Synthesis Javed Khan Biotechnology Department PGCSTR, Jalgaon

Introduction üThere are three systems for the synthesis of fatty acids 1. De novo synthesis of FAs in cytoplasm 2. Chain elongation in mitochondria 3. Chain elongation in microsomes

De novo synthesis of FAs ü In mammals fatty acid synthesis occurs primarily in the cytosol of the liver and adipose tissues. It also occurs in mammary glands during lactation. ü Acetyl-Co. A is the starting material for FA synthesis. However, most acetyl-Co. A in mitochondria(from the breakdown of sugars, some amino acids and other fatty acids). So, acetyl-Co. A must be transferred from the mitochondria to the cytosol BUT Mitochondria not permeable to acetyl Co. A

Continue. . ü Citrate-malate-pyruvate shuttle provides cytosolic acetyl Co. A and reducing equivalents NADPH for fatty acid synthesis. ü Acetyl–Co. A units are shuttled out of the mitochondrial matrix as citrate.

Continue. . RULE: Fatty acid synthesis is a stepwise assembly of acetyl Co. A unit (mostly as malonyl Co. A) ending with palmitate (16 C saturated) ü 4 Steps repeating cycle, extension 2 C: Condensation Reduction Dehydration Additional reduction

Formation of Malonyl-coenzyme A (Activation of acetate) ü Is the committed step in fatty acid synthesis (Rate Limiting Reaction) ü It takes place in two steps: 1. Carboxylation of biotin (involving ATP) 2. Transfer of the carboxyl to acetyl-Co. A to form malonyl-Co. A ü Reactions are (multienzyme) catalyzed by acetyl-Co. A carboxylase

Fatty acid synthase ü It is a multi-enzyme complex consist of 7 enzymes linked covalently in a single polypeptide chain. ü It is a dimer, and each monomer is identical, consisting of one chain (250 k. D) containing all seven enzyme activities of fatty acid synthase and an acyl carrier protein (ACP) ü ACP contains the vitamin pantothenic acid in the form of 4'-phosphopantetheine (Pant). ACP is the part that carry the acyl groups during fatty acid synthesis

Continue. . 1 - A molecule of acetate is transferred from Acetyl Co. A to the – SH group of ACP by acetyl Co. A-ACP transacylase (initiation or priming). 2 - Next, this 2 C fragment is transferred to a cysteine residue in the active site of the condensing enzyme. 3 -The now-empty ACP accepts a 3 C malonate unit from malonyl Co. A, malonyl Co. A-ACP transacylase catalyzes this

Continue. . 4 - Acetyl unit (on the condensing enzyme) condenses with 2 carbon portion of malonyl unit on ACP forming acetoacetyl-SACP with release of CO 2. This reaction is catalyzed by β-ketoacyl –ACP synthase Active site on the condensing enzyme is free.

Continue. . 5 -The β-ketone is reduced to an alcohol by e- transfer from NADPH. 6 - Dehydration yields a trans double bond. 7 - Reduction by NADPH yields a saturated chain.

Continue. . 8 - Following transfer of the growing fatty acid from Pant to the Condensing Enzyme's cysteine sulfhydryl, the cycle begins again, with another malonyl-Co. A. Note: Acetyl residue successively added is derived from the 2 C atoms of malonyl Co. A with the release of the third C as CO 2 EXCEPT the 2 donated by the original acetyl Co. A which are found at the methyl group end of the fatty acid.

Product Release ü When the fatty acid is 16 carbon atoms long, a Thioesterase domain catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine. ü The 16 -C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex (but it may produce short chain FAs) ü Further elongation and insertion of double bonds are carried out by other enzyme system.

ü Palmitate, a 16 -C saturated fatty acid, is the final product of the Fatty Acid Synthase reactions. 1 - a. How many acetyl-Co. A used for initial priming of enzyme? 1 b. How many acetyl-Co. A used for synthesis of each malonate? 1 c. How many malonate used (how many reaction cycles) per synthesis of one 16 -C palminate? 7 d. Total acetyl-Co. A used for priming & for syntheisis of malonate, a + b(c): 8 2 - a. How many ~P bonds of ATP used for synthesis of each malonate? 1 b. Total ~P bonds of ATP used for synthesis of one 16 -C palmitate, 2 a(1 c): 7 3 - a. How many NADPH used per reaction cycle? 2 b. Total NADPH used per synthesis of one 16 -C palmitate, 3 a(1 c): 14 No. of cycles = (C/2) – 1 No. of Malonate molecules = (C/2) – 1 No. of Acetyl Co. A molecules= [(C/2) – 1] +1 No. of NADPH molecules = [(C/2) – 1] x 2

Regulation of FA Synthesis ü Allosteric regulation • Acetyl Co. A carboxylase, which catalyzes the committed step in fatty acid synthesis, is a key control site. • End-product fatty acid is a feedback inhibitor (palmitoyl-Co. A) • Activated by citrate, which increases in well-fed state and is an indicator of a plentiful supply of acetyl-Co. A • Inhibited by long-chain acyl-Co. A

Regulation of FA Synthesis ü Glucagon inhibits fatty acid synthesis while increasing lipid breakdown and fatty acid βoxidation. ü Acetyl Co. A cayboxylase is inactivated by phosphorylation. ü Insulin prevents action of glucagon Inhibits lipases/activates acetyl Co A cayboxylase

Further Processing of C 16 Fatty Acids Additional Elongation In mammalian systems FA elongation can occur either in : • Microsomes • Mitochondria

Chain Elongation in Microsomes ü The reactions are similar to that which occurs in the cytosolic FA synthase in that: a) The source of the 2 carbon units is malonyl Co. A. b) NADPH is used as reducing power. ü In contrast to denovo synthesis of Fatty Acids, the intermediates in the subsequent reactions are Co. A esters, indicating that the process is carried out by separate enzymes rather than a complex of FA synthase type. (uses Co. A instead of ACP as the acyl carrier) ü It is the main site for elongation of existing long chain FAs

Chain Elongation in Mitochondria ü It differs from the microsomal system in that acetyl Co. A is the source of the added 2 C atoms (instead of malonyl Co. A) ü NADH and NADPH are sources of reducing agents ü This system operate by simple reversal of the pathway of FA oxidation with the exception that, NADPH-linked α, β-unsaturated acyl Co. A reductase replaces FAD linked acyl Co. A dehydrogenase. ü The mitochondrial system serves in the elongation of shorter chain fatty

Biosynthesis of Unsaturated Fatty Acids ü Desaturases introduce double bonds at specific positions in a fatty acid chain. ü Mammalian cells are unable to produce double bonds at certain locations, e. g. , ∆ 12. ü Thus some polyunsaturated fatty acids are dietary essentials, e. g. , linoleic acid, 18: 2 cis ∆ 9, 12 (18 C atoms long, with cis double bonds at carbons 9 -10 & 12 -13)

Continue. . ü Formation of a double bond in a fatty acid involves the following endoplasmic reticulum membrane proteins in mammalian cells: • NADH-cyt b 5 Reductase, a flavoprotein with FAD as prosthetic group. • Cytochrome b 5, which may be a separate protein or a domain at one end of the desaturase. • Desaturase, with an active site that contains two iron atoms complexed by histidine residues

Continue. . ü The ∆9 desaturase in the endoplasmic reticulum catalyzes the conversion of stearate (18: 0) to oleate (18: 1 cis ∆ 9). ü the overall reaction is: stearate + NADH + H+ + O 2 oleate + NAD+ + 2 H 2 O ü Synthesis of polyunsaturated fatty acids involves desaturase and elongase systems

Differences in the oxidation and synthesis of FAs