13 - Fatty Acid Oxidation

TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Metabolic Biochemistry::13 - Fatty Acid Oxidation

Fatty Acid Oxidation — Fat Metabolism

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Overview: Fat Metabolism Pathway

Lipoprotein → [Lipoprotein lipase 3.1.1.34]
             → [Hormone-sensitive lipase 3.1.1.3]
             → Fatty acid
             → [Fatty acid-CoA ligase 6.2.1.3]
             → Fatty acyl-CoA
             → [Carnitine O-acyltransferase 2.3.1.21]
             → Mitochondrial β-oxidation
             → Acetyl-CoA
             → [Pyruvate carboxylase 6.4.1.2 (Biotin)]

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Activation of Fatty Acid to Acyl-CoA

Prerequisite Step

Conversion of a fatty acid to a fatty acyl-CoA is the essential prerequisite for all fatty acid reactions:

• Catabolism: β-oxidation
• Anabolism: biosynthesis of lipids

Carboxylic Acids — Nomenclature

Name	Carbons	Double Bonds	Notes
Formic acid	1:0	0	Not in lipids
Acetic acid	2:0	0	Not in lipids
Propionic acid	3:0	0
Butyric acid	4:0	0
Valerianic acid	5:0	0
Caproic acid	6:0	0
Caprylic acid	8:0	0
Capric acid	10:0	0
Lauric acid	12:0	0
Myristic acid	14:0	0
Palmitic acid	16:0	0
Stearic acid	18:0	0
Arachidic acid	20:0	0
Behenic acid	22:0	0

Nomenclature Convention

Fatty acids are named by: Number of carbons : Number of double bonds, with positions of double bonds specified separately (e.g., ${\Delta }^9$ for oleic acid).

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Fatty acids are named using the format {1:carbons:double bonds}, and the naming also specifies the {2:position} of double bonds.

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Coenzyme A Structure

Coenzyme A (CoA)

CoA functions in acyl group transfer reactions. The acyl group (e.g., acetyl or acetoacetyl) is attached via a thioester linkage to the -mercaptoethylamine moiety.

Components:

• 4-Mercaptoethylamine
• Pantothenic acid
• 3’-Phosphoadenosine diphosphate (3’-P-ADP)

NAD⁺ functions in hydride transfers; FAD (active form of vitamin B2/riboflavin) functions in electron transfers.

SCCNC(=O)CCNC(=O)[C@@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H]([C@H](O)[C@@H]1OP(=O)(O)O)n1cnc2c(N)ncnc12

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Coenzyme A links acyl groups via a {1:thioester} bond to its {2:4-mercaptoethylamine} moiety.

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Fatty Acid Activation: Fatty Acyl-CoA Synthetase

Mechanism (Two Steps)

Step 1: The carboxylate ion displaces the outer two phosphates ($\beta$ and $\gamma$) of ATP → forms fatty acyl-adenylate (mixed anhydride of carboxylic acid and phosphoric acid) + PPi.

$\text{Fatty acid}+\text{ATP}\to \text{Fatty acyl-adenylate}+{\text{PP}}_i$

Thermodynamic Pull

PPi is an excellent leaving group and is immediately hydrolyzed to 2 Pi by inorganic pyrophosphatase, pulling the reaction forward.

Step 2: The thiol group of CoA performs nucleophilic attack on the enzyme-bound mixed anhydride → displaces AMP → forms fatty acyl-CoA (thioester).

$\text{Fatty acyl-adenylate}+\text{CoA-SH}\to \text{Fatty acyl-CoA}+\text{AMP}$

$\Delta G_{\text{Step 1}}^{\circ \prime }=-19\text{ kJ/mol},\Delta G_{\text{overall}}^{\circ \prime }=-19\text{ kJ/mol}$

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In fatty acid activation, {1:PPi} is hydrolyzed by {2:inorganic pyrophosphatase}, thermodynamically pulling the reaction forward.

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The fatty acid activation reaction requires {1:2} high-energy phosphate bonds (from ATP), producing {2:AMP} and {3:PPi} as byproducts.

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Transport into the Mitochondrion: The Carnitine Shuttle

Compartmentalization

Fatty acyl-CoA esters formed at the cytosolic side of the outer mitochondrial membrane can either:

• Be transported into the mitochondrion → oxidized to produce ATP
• Remain in cytosol → used for lipid biosynthesis

L-Carnitine

Info

L-Carnitine ([-], β-hydroxy-γ-N-trimethylaminobutyric acid) is a highly polar, water-soluble quaternary amine that exists as a zwitterion under physiological conditions.

C[N+](C)(C)CC(O)CC([O-])=O

Steps of the Carnitine Shuttle

Step	Location	Reaction
1	Outer mitochondrial membrane	Fatty acyl-CoA + Carnitine → Fatty acyl-carnitine + CoA (catalyzed by Carnitine acyltransferase I)
2	Inner membrane	Fatty acyl-carnitine transported into matrix via acyl-carnitine/carnitine transporter
3	Mitochondrial matrix	Fatty acyl-carnitine + CoA → Fatty acyl-CoA + Carnitine (catalyzed by Carnitine acyltransferase II)

Regulation — Malonyl-CoA Inhibition

Carnitine acyltransferase I is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis. This prevents simultaneous synthesis and degradation of fatty acids.

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The carnitine shuttle is inhibited by {1:malonyl-CoA}, which blocks {2:carnitine acyltransferase I} at the outer mitochondrial membrane.

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Conversion of a fatty acyl-CoA to its {1:fatty acyl-carnitine} ester commits the fatty acid to {2:mitochondrial oxidation}.

Two Separate CoA Pools

Info

The carnitine shuttle maintains two functionally separate pools of CoA and fatty acyl-CoA:

• Mitochondrial matrix CoA: oxidative degradation of pyruvate, fatty acids, amino acids
• Cytosolic CoA: biosynthesis of fatty acids and membrane lipids

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Stages of Fatty Acid Oxidation

Three Stages

• Stage 1: Long-chain fatty acid → acetyl residues as acetyl-CoA via β-oxidation
• Stage 2: Acetyl-CoA → CO₂ via the citric acid cycle
• Stage 3: Electrons from stages 1 & 2 → O₂ via the mitochondrial respiratory chain → ATP via oxidative phosphorylation

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The three stages of fatty acid oxidation are: {1:β-oxidation} → {2:citric acid cycle} → {3:oxidative phosphorylation}.

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β-Oxidation of Saturated Fatty Acids: Four Reactions

Example substrate: Palmitoyl-CoA (C16)

Reaction 1 — Acyl-CoA Dehydrogenase (FAD-dependent)

$\text{Acyl-CoA}+\text{FAD}\to \text{trans-}{\Delta }^2\text{-Enoyl-CoA}+{\text{FADH}}_2$

• Introduces a trans double bond between $C_{\alpha }$ (C-2) and $C_{\beta }$ (C-3)
• Naturally occurring unsaturated fatty acids have cis double bonds
• Three isozymes based on chain length:

Isozyme	Chain Length
VLCAD (Very Long Chain)	12–18 carbons
MCAD (Medium Chain)	4–14 carbons
SCAD (Short Chain)	4–8 carbons

• All are flavoproteins with FAD as prosthetic group
• Electrons transferred to electron-transferring flavoprotein (ETF) → mitochondrial respiratory chain

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In the first step of β-oxidation, acyl-CoA dehydrogenase introduces a {1:trans} double bond between {2:C-2 (Cα)} and {3:C-3 (Cβ)}, using {4:FAD} as electron acceptor.

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VLCAD acts on fatty acids of {1:12–18} carbons; MCAD on {2:4–14} carbons; SCAD on {3:4–8} carbons.

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Reaction 2 — Enoyl-CoA Hydratase

$\text{trans-}{\Delta }^2\text{-Enoyl-CoA}+H_{2}O\to \text{L-}\beta \text{-Hydroxyacyl-CoA}$

• Water is added across the double bond
• Produces specifically the L stereoisomer of β-hydroxyacyl-CoA (3-hydroxyacyl-CoA)

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Enoyl-CoA hydratase adds {1:water} to the trans-Δ² double bond, producing the {2:L stereoisomer} of β-hydroxyacyl-CoA.

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Reaction 3 — β-Hydroxyacyl-CoA Dehydrogenase (NAD⁺-dependent)

$\text{L-}\beta \text{-Hydroxyacyl-CoA}+{\text{NAD}}^{+}\to \beta \text{-Ketoacyl-CoA}+\text{NADH}+H^{+}$

• Enzyme is absolutely specific for the L stereoisomer
• NADH donates electrons to NADH dehydrogenase → respiratory chain → ATP synthesis

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β-Hydroxyacyl-CoA dehydrogenase is absolutely specific for the {1:L stereoisomer} and uses {2:NAD⁺} as electron acceptor, producing {3:NADH}.

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Reaction 4 — Acyl-CoA Acetyltransferase (Thiolase)

$\beta \text{-Ketoacyl-CoA}+\text{CoA-SH}\to {\text{Acyl-CoA}}_{(n-2)}+\text{Acetyl-CoA}$

• Cleaves the β-ketoacyl-CoA, releasing a 2-carbon acetyl-CoA unit from the carboxyl terminus
• Remaining fatty acyl-CoA is shortened by 2 carbons

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Thiolase cleaves β-ketoacyl-CoA using {1:free CoA}, releasing {2:acetyl-CoA} and a fatty acyl-CoA shortened by {3:two} carbons.

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Summary: One Full Cycle on Palmitoyl-CoA (C16)

Net products per cycle of β-oxidation

Each pass yields:

• 1 × FADH₂
• 1 × NADH
• 1 × Acetyl-CoA
• Acyl-CoA shortened by 2 carbons

For palmitoyl-CoA (C16): 7 cycles → 8 acetyl-CoA, 7 FADH₂, 7 NADH

$\text{Palmitoyl-CoA}+23O_2+108P_i+108ADP\to 108(106)ATP+16C{O}_2+23H_{2}O$

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Complete oxidation of palmitoyl-CoA (C16) requires {1:7} cycles of β-oxidation, yielding {2:8} molecules of acetyl-CoA.

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Analogy: β-Oxidation and the Citric Acid Cycle

Structural Parallel

Three steps of β-oxidation parallel the conversion of succinate → oxaloacetate in the TCA cycle:

β-Oxidation Step	TCA Cycle Equivalent
Acyl-CoA dehydrogenase (FAD)	Succinate dehydrogenase (FAD)
Enoyl-CoA hydratase (H₂O addition)	Fumarase (H₂O addition)
β-Hydroxyacyl-CoA dehydrogenase (NAD⁺)	Malate dehydrogenase (NAD⁺)

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The three steps of β-oxidation from acyl-CoA to β-ketoacyl-CoA are analogous to the TCA cycle steps from {1:succinate} to {2:oxaloacetate}.

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Oxidation of Unsaturated Fatty Acids

Monounsaturated Fatty Acids (e.g., Oleoyl-CoA, ${\Delta }^9$)

Info

Normal β-oxidation proceeds for 3 cycles (removing 6 carbons), leaving a cis-Δ³ intermediate.
An additional enzyme is required: enoyl-CoA isomerase → repositions the cis double bond to a trans-Δ² configuration → normal β-oxidation substrate.

CCCCCCCC/C=C\CCCCCCCC(=O)SCCNC(=O)CCNC(=O)[C@@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H]([C@H](O)[C@@H]1OP(=O)(O)O)n1cnc2c(N)ncnc12

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Oxidation of monounsaturated fatty acids requires the auxiliary enzyme {1:enoyl-CoA isomerase}, which converts a {2:cis-Δ³} intermediate to a {3:trans-Δ²} enoyl-CoA.

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Polyunsaturated Fatty Acids (e.g., Linoleoyl-CoA, ${\Delta }^{9,12}$)

Info

Requires two auxiliary enzymes:

1. enoyl-CoA isomerase
2. NADPH-dependent 2,4-dienoyl-CoA reductase

Together they convert a trans-2,cis-4-dienoyl-CoA intermediate → trans-Δ²-enoyl-CoA → normal β-oxidation.

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Oxidation of polyunsaturated fatty acids requires two auxiliary enzymes: {1:enoyl-CoA isomerase} and {2:NADPH-dependent 2,4-dienoyl-CoA reductase}.

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Oxidation of Odd-Chain Fatty Acids: Propionyl-CoA

Info

β-Oxidation of odd-numbered fatty acids produces propionyl-CoA (C3) as the final product.

Conversion of Propionyl-CoA → Succinyl-CoA

$\text{Propionyl-CoA}\stackrel{\text{propionyl-CoA carboxylase (Biotin, ATP)}}{\to }\text{D-methylmalonyl-CoA}\stackrel{\text{methylmalonyl-CoA epimerase}}{\to }\text{L-methylmalonyl-CoA}\stackrel{{\text{methylmalonyl-CoA mutase (Vit B}}_{12}\text{)}}{\to }\text{Succinyl-CoA}$

$\text{Propionyl-CoA}+C{O}_2+ATP\to \text{D-methylmalonyl-CoA}+ADP+P_i$

• Succinyl-CoA enters the citric acid cycle

Vitamin B₁₂ Dependency

Methylmalonyl-CoA mutase requires vitamin B₁₂ (cobalamin) as a cofactor. Deficiency leads to methylmalonic aciduria.

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The conversion of propionyl-CoA to succinyl-CoA proceeds via: propionyl-CoA → {1:D-methylmalonyl-CoA} → {2:L-methylmalonyl-CoA} → {3:succinyl-CoA}.

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Methylmalonyl-CoA mutase requires {1:vitamin B₁₂ (cobalamin)} as a cofactor.

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Propionyl-CoA carboxylase requires {1:biotin} and {2:ATP} to carboxylate propionyl-CoA to D-methylmalonyl-CoA.

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Regulation of Fatty Acid Oxidation

Malonyl-CoA and the Fed State

Important

Malonyl-CoA (first intermediate of cytosolic fatty acid synthesis) inhibits carnitine acyltransferase I → blocks fatty acid entry into mitochondria.

This occurs when:

• Carbohydrate intake is high
• Blood glucose is elevated → insulin ↑ → malonyl-CoA ↑

Purpose: prevents futile cycle of simultaneous fatty acid synthesis and oxidation.

Energy Sufficiency Signals

Allosteric Regulation

• High [NADH]/[NAD⁺] ratio → inhibits β-hydroxyacyl-CoA dehydrogenase (step 3)
• High [acetyl-CoA] → inhibits thiolase (step 4)

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When the [NADH]/[NAD⁺] ratio is high, {1:β-hydroxyacyl-CoA dehydrogenase} is inhibited, slowing β-oxidation.

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High concentrations of {1:acetyl-CoA} inhibit {2:thiolase}, preventing excess acetyl-CoA production from β-oxidation.

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In the well-fed state, elevated malonyl-CoA inhibits {1:carnitine acyltransferase I}, preventing {2:fatty acid} entry into the mitochondria for oxidation.

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Ketone Bodies

Overview

Definition

In the liver, acetyl-CoA from β-oxidation can enter the citric acid cycle or be converted to ketone bodies for export to other tissues.

Three ketone bodies:

Ketone Body	Notes
Acetoacetate	Primary exported form
D-β-Hydroxybutyrate	Reduced form; major fuel for extrahepatic tissues
Acetone	Minor, volatile; formed by spontaneous decarboxylation

CC(=O)CC(=O)O

(Acetoacetate)

C[C@@H](O)CC(=O)O

(D-β-Hydroxybutyrate)

CC(C)=O

(Acetone)

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The three ketone bodies are {1:acetoacetate}, {2:D-β-hydroxybutyrate}, and {3:acetone}.

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Formation of Ketone Bodies (Liver Mitochondrial Matrix)

Conditions promoting ketogenesis

• Starvation
• Untreated diabetes mellitus
• (Any condition where acetyl-CoA accumulates and OAA is drawn off for gluconeogenesis)

Pathway:

$2\text{Acetyl-CoA}\stackrel{\text{thiolase}}{\to }\text{Acetoacetyl-CoA}+\text{CoA}$

$\text{Acetoacetyl-CoA}+\text{Acetyl-CoA}+H_{2}O\stackrel{\text{HMG-CoA synthase}}{\to }\text{HMG-CoA}+\text{CoA}$

$\text{HMG-CoA}\stackrel{\text{HMG-CoA lyase}}{\to }\text{Acetoacetate}+\text{Acetyl-CoA}$

$\text{Acetoacetate}+\text{NADH}+H^{+}\stackrel{\text{β-hydroxybutyrate dehydrogenase}}{\to }\text{D-}\beta \text{-Hydroxybutyrate}+{\text{NAD}}^{+}$

$\text{Acetoacetate}\stackrel{\text{acetoacetate decarboxylase}}{\to }\text{Acetone}+C{O}_2$

HMG-CoA Dual Role

HMG-CoA is an intermediate in both ketone body synthesis (mitochondrial matrix) and sterol/cholesterol biosynthesis (cytosol). Different enzyme isoforms are responsible in each compartment; HMG-CoA lyase is present only in the mitochondrial matrix.

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HMG-CoA is an intermediate in both {1:ketone body synthesis} (mitochondria) and {2:sterol/cholesterol biosynthesis} (cytosol).

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Ketogenesis is promoted when {1:oxaloacetate} is drawn off for {2:gluconeogenesis}, slowing the citric acid cycle and causing acetyl-CoA to accumulate.

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Utilization of D-β-Hydroxybutyrate in Peripheral Tissues

Info

D-β-Hydroxybutyrate exported from liver → oxidized in extrahepatic tissues in three steps:

$\text{D-}\beta \text{-Hydroxybutyrate}+{\text{NAD}}^{+}\stackrel{\text{D-}\beta \text{-hydroxybutyrate dehydrogenase}}{\to }\text{Acetoacetate}+\text{NADH}+H^{+}$

$\text{Acetoacetate}+\text{Succinyl-CoA}\stackrel{\beta \text{-ketoacyl-CoA transferase}}{\to }\text{Acetoacetyl-CoA}+\text{Succinate}$

$\text{Acetoacetyl-CoA}+\text{CoA}\stackrel{\text{thiolase}}{\to }2\text{Acetyl-CoA}$

Liver Cannot Use Its Own Ketone Bodies

The liver lacks β-ketoacyl-CoA transferase (succinyl-CoA:3-oxoacid-CoA transferase), so it cannot reactivate acetoacetate for its own use. Ketone bodies are exclusively an export fuel.

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The liver cannot use its own ketone bodies because it lacks {1:β-ketoacyl-CoA transferase (succinyl-CoA:3-oxoacid-CoA transferase)}.

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Activation of acetoacetate in peripheral tissues requires a CoA group donated by {1:succinyl-CoA}, producing {2:succinate} and {3:acetoacetyl-CoA}.

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β-Oxidation Enzyme Summary (EC Numbers)

Enzyme	EC Number	Reaction
Acyl-CoA dehydrogenase	1.3.99.3	Acyl-CoA → trans-Δ²-Enoyl-CoA (FAD)
Enoyl-CoA hydratase	4.2.1.17	Enoyl-CoA → L-β-Hydroxyacyl-CoA
3-Hydroxyacyl-CoA dehydrogenase	1.1.1.35	L-β-Hydroxyacyl-CoA → β-Ketoacyl-CoA (NAD⁺)
Acetyl-CoA acyltransferase (thiolase)	2.3.1.16	β-Ketoacyl-CoA → Acyl-CoA + Acetyl-CoA
ETF dehydrogenase	1.5.5.1	Re-oxidizes FADH₂ via ETF [FAD, Fe₄S₄]

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Detailed Summary

• Activation: Fatty acids are activated to fatty acyl-CoA by fatty acyl-CoA synthetase in two ATP-consuming steps, driven forward by hydrolysis of PPi.
• Transport: Fatty acyl-CoA crosses the inner mitochondrial membrane as fatty acyl-carnitine, via the carnitine shuttle (acyltransferases I & II + transporter). Malonyl-CoA inhibits acyltransferase I.
• β-Oxidation (4 reactions per cycle):
  1. FAD-dependent dehydrogenation → trans-Δ²-enoyl-CoA (FADH₂ → ETF → respiratory chain)
  2. Hydration → L-β-hydroxyacyl-CoA
  3. NAD⁺-dependent dehydrogenation → β-ketoacyl-CoA (NADH → Complex I → ATP)
  4. Thiolysis → acetyl-CoA + shortened acyl-CoA
• C16 palmitoyl-CoA → 7 cycles → 8 acetyl-CoA + 7 FADH₂ + 7 NADH → ~108 ATP total
• Unsaturated fatty acids require enoyl-CoA isomerase (monounsaturated) and additionally 2,4-dienoyl-CoA reductase (polyunsaturated).
• Odd-chain fatty acids produce propionyl-CoA → D-methylmalonyl-CoA (biotin, ATP) → L-methylmalonyl-CoA → succinyl-CoA (vitamin B₁₂) → TCA cycle.
• Regulation: High NADH inhibits β-hydroxyacyl-CoA dehydrogenase; high acetyl-CoA inhibits thiolase; malonyl-CoA inhibits carnitine acyltransferase I (fed-state brake).
• Ketone bodies (acetoacetate, D-β-hydroxybutyrate, acetone) are formed in liver mitochondria when acetyl-CoA accumulates; exported as fuel. Peripheral utilization requires succinyl-CoA–dependent reactivation. Liver itself lacks the reactivation enzyme.