Fatty Acids: Synthesis and Degradation

• Fatty acids have 4 major physiological roles:
• Building blocks of phospholipids and glycolipids
• Covalent attachment modifies many proteins, targeting them to membrane locations
• Fuel molecules
• Fatty acid derivatives are hormones and intracellular messengers

Synthesis vs Degradation

Fatty Acid Synthesis Overview

• Glucose is converted to acetyl-CoA
• Acetyl-CoA is carboxylated to malonyl-CoA by acetyl-CoA carboxylase (ACCase) with CO2 and ATP
• Malonyl-CoA is used to synthesize fatty acids, alkanes, or alkenes, and triacylglycerols (TAG)
• Malonyl-CoA is converted to malonyl-ACP by Malonyl-CoA: ACP transferase
• Acetyl-CoA condenses with malonyl-ACP, releasing CO2
• The 3-ketoacyl-ACP is reduced by 3-ketoacyl-ACP reductase using NADPH + H+
• 3-hydroxybutyryl-ACP is dehydrated by 3-hydroxyacyl-ACP dehydratase
• trans-Δ2-Butenoyl-ACP is reduced by Enoyl-ACP reductase using NADPH + H+ to form butyryl-ACP

Fatty Acid Synthesis Essentials

• Glucose is converted to 2 pyruvate molecules via glycolysis in the cytoplasm
• Pyruvate is converted to Acetyl CoA in the mitochondria
• Acetyl CoA combines with oxaloacetate to form citrate (6C)
• This reaction is catalyzed by citrate synthase
• Citrate is transported into the cytoplasm
• In the cytoplasm, citrate is cleaved by Citrate Lyase into Acetyl CoA (2C) and oxaloacetate (4C)
• This process requires ATP, which is converted to ADP
• Pyruvate is formed from pyruvate
• Pyruvate is converted to acetyl-CoA
• This requires acetyl-CoA carboxylase and pyruvate
• Acetyl CoA can also be converted to malonyl CoA
• It requires biotin (B7)
• Fatty acids are synthesized in the cytoplasm
• The synthesis requires acetyl CoA, malonyl CoA, NADPH, and fatty acid synthase
• The process is stimulated by insulin

Important Fatty Acid Properties

• The Fatty Acid consists of carboxylic acid and a chain of hydrocarbons
• The synthesis requires acetyl CoA in the cytoplasm
• It also requires NADPH
• This occurs in the cytosol
• Fatty acids are stored as triacylglycerols (triglycerides) in adipose tissue
• This is anhydrous and helps with energy/mass

Fatty Acid Biosynthesis

• Fatty acid biosynthesis is a stepwise assembly of acetyl-CoA units, in the form of malonyl-CoA units
• It ends with palmitate (C-16)
• It involves three steps:
• Activation
• Elongation
• Termination
• Fatty acid synthesis occurs in the liver, kidney, adipose tissue, and lactating mammary glands

Fatty Acid Synthesis Phases

• Fatty acid synthesis occurs in three phases:
• Transport of acetyl-CoA from mitochondria to cytosol
• Carboxylation of acetyl-CoA to malonyl-CoA
• Reactions of fatty acid synthase complex
• De novo synthesis of FAs occurs in the cytoplasm
• Chain elongation occurs in the mitochondria and microsomes

Transferring Acetyl CoA

• Acetyl CoA is transferred from the mitochondria to the Cytosol
• Acetyl CoA and oxaloacetate convert to citrate in the mitochondria
• Citrate converts to Acetyl CoA and oxaloacetate in the cytosol
• Oxaloacetate converts to malate, which is converted to pyruvate via NADPH to NADP+

Acetyl-CoA Carboxylase

• Acetyl-CoA Carboxylase (ACC) is a committed step in fatty acid synthesis
• It is inhibited by epinephrine, palmitoyl-CoA, and AMPK
• It is also affected by glucagon and fasting
• It is stimulated by insulin and citrate and high carbohydrate caloric intake
• ATP Citrate Lyase converts Citrate to Acetyl-CoA
• This also produces oxaloacetate
• Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA
• This requires Carboxybiotin (Vit. B7) and ATP.
• Malonyl CoA Decarboxylase (MCD) converts Malonyl-CoA back to Acetyl-CoA
• Acetyl-CoA is the precursor for fatty acid catabolism and carbon dioxide
• Carboxybiotin (Vit. B7) is needed

TRANSPORTATION OF ACETYL CoA

• Glucose is converted to pyruvate
• Pyruvate is converted to Amino Acids
• Citrate and oxaloacetate can combine

Key Enzymes and Cofactors

• The 2 main enzymes involed in synthesis are:
• Acetyl CoA carboxylase
• Fatty acid synthase
• Both enzymes are multi enzyme complexes
• Coenzymes and cofactors involed are
• Biotin
• NADPH
• Mn++
• Mg++

Carboxylation of Acetyl CoA

• Acetyl-CoA is converted to Malonyl CoA
• This requires ATP, Acetyl-CoA carboxylase, and occurs in the presence of biotin
• Bicarbonate is also involved

Fatty acid synthase complex

• The Fatty acid synthase complex:
• Is a polypeptide containing seven enzyme activities, and acyl carrier protein (ACP) segment
• Includes: Acetyl transacylase (AT), Malonyl transacylase (MT), 3-ketoacyl synthase (KS), 3-ketoacyl reductase (KR), Enoyl reductase (ER), and Thioesterase
• ACP contains the vitamin pantothenic acid in the form of 4'-phosphopantetheine (Pant); it carries the acyl groups

Reactions of fatty acid synthase complex

• Fatty acid synthase (FAS) is a multifunctional enzyme
• In eukaryotic cells, FAS exists as a dimer with two identical units
• Each monomer has activities of seven different enzymes and an acyl carrier protein (ACP) bound to 4'-phosphopantetheine
• Fatty acid synthase functions as a single unit, catalyzing all seven reactions

Fatty Acid Synthase Components

• Fatty acid synthase contains: Condensing enzyme, reducing enzyme
• Contains subunits, a functional division, and the following:
• Acetyl transacylase, Malonyl transacylase, Ketoacyl synthase, dehydratase, Enoyl reductase, Ketoacyl reductase, ACP Thioesterase
• Uses Cys and SH 4'-phospho-pantetheine

First Round in Fatty Acid Synthesis

• To initiate FA biosynthesis, malonyl and acetyl groups activate onto the enzyme fatty acid synthase
• Initially, a priming molecule of acetyl-CoA combines with a cysteine —SH group catalyzed by acetyl transacylase
• Malonyl-CoA combines with the adjacent —SH on the 4'-phosphopantetheine of ACP of the other monomer
• This is catalyzed by malonyl transacylase to form acetyl (acyl)-malonylenzyme

Activation of the Molnyl Group

• The acetyl group, from acetyl-CoA, transfers to the Cys-SH group of the-ketoacyl ACP synthase
• The reactions are catalyzed by acetyl- CoA transacetylase

Fatty Acid Synthesis Steps

• Fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP
• Acetyl transacylase and malonyl transacylase catalyze these reactions

Condensation reaction

• Acetyl ACP and malonyl ACP react to form acetoacetyl ACP
• The enzyme is acyl-malonyl ACP condensing enzyme

Reduction Reaction

• Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP
• NADPH is the reducing agent
• The enzyme is β-ketoacyl ACP reductase

Dehydration

• D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP (trans-Δ2-enoyl ACP)
• The enzyme is 3-hydroxyacyl ACP dehydratase

Reduction Reaction

• Is the final step in the cycle
• Reduces crotonyl ACP to butyryl ACP
• NADPH is reductant
• The enzyme is enoyl ACP reductase

Step 4: Reduction

• Reduction of the double bond to form butyryl-ACP
• Reaction is catalyzed by enoyl-reductase
• Reaction is NADPH dependent

Elongation in Fatty Acid Synthesis

• In the second round, butyryl ACP condenses with malonyl ACP to form a C6-β-ketoacyl ACP
• Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl ACP into a C6-acyl ACP, ready for a third round of elongation.

Termination

• Synthesis continues until a C16 palmitoyl group forms
• Palmitoyl-ACP is hydrolyzed by a thioesterase to give palmitate + HS-ACP

Net Production

• The net reaction is: 8 acetyl-CoA + 14NADPH + 7 ATP → palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi
• The overall net reaction is: acetyl-CoA + 7 malonyl-CoA + 14 NADPH → palmitate + 7 CO2 + 14 NADP+ + 8 CoA

De Novo Synthesis of Fatty Acids

• Involves multiple steps with Cys-SCOA, ACP, and COASH to create Acylmalonyl enzyme

Chain Elongation

• During chain elongation
• A new malonyl-CoA molecule combines with the -SH of 4'-phosphopantetheine
• It displaces the saturated acyl residue onto the free cysteine —SH group
• This sequence of reactions repeats until a saturated 16-carbon acyl radical (Palmityl) has been assembled
• The enzyme, Thioesterase (deacylase), catalyzes this action

Result of Fatty Acid Synthesis

• Seven condensation and reduction cycles produce the 16-carbon saturated palmitoyl group while still bound to ACP
• Chain elongation ends at this point and free palmitate releases from the ACP molecule as chain elongation stops
• Stearate (18:0), is formed from smaller amounts of longer fatty acids such as

Fatty Acid Elongation

• Palmitate, found in animal cells, is the precursor of other long-chained FAs
• Through FA elongation systems, by further fatty chains, the length is further elongated
• The system is found in the same endoplasmic reticulum and in the mitochondria

Regulation of Fatty Acid Biosynthesis

• Acetyl-CoA carboxylase-catalyzed reactions is the rate-limiting step during fatty acid synthesis
• Long-chain fatty acid synthesis is controlled: -Short term: allosteric and covalent modification of enzymes -Long term: changes in gene expression

Conclusion

• Fatty acid biosynthesis occurs in the cytosol and its primary substrate is acetyl CoA
• Free palmitate is the end product
• It is an ATP and NADPH dependent reaction
• The reaction rate is regulated by acetyl CoA carboxylase
• Net Reaction: 8 Acetyl CoA + 7ATP + 14NADPH → Palmitate + 14NADP+ + 8CoA + 6H2O + 7ADP + 7Pi
• Lipogenesis (long-chain fatty acid synthesis) involves two enzymes:
• Acetyl-CoA carboxylase
• Fatty acid synthase
• The pathway converts acetyl-CoA to palmitate
• This pathway uses NADPH, ATP, Mn2+, biotin, pantothenic acid, and HCO3 − as cofactors
• Acetyl-CoA carboxylase converts acetyl CoA to malonyl-CoA
• Fatty acid synthase is a multienzyme complex of one polypeptide chain with seven enzymatic activities
• It catalyzes the assembly of palmitate from one acetyl-CoA and seven malonyl-CoA molecules
• Lipogenesis regulation - acetyl-CoA carboxylase step
• It is regulated by:
• Allosteric modifiers
• Phosphorylation/dephosphorylation
• Induction and repression of enzyme synthesis
• Citrate activates the enzyme
• Long-chain acyl-CoA inhibits activity
• Insulin activates acetyl-CoA carboxylase
• Glucagon and epinephrine have opposite impacts