BIOSYNTHESIS OF FATTY ACIDS doc. Ing. Zenóbia Chavková, CSc.
The pathway for the of FAs is not the reversal of the oxidation pathway Both pathways are separated within different cellular compartments In humans the pathway for FA synthesis occurs primarily in the cytoplasm of the liver and adipose tissue, to a lesser extend in lactating mammary glands, brain, lungs, and kidneys whereas, oxidation occurs in the mitochondria
The other major difference is the use of nucleotide co-factors Oxidation of fats involves the reduction of FAD, NAD + Synthesis of fats involves the oxidation of NADPH Both oxidation and synthesis of fats utilize an activated 2C intermediate, acetyl-coa
Acetyl-CoA must be first transported out of mitochondria using citrate shuttle transport system The total energy requirement for converting mitochondrial acetyl-coa into cytoplasmic acetyl-coa is 1 ATP
Origin of Cytoplasmic Acetyl-CoA Acetyl-CoA is generated in the mitochondria primarily from the sources: The pyruvate dehydrogenase (PDH) reaction (glycolysis glucose pyruvic acid acetyl-coa) Fatty acid oxidation AAs degradation and ketone bodies In order to be utilized for fatty acid synthesis they must be present in the cytoplasm The shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energy diminishes
Source of reducing equivalents The origin of NADPH for FA biosynthesis depends on cell type In liver, the 2 NADPHs come from the pentose phosphate pathway In adipose tissue, NADPH is generated by malic enzyme - OOC-CH 2 -CH-COO - CH 3 -C-COO - OH O The pentose phosphate pathway
Another source for NADPH for these reactions is the isocitrate shuttle
The synthesis of malonyl-coa is the first committed step of FAs synthesis Acetyl-CoA carboxylase (ACC), is the major site of regulation of FAs synthesis ACC requires a biotin co-factor
First, CO 2 is covalently bound to biotin using energy from hydrolysis of ATP Then, the CO 2 is transferred to acetyl-coa producing malonyl-coa The biotinyl group serves as a temporary carrier of CO 2
The carboxylation of acetyl-coa to form malonyl-coa catalyzed by acetyl-coa carboxylase O ll CH 3 -C-SCoA acetyl-coa Enzyme-biotin HCO - 3 + ATP 1 ADP + P i Enzyme-biotin-CO 2-2 O - O 2 C-CH 2 -C-SCoA malonyl-coa ll Enzyme-biotin is the rate-limiting step of FA biosynthesis The overall reaction may be summarized as:
Acetyl-CoA Carboxylase activity, in the mammals is regulated by phosphorylation allosteric regulation by local metabolites The active conformation of the enzyme associates in multimeric filamentous complexes The inactive conformation of the enzyme exists as individual protomers
The rate of fatty acid synthesis is controlled by the equilibrium between monomeric and polymeric acetyl-coa carboxylase (ACC) The activity of ACC requires polymerization This conformational change is controlled by local metabolites (citrate, palmitoyl-coa and other long-chain fatty acyl-coas)
Regulation by local metabolites The equilibrium between monomeric and polymeric acetyl-coa carboxylase is inhibited by palmitoyl- CoA (product of FA synthase) other long-chain fatty acyl-coas enhanced by citrate (promoting enzyme polymerization)
Regulation of Acetyl-CoA carboxylase activity through hormone mediated phosphorylation Glucagon and epinephrine promote phosphorylation and decrease the enzymatic activity ( ) Insulin promotes dephosphorylation and increases the activity ( )
With Acetyl-CoA Carboxylase inhibited, acetyl-coa remains available for ketone bodies synthesis the alternative metabolic fuel used when blood glucose is low
Changes in diet affect the amount of fatty acid biosynthesis by affecting the amount of acetyl-coa carboxylase A diet rich in carbohydrate or low in fat increases the biosynthesis of the enzyme by affecting the rate of transcription Starvation or diet high in fat has the opposite effect and reduces the rate of synthesis of acetyl-coa carboxylase
Synthesis of the Acyl chain The reactions of FA biosynthesis take place on a multifunctional protein, called fatty acid synthase (fatty acid synthase complex) A polyprotein is a single protein with more then 1 activity, and fatty acid synthase is formed from 2 chains of this protein The active enzyme is a dimer of identical subunits
There is some evidence that the 2 copies of the multi-domain enzyme are aligned antiparallel, as below Pant-SH HS-Cys Cys-SH HS-Pant Fatty Acid Synthase dimer
Fatty Acid Synthase prosthetic groups: The thiol of the sidechain of a cysteine residue of condensing enzyme domain The thiol H 3 N + C COO CH 2 SH of phosphopantetheine, equivalent in structure to part of coenzyme A H cysteine
The fatty acid synthase complex contains 2 types thiol groups The central thiol, made up of 4 phosphopantetheine a derivative of coenzyme A, covalently linked by a phosphodiester bond to serine residue of acyl carrier protein, or ACP The peripheral thiol, belongs to a cysteinyl residue on ketoacyl-acp synthase
Like fat oxidation, fat synthesis involves 4 enzymatic activities -keto-acp synthase, -keto-acp reductase, 3-OH acyl-acp dehydratase Enoyl-CoA reductase The two reduction reactions require NADPH oxidation to NADP +
The acetyl-coa are transferred to ACP + malonyl CoA by the action of: Acetyl-CoA transacylase Malonyl-CoA transacylase The attachment of these carbon atoms to ACP allows them to enter the fatty acid synthesis cycle During the sequence of reaction, the growing FA takes the form of a thioester attached to the: Peripheral SH group of a cysteine residue of the protein or to the central SH group of a protein-bound phosphopantetheine
The biosynthetic intermediates do not diffuse away from the polyprotein but are passed from one enzyme active site to the next active site by acyl carrier protein (ACP)
Individual steps of the Fatty Acid Synthase reaction pathway In the first reaction, (to initiate biosynthesis) acetyl-coa is transferred: From CoA To the central SH (thiol) group of phosphopantetheine to form a covalent bond with release of CoA
Then, Central thiol the acetyl group is transferred to the peripheral SH (thiol) group of a cysteine Peripheral thiol Next, the malonyl group is transferred to the pantetheine central SH group of ACP, just vacated by the acetyl group Now the reactants are poised for the first condensation reaction
In the first step, the acetyl group and malonyl groups are condensed, with the release of CO 2 This forms CONDENSATION acetoacetate attached to the pantetheine (central) SH group The condensation reaction of fatty acid biosynthesis is catalyzed by -ketoacyl-acp synthase
REDUCTION, step 2. Using NADPH, acetoacetyl-acp undergoes a reduction, yielding -hydroxybutyryl-acp and NADP + in reaction The ketone is reduced to a hydroxyl group, mediated by -ketoacyl-acp reductase
DEHYDRATION, step 3. Then the compound is dehydrated to 2,3-trans-butenoyl-ACP (crotonyl-acp) catalyzed by -hydroxyacyl-acp-dehydratase
REDUCTION, step 4. The double bond is reduced by NADPH + H + in reaction catalyzed by 2,3 trans-enoyl-acp reductase to form butyryl-s-acp
Lengthened fatty acid chain is then translocated to the peripheral SH (thiol) group of a cysteine ketoacyl ACP synthase Another malonyl group is added to the central SH (thiol) group of ACP H This series of reactions form a 4-carbon acyl group still attached to the phosphopantetheine (central -SH)
In the next reaction: The growing fatty acyl chain is transferred to the cysteine (peripheral thiol), Another malonyl group is added to the pantetheine -SH (central thiol) and cycle begins again
This cycle of condensation, reduction, dehydration, reduction and transfer of the acyl group continues until chain of 16 carbons has been created The resulting palmitoyl group - palmitate is released from the fatty acid synthase complex by an exergonic hydrolysis reaction Palmitate, a 16-C saturated fatty acid, is the final product of the FA synthase reactions
Therefore: Acetate group is added at the beginning Then need 1 malonate to extend the chain by 2 carbons 3C 2C = malonate = acetate 1C = CO 2 2C 3C 2C FAS 2C 2C FAS 3C 2C 2C 2C FAS 3C 1C 1C 1C
That fatty acid synthesis by multienzyme complex stops at palmitate is probably due to limitation in the size of an active site of fatty acid synthase Palmitate can then undergo separate elongation and/or unsaturation to yield other fatty acid molecules
Fatty acid biosynthesis is energetically expensive however, occurs when is abundant precursor to provide both the mass the energy
BIOENERGETICS OF FA BIOSYNTHESIS 1 mole ATP is required for the generation of 1 mole of acetyl-coa from citrate 7 moles of ATP are required for the transport of acetyl-coa from mitochondria into cytosol, as a substrate for the synthesis of malonyl-coa 7 additional moles of ATP are required for the synthesis of 7 moles of malonyl-coa from acetyl-coa and CO 2 A total of 15 ATP equivalents are required for the synthesis of palmitate from citrate 14 moles of NADPH are required for the biosynthesis of 1 mole of palmitate
THE REGULATION OF FAT METABOLISM Occurs via two distinct mechanisms One is short term regulation which is regulation effected by events such as substrate availability, allosteric effectors and/or enzyme modification Control of a given pathways' regulatory enzymes can also occur by alteration of enzyme synthesis and turn-over rates of synthesis These changes are long term regulatory effects
Insulin stimulates lipogenesis by several mechanisms It increases the transport of glucose into cell (in adipose tissue) and thereby increases the availability of both: pyruvate for FAs synthesis glycerol-3-p for esterification of the newly formed FAs Insulis converts the inactive form of pyruvate dehydrogenase to the active form (in adipose tissue but not in liver!) Insulin activates acetylco-a carboxylase. It involves dephosphorylation by a protein phosphatase accompanied by change in aggregation of monomers to a more polymeric state
Insulin by its ability to depress the level of intracellular camp, inhibits lipolysis in adipose tissue and thereby reduces the concentration of plasma free FAs and long-chain acyl-coa, an inhibitor of lipogenesis By this same mechanism insulin antagonizes the action of glucagon and epinephrine, which inhibit acetyl-coa carboxylase and therefore lipogenesis, by increasing camp, allowing camp dependent protein kinase to inactivate the enzyme by phosphorylation
Regulation of fat metabolism also occurs through malonyl-coa induced inhibition of carnitine acyltransferase I. This functions to prevent the newly synthesized FAs from entering the mitochondria and being oxidized
ELONGATION AND DESATURATION Stearic Oleic acids are major constituent of FAs found in human cells The fatty acid product released from fatty acid synthase (FAS) is palmitate which is a 16:0 fatty acid, (16 carbons and no sites of unsaturation)
Although the FA synthase complex stops at 16 C atoms, human cells: Can extend the length of the FA chain Posseses the machinery for converting saturated to unsaturated FAs 2-carbon units can be added: To endogenously synthesized or dietary fatty acids by elongation reactions
ELONGATION AND UNSATURATION of fatty acids occurs after palmitate (16C) in both the mitochondria and endoplasmic reticulum (microsomal membranes) The endoplasmic reticulum pathway is quantitatively more important This strategy agrees with the role of mitochondria functioning as a catabolic organell The substrate for the elongation reactions is fatty acyl-coa and not fatty acyl-acp
The endoplasmic reticulum contains the enzyme activities found in the FA synthase complex, that succesively reduce, dehydrate, and reduce the compound to produce fatty acyl-coa containing 2 additional carbon atoms The reactions are analogous to the condensation reactions that occurs during conventional FA biosynthesis
The resultant product is 2C longer The fatty acyl-coa substrate for the elongation reaction is malonyl-coa More then 1 elongation reaction can occur, and fatty acids up to 26 C atoms can be synthesized The reduction reactions of elongation require NADPH as co-factor just as for the similar reactions catalyzed by FAS
Mitochondrial elongation Involves acetyl-coa units is a reversal of oxidation Except that the final reduction utilizes NADPH instead of FADH 2 as co-factor Acetyl-CoA, not malonyl-coa donates the 2C units in mitochondria
C-C-C-C-C-C=C-C-C=C-C-C=C-C-C=C-C-C-C-COOH 18 18 n : = ( x,y.. ) 14 12 11 9 C-C-C-C-C-C=C-C-C=C-C-C-C-C-C-C-C-COOH 8 5 1 2 1 Animals cannot put double bonds in this part of the molecule, plants can! Essential fatty acids: Linoleate 18:2(Δ 9,12 ) Arachidonate 20:4(Δ 5,8,11,14 )
Since these enzymes cannot introduce sites of unsaturation beyond C9 they cannot synthesize either linoleate (18:2 D9, 12 ) linolenate (18:3 D9, 12, 15 ) 17 15 12 9 7 5 3 COOH CH 3 Linoleic acid (cis 9,12 octadecadienoic acid) These fatty acids must be acquired from the diet and are, therefore, referred to as essential fatty acids
These essential FAs are necessary for normal membrane structure Linoleic acid especially important, serves as a precursor for the synthesis of arachidonic acid, from which the eicosanoids (the prostaglandins, thromboxanes) are formed is also a constituent of epidermal cell sphingolipids that function as the skins water permeability barrier
It is this role of FAs in eicosanoid synthesis that leads to poor growth, wound healing dermatitis in persons on fat free diets
Desaturation occurs in the ER membranes in mammalian cells involves 4 broad specificity fatty acyl-coa desaturases (non-heme iron containing enzymes) These mixed-function oxidase require NADPH and molecular oxygen to add a hydroxyl group to the fatty acid
Arachidonic acid is produced by elongation and the addition of 2 double bonds as shown in Fig. Common in healthy diet desaturation Some available from meat and eggs
-6 Pathway Linoleic acid (18:2) -Linolenic acid (18:3) Anti-inflammatory metabolites Pro-inflammatory metabolites Dihomo- -linolenic acid (20:3) (Slow) Arachidonic acid (20:4) Meat and eggs Released from stores
Acetyl CoA carboxylase O - O C CO 2 Biotin carboxylase BIOTIN Irreversible two-step reaction C=O H-N Lys Biotin carrier protein H-N Lys C=O BIOTIN O = C - O Transcarboxylase
AMP-Activated Kinase catalyzes phosphorylation of Acetyl-CoA Carboxylase causing inhibition ( ) Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate Dephosphorylated, e.g., by insulinactivated Protein Phosphatase Palmitoyl-CoA Phosphorylated, e.g., via AMP-activated Kinase when cellular stress or exercise depletes ATP. Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) Regulation of Acetyl-CoA Carboxylase The primary phosphorylation of ACC occurs through the action of AMP-activated protein kinase, AMPK This is not the same as camp- dependent protein kinase, PKA! Phosphorylation causes the filamentous enzyme to dissociate into inactive mononomers