Fatty Acid Synthesis Shuttle And Synthesis Steps

Overview of beta-reduction: Building FAs in the cytosol

• De novo fatty acid synthesis, metabolic process by which cells build FA from scratch starting with acetyl-CoA as the carbon source

• Occurs in liver and adipose tissue with rounds of chain lengthening and beta-reduction

• Occurs in the cytosol using acetyl-CoA tagged with CO2 to form malonyl-CoA for synthesis

• Acetyl groups needed in the cytosol are exported from the mitochondrial matrix carried by oxaloacetate in a citrate shuttle

De Novo synthesis Location and Features

• Occurs exclusively in the cytosol in liver and adipocytes , primarily produces palmitate (16:0)

• Mitochondria doesn't perform de novo synthesis, only specialized pathways like 8-carbon Fas for lipoic acid

• ER does not perform de novo fatty acid synthesis

Elongation and Desaturation

• Imported FAs (greater and equal to 12) elongate in the smooth ER

• Adds 2 carbon units from acetyl CoA

Reducing Equivalent

• Cytosolic NADP drives de novo fatty acid synthesis, supplies reducing power for biosynthesis without disrupting NADH-dependent energy metabolism

• NADPH is generated by 2 pathways

  1. Pentose Phosphate Pathway, Primary source

     1. Oxidizes glucose 6-phosphate to Ribulose 5-Phosphate to generate 2 NADPH

  2. Malic enzyme, secondary NADPH source

     1. Converts malate to pyruvate while reducing NADP+ to NADPH

Rationale

• High cytosolic NADPH/NADP+ ratio

• High NADPH drives biosynthetic reactions (fatty acid synthesis) forward

Why cells use separate NADH and NADPH redox currencies

1. Classic redox regulation

   1. High NADH/NAD+ = times of plenty: inhibits catabolism and drives anabolic fuel-storage pathways

   2. High NAD+/NADH = times of need: activates catabolism and suppresses biosynthetic pathways

2. Cell growth creates conflict

   1. Growing cells catabolize fuels for ATP and run anabolic pathways

   2. Catabolic and anabolic redox demands cannot be mets by the same NAD+/NADH pool

3. Solution

   1. NADH/NAD+ is for catabolism (low ratio = oxidation proceeds)

   2. NADPH/NADP+ is for anabolism (high ratio = reductive synthesis proceeds

   3. Cells burn fuels (low ratio = oxidation occurs NADH/NAD+) while building biomolecules (high NADPH/NADP) at the same time

• 2 currencies allows simultaneous catabolism and anabolism without redox interference

NADH vs NADPH

• NADP+ has a phosphate on 2C of the ring

• Low NAD+/NADH in mito drives catabolism (ETC). High NADPH/NADP+ in cytosol drives anabolism (FA synthesis)

• NADH inhibits anabolism, NAD+ acts as an oxidizing agent for anabolic pathways so high NADH means NAD+ is low. NADH inhibits anabolic enzymes which slows down production of precursors for biosynthesis. High NADPH drives anabolism for FA/sterol synthesis, nucleotide synthesis and ROS neutralization

• NAD/NADH is from fuel oxidation like TCA. NADPH is from primarily PPP and secondarily malic enzyme

Pentose Phosphate pathway makes NADPH

• Relies on glucose as primary source of cytosolic NADPH for PPP

  • NADPH is required for biosynthesis, therefore all human cells need glucose so it can oxidize other fuels for energy

• Input and Output

  • Uses glucose-6-phospahte (G6P) to generate 2 NADPH per G6P molecule

• Commitment step

  • Catalyzed by G6PDH which is the rate limiting and regulated step, NADPH inhibit it signalling there is enough currency. Feedback inhibition

• Anabolic role

  • It does lose a carbon atom as CO2, but PPP is anabolic as it provides reducing power for biosynthesis

  • Activated in time of plenty when G6P accumulates and glycolysis is inhibited

Additional Output

• Produces ribose-5-phosphate, essential for nucleotide and nucleic acid synthesis

Explain how acetyl-CoA is exported from mitochondria to the cytosol, detailing each step of

the citrate shuttle system.

Step 1: In the mitochondria

• Acetyl-CoA condenses with OAA to form citrate in the first step of TCA (citrate synthase)

Step 2

• When citrate exceeds TCA needs, it is exported to the cytosol by citrate/malate antiporter

• Exchanges citrate3- for malate2-, coupling citrate export to malate import to maintain metabolic balance

• Since citrate carries an extra charge, a proton is co-transported with citrate to preserve electroneutrality across the membrane

Step 3: commitment step

• In the cytosol, citrate lyase cleave citrated back into Acetyl-CoA and OAA, it consumes 1 ATP

• Acetyl-CoA becomes a substrate in FA synthesis

Step 4

• OAA is reduced to malate by cytosolic malate DH to facilitate return to mitochondria

Step 5

• Malate is imported by malate/alpha-KG antiporter, malate turns back into OAA using malate DH

Step 6

• Some malate is converted to pyruvate by malic enzyme in the cytosol, generating NADPH for FA synthesis

• Pyruvate is transported back into mitochondria and reconverted to OAA by pyruvate carboxylase to complete the shuttle

• Couples carbon export with NADPH production, ensures both substrate (acetyl-CoA) and reducing power (NADPH) are available lipid biosynthesis

 

Citrate Shuttle

• Mitochondrial citrate (Acetyl-CoA + OAA) is exported to cytosol by citrate/malate antiporter

• ATP-citrate lyase cleaves citrate > acetyl-CoA + OAA

• OAA > malate which returns to the mitochondria by the malate/anti-KG antiporter) is converted to pyruvate and generates NADPH

• Pyruvate re-enters mitochondria and is carbonylated back to OAA completing the cycle

• ATP cost of 1 per citrate cleavage

• NADP generation is by malic enzyme in the cytosol to support fatty acid synthesis

• Supplies cytosolic acetyl-COA for lipid biosynthesis

• Generates NADPH for reductive biosynthesis and redox balance

• Integrates mitochondrial TCA cycle with cytosolic anabolism

• Antiporters maintain electroneutral transport and metabolite flux

FAS beta-reduction

• Distinct cytosolic enzymes carry out reactions to avoid highly exergonic steps of catabolism

• 2 key steps to bypass catabolic thiolase

• Acetyl-CoA carboxylase (ACC) perform ATP=dependent carboxylation of acetyl-CoA, creates a new committed step

• Then beta-ketoacyl synthase uses malonyl-CoA as a two carbon donor

• Fatty acid synthesis relies on NADPH instead of NADH and FADH2, separation of redox pools allows high cytosolic [NADPH]/[NADP+] to drive anabolism an dlow cytoplasmic [NADH/NAD+] support catabolic glucose metabolism

Difference between beta-oxidation and beta-reduction

• Location

  • Mitochondria/peroxisomes vs cytosol

• Enzymes

  • Separate soluble enzymes vs multidomain FAS (single polypeptide)

• Redox

  • NADH/FADH2 vs NADPH (2 per cycle)

• Thiolase bypass

  • Exergonic thiolase, free energy is -33kJ/mol vs Malonyl-COA (ACC carboxylation) + KS decarboxylation (irreversible)

• Direction

  • Breaks 2C units (acetyl-COA vs builds 2 units via malonyl

Rationale

• Avoids futile cycling

• Uses NADPH for anabolism

• Malonyl decarboxylation drive C-C bond formation

Acetyl-COA carboxylate catalyzes the committed step: Formation of malonyl-CoA

Committed step in Fa synthesis: the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA

 

Purpose/Why Required

• Converts a 2C acetyl into a 3C activated building block

• Decarboxylation drive fatty-acid chain elongation, bypasses catabolic thiolase

• Regulatory: committing carbon to FA synthesis while inhibiting CPT-1 to prevent futile cycle with beta-oxidation

Reaction

• Acetyl-CoA + HCO3- + ATP > malonyl-COA + ADP + Pi

• Acetyl-CoA carboxylase

 

Mechanism is  similar to pyruvate carboxylate (PC)

• A biotin carrier protein

  • Biotin carboxylase uses ATP to active and load CO2 onto biotin

  • Performs transcarboxylase to transfer the activated CO2 to acetyl-CoA

  • ATP driveCO2 activation on biotin, carboxyl transfer to acyl-COA

• Conserved metabolic strategy for building carboxylate intermediates

FAS, Fatty Acid Synthesis

• Multidomain homodimer, single polypeptide, synthesizes palmitate (16:0) through 7 iterative elongation each adding 2 carbons

  • Enabled efficient substrate channeling and coordinated reaction

• Domains: MAT, AT, KS, KR, DH, ER, TE, ACP

  • ACP is the swinging arm shuttle

  • KS and ACP act crosswise, monomers cooperate than function independently

• 6 steps per elongation Round

Step 0: Loading/Thioester Activation (MAT, AT Domains)

• Loading substrates on FAS by 2 acyltransferase domains

• Acetyl-CoA transacylase (AT) transfers the acetyl group from Acetyl-CoA onto the active site cysteine of beta-ketoacyl synthase (KS) domain

  • Covalent thioester to primes FAS, initiates 2C primer

• Malonyl-COA transacylase (MT) domain transfers malonyl group from malonyl-CoA to 4'-phosphopantetheine thiol on ACP, forming malonyl-S-ACP

  • Malonyl is an acetyl-CoA with carboxyl on the methyl group

Mechanism

• Nucleophilic acyl substitution

  • Thiolate (S-) attacks thioester carbonyl of incoming acyl-COA to release CoA-SH

  • Forms new thioester

Purpose

• Positions acetyl on CS and malonyl on ACP for the first condensation

• The chain on KS reacts with malonyl group on ACP, malonyl goes through decarboxylation

  • Makes it more reactive and drives the C-C bond formation forward, Claisen condensation

• Malonyl sits on ACP, the growing chain sits on KS

Fatty Acid tethering

• Growing fatty acyl chain and incoming malonyl group are transferred from CoA onto thiol group within FAS

• Nascent acyl chain (acetyl at the start) is loaded onto the KS domain and malonyl group is loaded onto the ACP domain

  • Serves as a flexible swinging arm to deliver substrates to each active site

• Analogy: FAS function like ribosome

  • Like the P site, ACP holds the growing peptide

  • Like the A site, KS domain accepts the incoming aminoacyl-tRNA

    • Holds incoming acyl-group for condensation

    • CO2 leaves malonyl-ACP exposing an enolate nucleophile that attacks the nascent acyl-KS

  • Products = HS-KS and beta-ketoacyl-S-ACP (2C longer)

Step 1 Chain elongation: Condensation reaction of FAS

Fatty acid elongation starts withs KS-catalyzed condensation of malonyl-ACP with the acyl-KS thioester

Beta-ketoacyl synthase (KS)

• Acyl-S-KS + malonyl-S-ACP > beta-ketoacyl-S-ACP + CO2

• Beta-ketoacyl-ACP extends by 2 carbon

• Malonyl provides the both activated nucleophile

Mechanism

• Decarboxylation generates a nucleophilic enolate

• Enolate attacks acyl-KS thioester carbonyl, forms a new C-C bond (attacks carbonyl

• CO2 release provides thermodynamic drive (irreversible)

  • Activated nucleophile (via decarboxylation) and the thermodynamic push needed for C-C bond

  • Acyl group on KS is the correct electrophile

Key

• Bypasses catabolic thiolase; malonyl provides activated 2C donor

• Backbone for subsequent reduction-dehydration-reduction sequence to build chain

 

Step 2 - Reduction of Beta-Keto Reduction (KR domain)

Beta-Ketoacyl-ACP reductase (KR

• Beta-ketoacyl-S-ACP + NADPH > beta-hydroxyacyl-S-ACP + NADP+

• Reduced using NADPH as the electron donor, turns a ketone to hydroxy

Thermodynamics

• -25kJ/mol

• Highly exergonic from high cytosolic NADPH/NADP+ ratio, controlled stepwise elongation for the fatty acid

• Anabolic nature of fatty acid synthesis, links reaction to cells reducing power

Mechanism

• Hydride transfer from NADPH to beta-carbonyl

• Protonation of oxygen = hydroxy group

• Prepare for dehydration and enoyl reduction steps

Purpose

• Convert reactive beta-keto to stable alcohol from dehydration

• Remains tethered to ACP

 

Step 3 - Dehydration of beta-hydroxyacyl-ACP

Dehydratase (DH)

• beta-hydroxyacyl-S-ACP > trans-enoyl-S-ACP + H2O

• Removes H2O to form a trans double bond between alpha-beta carbon

Mechanism

• Acid/base catalysis: DH positions B-hydroxyacyl intermediate to facilitate proton abstraction from the alpha-carbon,

• B-hydroxyl elimination forms trans doble bond

Outcome

• Reaction is favourable as it generates a conjugated system that stabilizes the intermediate for the next enoyl reduction

• Prepares intermediate for full saturation

• Growing chain remains tethered to ACP

 

Step 4 - NADPH-driven reduction of enoyl-ACP to a saturated fatty Acid, Enoyl Reduction

Enoyl-ACP reductase (ER)

• Trans-enoyl-S-ACP + NADPH > saturated acyl-S-ACP + NADP+

• Enoyl-ACP intermediate is fully saturated acyl-ACP by the ER domain

• Uses NADPH as electron donor, C=C between beta-alpha C to single bond

• After the step the chain is ready for another round of elongation or release when it has reached 16 carbons

Mechanism

• Hydride donor form NADPH to beta-carbon, shift the electrons of double bond to carbonyl oxygen of thioester

• Oxyanion collapse to reform carbonyl when a proton adds to the alpha-carbon, yields saturated acyl chain

Thermodynamics

• Exergonic due to NADPH oxidation

Step 5: Chain Transfer, priming the next cycle (KS domain)

• The next FAS cycle, the four-carbon acyl group transfer to the KS thioester

B-ketoacyl synthase catalyzes transesterification

• Active site cysteine attack the thioester carbonyl of butyryl-ACP

• Forms KS-bound thioester, releasing ACP

• New malonyl unit is loaded onto ACP

Cycle repeats for 7 rounds

Chain termination

• Elongation continues until 7 rounds produce the 16 carbon saturated palmitoyl-ACP

• De novo FA elongation terminates at at 16:0

  • Plants like coconut terminate earlier at 8-14C

• Thioesterase(TE) domain of FAS hydrolyze the acyl-ACP, releases free palmitate

  • palmitoyl-S-ACP + H2O > free palmitate + HS-ACP

To build per palmitate

7 malonyl-CoA + 1 acetyl-CoA; 14 NADPH (2/cycle)

Summary

• De novo FA synthesis occurs in liver and adipose tissue with rounds of chain lengthening and beta-reduction, reverse of FA catabolism

• FAS is located in the cytosol

• Uses  acetyl-CoA that is tagged with CO2 in the form of malonyl-CoA to be reserved for synthesis

• Acetyl-CoA group are supplied by a citrate shuttle to transport acetyl-CoA from matrix to cytosol

• NADPH-drive FA synthesis in cytosol (for export or storage)

  • Able to run simultaneously with NAD+ driven glucose oxidation

Carbon origin in palmitate

• 1 acetyl-CoA contributes to 2 carbons: acts as a primer (carbon 15-16, omega end)

• 7 malonyl-CoA contribute to 14 carbons: elongating units (carbon 1-14)

• Each malonyl-CoA

  • Derived from acetyl-CoA + HCO3- + ATP by ACC

  • Decarboxylates during KS condensation, losses CO2

  • Adds 2 carbons per cycle

 

Reducing equivalents: FAS vs B-oxidation

FAS elongation Cycle, Anabolism (cytosol)

Per cycle add 2 C, 2 NADPH

1. KR, B-keto > B-hydroxyl

   1. 1 NADPH

2. ER, enoyl > saturated

   1. 1 NADPH

Total for palmitate: 14 NADPH  (7 cycles*2)

Sources of NADPH

• Primarily PPP

• Secondarily malic enzyme

 

B-oxidation (mitochondria)

Per cycle, removes 2C: uses 1NADH and 1 FADH2

1. 3-hydrxyacyl-CoA dehydrogenase

   1. 1NADH

2. Enoyl-CoA hydratase > acyl-CoA hydrogenase

   1. 1 FADH2

 

Total for palmitate

• 8 acetyl-CoA: 7 NADH + 7 FADH2