23b-Biochemistry-Lecture23b | Week 13 - Lecture 1

Lipid Metabolism and Fatty Acid Biosynthesis

Key Concepts in Fatty Acid Metabolism

  • Pathway Distinction:

    • Catabolism (oxidation) of fatty acids (breakdown):

      • Occurs in mitochondria

      • Forms acetyl-CoA and produces NADH

    • Anabolism (synthesis):

      • Takes place in cytosol of animals (and chloroplasts of plants)

      • Requires both acetyl-CoA and malonyl-CoA as starting materials

      • Utilizes NADPH as reducing power

Fatty Acid Biosynthesis Steps

  • Fatty acids are built in several passes, adding one acetate unit at a time by the fatty acid synthase complex (in the cytoplasm, where NADPH/NADP⁺ is high).

  • Acetate is converted to malonyl-CoA (a 3-carbon intermediate) using acetyl-CoA.

  • Malonyl-CoA formation is catalyzed by Acyl CoA Carboxylase (ACC).

  • Each pass reduces a carbonyl carbon to a methylene carbon.

  • Synthesis is similar to β-oxidation in reverse, but uses:

    • Different enzymes

    • Different cofactors

    • Occurs in a different location

  • The end product is palmitoyl-CoA, which inhibits ACC (negative feedback).

Comparison: Fatty Acid Synthesis vs β-Oxidation

  • β-Oxidation: Repeated cycles of oxidation, hydration, oxidation, cleavage.

  • Synthesis: Repeated cycles of condensation, reduction, dehydration, reduction.

  • Opposite reactions but with different enzymes and energy flow.

Acetyl-CoA is Transported from Mitochondria into the Cytosol as Citrate

  • Acetyl-CoA is made in the mitochondria.

  • Lipid synthesis occurs in the cytosol, but acetyl-CoA cannot cross the mitochondrial membrane.

  • Solution: Convert acetyl-CoA to citrate:

    • Acetyl-CoA + oxaloacetate → citrate (same as CAC reaction, catalyzed by citrate synthase).

    • Citrate crosses to the cytosol via the citrate transporter.

Citrate in Cytosol is Cleaved to Regenerate Acetyl-CoA

  • Citrate is cleaved by citrate lyase to regenerate:

    • Acetyl-CoA

    • Oxaloacetate

  • Reaction requires 1 ATP.

  • Acetyl-CoA can now be used to make malonyl-CoA.

Oxaloacetate is converted to Malate, which has two fates

  • Converted to malate by malate dehydrogenase.

  • Malate has 2 fates:

    1. Converted to pyruvate + NADPH (via malic enzyme):

      • Pyruvate enters mitochondria and is converted to oxaloacetate.

      • NADPH is used for fatty acid synthesis.

    2. Transported back to mitochondria as malate via malate-α-ketoglutarate transporter, then reoxidized to oxaloacetate.

SHUTTLE FOR TRANSFER OF ACETYL GROUPS FROM MITOCHONDRIA TO THE CYTOSOL

  • The mitochondrial outer membrane is freely permeable to all these compounds.

  • Pyruvate, derived from amino acid catabolism in the mitochondrial matrix or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the matrix.

  • Acetyl groups pass out of the mitochondria as citrate; in the cytosol they are delivered as acetyl-CoA for fatty acid synthesis.

  • Oxaloacetate is reduced to malate, which can return to the mitochondrial matrix and be converted to oxaloacetate.

  • The major fate for cytosolic malate is oxidation by malic enzyme to generate cytosolic NADPH; the pyruvateproduced returns to the mitochondrial matrix.

ACETYL-CoAᶜʸᵗ AND BICARBONATE FORM MALONYL-CoA

  • Reaction carboxylates acetyl-CoA.

  • Catalyzed by Acetyl-CoA Carboxylase (ACC) (rate limiting step of fatty acid synthesis):

    • Enzyme has three subunits:

      • One unit has Biotin to carry CO₂.

      • HCO₃⁻ (bicarbonate) is the source of CO₂.

      • Uses one ATP molecule.

      • In animals, all three subunits are on one polypeptide chain.

  • Therefore, the cost of malonyl-CoA formation (precursor for fatty acid synthesis) is 3 ATPs per 2-carbon unit (2 for shuttling acetyl-CoA to the cytosol and 1 for making malonyl-CoA).

A REMINDER: BIOTIN CARRIES CO₂

  • Two-step reaction similar to carboxylations catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase.

  • CO₂ binds to biotin:

    • Bicarbonate reacts with ATP to produce carboxyphosphate as an intermediate.

    • Carboxyphosphate breaks down to CO₂.

    • CO₂ is attached to N in ring of biotin.

    • Biotin transports CO₂ to acetyl-CoA on another active site.

THE ACETYL-CoA CARBOXYLASE (ACC) REACTION

Malonyl-CoA is formed from Acetyl-CoA and Bicarbonate

  • ACC has 3 functional regions:

    • Biotin carrier protein

    • Biotin carboxylase, which activates CO₂ by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction

    • Transcarboxylase, which transfers activated CO₂ (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA.

  • The long, flexible biotin arm carries the activated CO₂ from the biotin carboxylase region to the transcarboxylase active site.

  • The active enzyme in each step is shaded blue in the diagram.

FATTY ACID SYNTHESIS OCCURS IN CELL COMPARTMENTS WHERE NADPH LEVELS ARE HIGH

  • Cytosol for animals (and yeast)

  • Major sources of NADPH:

    1. In adipocytes: pentose phosphate pathway and malic enzyme:

      • NADPH made as malate converts to pyruvate + CO₂

    2. In hepatocytes and mammary gland: pentose phosphate pathway

      • NADPH made as glucose-6-phosphate converts to ribulose-5-phosphate

    3. To a lesser extent, NADP dependent isocitrate dehydrogenase reaction → isocitrate is oxidatively decarboxylated to α-ketoglutarate (depends on mitochondria)

SYNTHESIS OF FATTY ACIDS IS CATALYZED BY FATTY ACID SYNTHASE (FAS)

  • Catalyzes a repeating four-step sequence that elongates the fatty acyl chain by two carbons at each step.

    • Uses NADPH as the electron donor

    • Uses two enzyme-bound –SH groups as activating groups

  • FAS I in vertebrates:

    • Single polypeptide chain with several enzyme activities

    • Leads to single product: palmitate 16:0

    • C-15 and C-16 are from the acetyl CoA used to prime the reaction

FATTY ACID SYNTHESIS

  • Overall goal: attach two-carbon acetate unit from malonyl-CoA to a growing chain and then reduce it.

  • Reaction involves cycles of four enzyme-catalyzed steps:

    • Condensation of the growing chain with activated acetate

    • Reduction of carbonyl to hydroxyl

    • Dehydration of alcohol to trans-alkene

    • Reduction of alkene to alkane

  • The growing chain is initially attached to the enzyme via a thioester linkage.

  • During condensation, the growing chain is transferred to the acyl carrier protein (ACP)

  • After the second reduction step, the elongated chain is transferred back to fatty acid synthase.

ACYL CARRIER PROTEIN (ACP) SERVES AS A SHUTTLE IN FATTY ACID SYNTHESIS

  • Contains a covalently attached prosthetic group 4’-phosphopantetheine.

    • Flexible arm to tether acyl chain while carrying intermediates from one enzyme subunit to the next.

  • Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase.

  • Shuttles the growing chain from one active site to another during the four-step reaction.

FATTY ACID SYNTHASE TYPE I (FAS I)

Mammalian FATTY ACID SYNTHASE (FAS I): a mega synthase with multiple active sites

  • Condensation with acetate → β-ketoacyl-ACP synthase (KS)

  • Reduction of carbonyl to hydroxyl → β-ketoacyl-ACP reductase (KR)

  • Dehydration of alcohol to alkene → β-hydroxyacyl-ACP dehydratase (DH)

  • Reduction of alkene to alkane → enoyl-ACP reductase (ER)

  • Chain transfer/charging → Malonyl/acetyl-CoA ACP transferase (MAT)

  • Thioesterase → TE: releases the palmitate product from ACP

  • ACP → Acyl Carrier Protein

FATTY ACID SYNTHESIS PROCEEDS IN A REPEATING REACTION SEQUENCE

Addition of two carbons to a growing fatty acyl chain: a four-step sequence

  • Each malonyl group (on Acyl Carrier Protein; ACP) and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, then:

    1. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) with two carbons derived from malonyl-CoA, with elimination of CO₂ from the malonyl group, extends the acyl chain by two carbon units.

    2. Reduction of the β-keto group to an alcohol.

    3. Dehydration (elimination of H₂O) → to create a double bond

      • Enzyme: DH (Dehydratase)

    4. Reduction of the double bond → to create a saturated fatty acyl group

      • Enzyme: ER (Enoyl-ACP Reductase)

    5. Translocation of the elongated product to an SH group on FAS → recharging the now free SH of ACP with another malonyl group

THE OVERALL PROCESS OF PALMITATE SYNTHESIS: SEVEN CYCLES OF CONDENSATION AND REDUCTION

  • The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO₂ at each step.

  • The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO₂ is shaded green.

  • After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on.

  • The final product is palmitate (16:0).

STOICHIOMETRY OF SYNTHESIS OF PALMITATE (16:0)

  1. Formation of 7 malonyl-CoA:
    7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs… using ATP:
    7 Acetyl-CoA + 7 CO₂ + 7 ATP → 7 Malonyl-CoA + 7 ADP + 7 Pi

  2. 7 Cycles of condensation, reduction, dehydration, and reduction:
    Uses NADPH to reduce the β-keto group and trans-double bond:
    Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 14 H⁺ → Palmitate + 7 CO₂ + 8 CoA + 14 NADP⁺ + 6 H₂O

  3. Sum:
    8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H⁺ → Palmitate + 8 CoA + 6 H₂O + 7 ADP + 7 Pi + 14 NADP⁺

  • Note: The total cost of fatty acid synthesis is 3 ATPs per 2-carbon unit
    (2 for shuttling acetyl-CoA to cytosol, and 1 for making malonyl-CoA) → Minus 1 ATP for the first acetyl-CoA (not from malonyl-CoA).

FATTY ACID SYNTHESIS IS TIGHTLY CONTROLLED VIA ALLOSTERIC REGULATION OF ACC

  • Acetyl-CoA Carboxylase (ACC) catalyzes the rate-limiting step

    • Inhibited by palmitoyl-CoA

    • Activated by citrate

      • Citrate is made from acetyl-CoA

      • When [acetyl-CoA]⁽ᵐᵗ⁾ ↑ (high ATP), citrate is exported → activates ACC

      • Citrate signals excess energy to be converted to fat

    • Note: Citrate also inhibits glycolysis by inhibiting PFK-1

ACC IS ALSO HORMONALLY REGULATED BY COVALENT MODIFICATION

  • ACC is inhibited when energy is needed (low glucose):

    • Glucagon and epinephrine:

      • ↓ Citrate activation sensitivity

      • → Phosphorylation & inactivation of ACC

      • (Phosphorylated monomers of ACC are inactive)

  • ACC is activated when energy is plenty (high glucose):

    • Insulin:

      • → Dephosphorylation of ACC monomers

      • → Polymerization into long active ACC filaments

RECAP: REGULATION OF FATTY ACID SYNTHESIS IN VERTEBRATES

  • Regulation of fatty acid synthesis:
    (a) In vertebrates, allosteric & hormone-dependent covalent regulation influence precursor flow to malonyl-CoA
    (b) ACC filaments (active, dephosphorylated form) are observed via electron microscopy

COORDINATED REGULATION OF FATTY ACID SYNTHESIS AND BREAKDOWN

  • Two enzymes are key to the coordination of fatty acid metabolism: acetyl-CoA carboxylase (ACC) (the first enzyme in the synthesis of fatty acids), and carnitine acyltransferase I (which limits the transport of fatty acids into the mitochondrial matrix for β-oxidation).

  • Synthesis: Acetyl-CoA Carboxylase (ACC)

  • Oxidation: Carnitine Acyl Transferase I (CAT I)

REGULATION TARGETS

  • When the diet provides a ready source of carbohydrate as fuel, β-oxidation of fatty acids is unnecessary and is therefore downregulated.

  • Ingestion of a high-carbohydrate meal raises the blood glucose level and thus triggers the release of insulin.

  1. Insulin triggers the release of protein phosphatase which dephosphorylates ACC, activating it.

  2. ACC catalyzes the formation of malonyl-CoA (the first intermediate of fatty acid synthesis).

  3. Malonyl-CoA inhibits carnitine acyltransferase I, thereby preventing fatty acid entry into the mitochondria.

  • When blood glucose levels drop between meals:

  1. Glucagon is released and activates cAMP-dependent protein kinase (PKA).

  2. PKA phosphorylates and inactivates ACC.

  3. The concentration of malonyl-CoA falls, the inhibition of fatty acid entry into mitochondria is relieved.

  4. Fatty acids enter the mitochondrial matrix.

  5. β-oxidation becomes the major fuel source.

  6. Because glucagon also triggers the mobilization of fatty acids in adipose tissue, a supply of fatty acids begins arriving in the blood.

  • Glucagon also triggers mobilization of fatty acids from lipid droplets in adipose tissue (through phosphorylation of HSL).

  • Also, transcriptional regulation of CAT, acyl CoA dehydrogenases, etc., contributes to long-term adaptation.

LONGER-CHAIN AND UNSATURATED FATTY ACIDS ARE SYNTHESIZED FROM PALMITATE

Routes of synthesis of other fatty acids:
Elongation of Palmitate (16:0) → Stearate (18:0)

  • Catalyzed by elongase

    • Adds two carbons at COOH end

    • Uses malonyl-CoA, just like fatty acid synthase (FAS)

Desaturation of Palmitate (16:0) → Palmitoleate (16:1Δ9)

Stearate (18:0) → Oleate (18:1Δ9)

  • Catalyzed by fatty acyl-CoA desaturase

    • Requires NADPH

    • Uses cytochrome b₅ and cytochrome b₅ reductase

This is a Δ9-desaturase! It reduces the bond between C-9 and C-10

  • Mammals cannot desaturate beyond Δ9 → cannot make linoleate or α-linolenate
    ➝ Must be obtained in diet → essential fatty acids

  • Conversion of α-linolenate (ALA) to:

    • EPA: 20:5 (Δ5,8,11,14,17)

    • DHA: 22:6 (Δ4,7,10,13,16,19)

    • Requires elongation/desaturation

PLANTS, BUT NOT MAMMALS, CAN DESATURATE POSITIONS BEYOND C-9

  • Humans have Δ4, Δ5, Δ6, and Δ9 desaturases

  • But cannot desaturate beyond Δ9

  • Plants can produce:

    • Linoleate (18:2, Δ9,12)

    • α-Linolenate (18:3, Δ9,12,15)

  • These are essential fatty acids for humans and are precursors to:

    • PUFAs (polyunsaturated fatty acids) → membrane fluidity, precursors to eicosanoids

VERTEBRATE FATTY ACYL DESATURASE: NON-HEME IRON-CONTAINING OXIDASE

  • O₂ accepts 4 electrons total:

    • 2 from saturated fatty acid

    • 2 from ferrous state of cytochrome b₅ / NADPH

  • Located in smooth ER

  • Electrons flow through:
    Fatty acyl-CoA → NADPH → desaturation pathway

BIOSYNTHESIS OF TRIACYLGLYCEROLS

  • Phosphatidic acid is the precursor for both TAGs and glycerophospholipids

  • Lipin (phosphatidic acid phosphatase) removes the phosphate:

    • Yields 1,2-diacylglycerol

  • Third carbon is acylated with another fatty acid:

    • Forms triacylglycerol (TAG)

BIOSYNTHESIS OF MEMBRANE LIPIDS
PHOSPHODIESTER BOND OF PHOSPHOLIPIDS FROM CDP

Two general strategies for forming the phosphodiester bond of phospholipids:

  • In both cases, CDP supplies the phosphate group of the phosphodiester bond.

Strategy 1: Diacylglycerol activated with CDP

  1. DAG is activated with CDP

  2. The head group is transferred

    • Example: Inositol → phosphatidylinositol

Strategy 2: Head group activated with CDP

  1. Headgroup is activated with CDP

  2. DAG is transferred

    • Example: CDP-choline → phosphatidylcholine

BIOSYNTHESIS OF MEMBRANE LIPIDS
SPHINGOLIPIDS ARE MADE IN FOUR STEPS

  1. Synthesis of sphinganine from palmitoyl-CoA and serine

  2. Attachment of fatty acid via amide linkage

  3. Desaturation of sphinganine

    • Yields N-acylsphingosine (ceramide)

  4. Attachment of head group

    • Can yield a cerebroside or ganglioside

Summary of Fatty Acid Biosynthesis and Degradation

  • Fatty acid synthesis and degradation consist of cycles to add or remove 2-carbon units, governed by distinct but coordinated pathways.

  • Key regulatory enzymes involved include Acetyl-CoA Carboxylase and Carnitine Acyltransferase I.