17.2: Oxidation of Fatty Acids

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Compartmentalization and Import of Fatty Acids for Oxidation

• Compare the roles of mitochondria, peroxisomes, and the endoplasmic reticulum in fatty acid oxidation, specifying the types of fatty acids oxidized in each compartment and the distinctive features of each pathway (e.g., FAD vs. O₂ as initial oxidant, production of FADH₂ vs. H₂O₂, and ATP-dependent vs. carnitine-dependent import).
• Explain the carnitine shuttle mechanism — including the sequential actions of CPT-1, CACT, and CPT-2 — that transports long-chain fatty acyl-CoA across the inner mitochondrial membrane, and explain why malonyl-CoA inhibition of CPT-1 coordinately prevents simultaneous fatty acid synthesis and oxidation.

Mitochondrial β-Oxidation: Mechanism and Energetics

• Describe the four enzymatic steps of mitochondrial β-oxidation (acyl-CoA dehydrogenase, enoyl-CoA hydratase, β-hydroxyacyl-CoA dehydrogenase, and thiolase), identifying the oxidizing agent used at each oxidative step, the stereochemistry of intermediates, the type of bond broken in the thiolytic cleavage, and the mechanistic rationale for each transformation.
• Explain why acyl-CoA dehydrogenase uses FAD rather than NAD⁺ as its oxidizing agent for forming the trans double bond between the α and β carbons, and compare the ATP equivalents generated from FADH₂ (1.5 ATP) and NADH (2.5 ATP) per oxidative step.
• Describe the additional enzymes required for the β-oxidation of unsaturated fatty acids — enoyl-CoA isomerase (cis-to-trans isomerization) and 2,4-dienoyl-CoA reductase (NADPH-dependent reduction) — and explain why these are necessary for fatty acids with double bonds at even or odd positions.
• Trace the three-step conversion of propionyl-CoA (the terminal product of odd-chain fatty acid oxidation) to succinyl-CoA via biotin-dependent propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, and adenosylcobalamin-dependent methylmalonyl-CoA mutase, explaining the chemical logic and cofactor requirement of each step.

Alternative Oxidation Pathways and Disease

• Describe the peroxisomal α-oxidation pathway for branched-chain fatty acids such as phytanic acid, explaining why β-oxidation cannot proceed directly on this substrate and how phytanyl-CoA hydroxylase initiates its degradation, and connect mutations in this pathway to Refsum disease.
• Identify the major diseases arising from defects in fatty acid oxidation pathways — including MCAD deficiency, Zellweger syndrome, X-linked adrenoleukodystrophy, and Refsum disease — and for each, explain the biochemical basis of the defect, the class of molecules that accumulates, and the key clinical manifestations.

Introduction

Fatty acids, esterified to glycerol in triacylglycerols, are organisms' major source of stored energy. As we burn fossil fuels to produce energy to drive our society, so can we "burn" fatty acids to ultimately produce heat and ATP to drive biosynthetic reactions and to do work. As discussed in an earlier chapter, fatty acids are highly reduced, so their oxidation by dioxygen is highly favored both enthalpically (exothermic reaction) and entropically. Of course, biological oxidation reactions occur stepwise, not directly using O_2, but with less potent oxidizing agents, such as NAD⁺ and FAD. We'll focus first on fatty acid oxidation in animals (humans).

As discussed in the previous section, fatty acids released from triglyceride stores in response to epinephrine and glucagon signaling during exercise and between meals are utilized for energy when glycogen stores are low, without breaking down muscle protein to produce energy. Some fatty acids are broken down in the normal process of membrane turnover and removal of xenobiotic lipids.

Most fatty acids are oxidized in the mitochondria, where the oxidation reaction occurs at the β-carbon of the acyl chain, as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): α and β carbons of fatty acids

This pathway is called β-oxidation. Fatty acids are oxidized in a step-wise fashion by this pathway. In each repetitive cycle of this pathway, acetyl-CoA and one CO₂ are released. In addition, oxidation can occur at α- and β-carbons when oxidized in the peroxisome. The α-oxidation pathway is used for fatty acids branched at the β-carbon (C3), releasing one CO₂ until the β-oxidation pathway can be used. The peroxisome degrades fatty acids that can't be oxidized in the mitochondria. These include very long-chain fatty acids (VLCFAs) such as 24:0 and 26:0, and in addition, branched-chain fatty acids (BRCHAs), including some fatty acids from dietary sources such as pristanic acid (an odd-chain 15:0 fatty acid methylated at carbons 2, 6, 10, and 14). The α-oxidation pathway cannot completely oxidize fatty acids in the peroxisome. At some point in the oxidative stepwise pathway, the resulting shorter fatty acids are exported to the mitochondria for β-oxidation. Additionally, enzymes in the endoplasmic reticulum facilitate the ω-oxidation pathway, which oxidizes fatty acids at the omega (terminal) carbon. The enzyme used is cytochrome P450 monooxygenase, which uses one oxygen atom from O₂ to hydroxylate the ω-carbon.

Peroxisomes - An underappreciated organelle

These organelles, initially called microbodies, are crucial to cellular metabolism and overall health. In people with Zellweger syndrome spectrum, there is a severe disorder in the formation of peroxisomes, which is often lethal. They are important metabolically in lipid metabolism, the synthesis of myelin sheath lipids, and the metabolism of reactive oxygen species like peroxides. The enzymes catalase and urate oxidase are found in such high concentrations that they often form crystal "bodies" in the peroxisome matrix. Additional roles include responses to pathogens and viruses. Effectively, they are a protective organelle.

In contrast to mitochondria, peroxisomes, like most other organelles, have a single bilayer and no DNA, from which transcription of RNA and translation of proteins occur. Hence, all proteins are imported from the cytoplasm after synthesis on free ribosomes. Imported proteins have a peroxisome targeting sequence (PTS) of serine-lysine-leucine (SKL) near their C-terminus, which facilitates the binding of these proteins to a PTS receptor in the peroxisome membrane. These organelles oxidize very long-chain fatty acids (VLFA), produce and decompose hydrogen peroxide (hence the name), and synthesize plasmalogens. The enzymes involved in the stepwise cycle of reactions in the peroxisomal β-oxidation pathway differ from those in the mitochondrial β-oxidation pathway.

For those more inclined towards chemistry than biology, another organelle with its structures and function may seem like one too many. However, this less-discussed organelle is critically important in its own right. Figure \(\PageIndex{2}\) shows the features of peroxisomes and their proteins.

 

Figure \(\PageIndex{2}\): Features of peroxisomes and their proteins. Islinger, M., Voelkl, A., Fahimi, H.D. et al. The peroxisome: an update on mysteries 2.0. Histochem Cell Biol 150, 443–471 (2018). https://doi.org/10.1007/s00418-018-1722-5. Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

One interesting feature is its relationship with different cell organelles, as shown in the left panel of Figure 2. Some proteins involved in organelle functions are also shown (right panel).

The peroxisome (PO, green) has binding interactions (red interfaces) with the endoplasmic reticulum (ER), lysosomes, mitochondria, lipid droplets (a pseudo-organelle), and also itself (left figure). Some of the key membrane proteins (which we have discussed previously) involved in peroxisome function include the ABC transporter proteins ABCD1-3 for fatty acids transport, OCTN3 for organic and cation/carnitine transport, and MCT1/2 for monocarboxylate transport. In addition, peroxisomes have receptors for protein import, mediated by PTSs, and for peroxisome movement along microtubules within the cell.

Mitochondrial β-Oxidation

Mitochondrial β-oxidation in muscle generates acetyl-CoA, which enters the citric acid cycle for subsequent ATP production via mitochondrial electron transport and oxidative phosphorylation. In the liver, the generated acetyl-CoA is used to produce ketone bodies during fasting. Figure \(\PageIndex{3}\) shows the β-oxidation pathway for palmitic acid (16:0), a saturated fatty acid, starting with its import from the cytoplasm.

Figure \(\PageIndex{3}\): Mitochondrial β-oxidation of palmitic acid (16:0)

The pathway involved the cyclic removal of 2-carbon units until 16:0 was cleaved 7 times, producing 8 2-carbon acetyl-CoAs. The net chemical equation for the beta-oxidation of 16:0 is shown below.

\begin{equation}
\mathrm{C}_{16}-\mathrm{CoA}+7 \mathrm{NAD}^{+}+7 \mathrm{FAD}+7 \mathrm{CoASH}+7 \mathrm{H}_{2} \mathrm{O} \rightarrow 8 \mathrm{Acetyl}-\mathrm{CoA}+7 \mathrm{NADH}+7 \mathrm{FADH}_{2}+7 \mathrm{H}^{+}
\end{equation}

Figure \(\PageIndex{4}\) shows an abbreviated comparison of the β-oxidation pathways in the mitochondria and peroxisomes. Peroxisomal beta-oxidation is used to metabolize very-long-chain fatty acids (VLCFAs), which are composed of 24-26 carbon units, and branched-chain fatty acids (BRCHAs).

Figure \(\PageIndex{4}\): Comparison and interplay of peroxisomal and mitochondrial fatty acid β-oxidation. Fatty acid β-oxidation, the NAD(H) redox shuttles, the tricarboxylic acid cycle, and the electron transfer chain are depicted in blue, purple, red, and pink, respectively. 1a, acyl-CoA oxidase; 1b, acyl-CoA dehydrogenase; 2, enoyl-CoA hydratase; 3, 3-hydroxyacyl-CoA dehydrogenase; 4, 3-ketoacyl-CoA thiolase. ABCD, ATP-binding cassette transporters of subfamily D; ADP, adenine dinucleotide phosphate; BRCFA, branched-chain fatty acid; CAC, carnitine-acylcarnitine carrier; FAD, flavin adenine dinucleotide; FADH₂, reduced FAD; LCFA, long-chain fatty acid; MCFA, medium-chain fatty acid; NAD, nicotinamide adenine dinucleotide; NADH, reduced NAD; NRS, NAD(H) redox shuttles; OXPHOS, oxidative phosphorylation; TCA, tricarboxylic acid; VLCFA, very-long-chain fatty acid. Fransen et al. International Journal of Molecular Sciences. 18. 1126. 10.3390/ijms18061126. DOI: 10.3390/ijms18061126. Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

It should be noted that a likely NAD⁺/NADH mitochondrial transporter, a multi-pass inner mitochondrial membrane protein, has just been identified. The transporter, MCART1, is also called SLC25A51,

β-Oxidation - Mechanisms

Fatty acids are imported into the matrix from the cytoplasm through their acyl-CoA derivatives. Two different proteins are required for their import. Carnitine palmitoyltransferase-1(CPT-1) transfers the acyl group from CoASH to a small molecule carrier called carnitine. The acylcarnitine is translocated through the inner membrane by the carrier protein carnitine-acylcarnitine translocase (CACT). Once inside the matrix, the acyl group is transferred back to CoASH by carnitine palmitoyltransferase-2 (CPT-2). This carnitine cycle is illustrated in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Carnitine cycle and the connections between major catabolic pathways.

At the outer mitochondrial membrane (OMM), fatty acyl-CoAs become linked to carnitine through carnitine palmitoyltransferase-1 (CPT-1). The complex is translocated across the inner mitochondrial membrane (IMM) via carnitine-acylcarnitine translocase (CACT). In the mitochondrial matrix, CPT-2 converts fatty acylcarnitines back to fatty acyl-CoAs, which enter the β-oxidation pathway. Free carnitine is transported back into the cytoplasm via exchange with acyl-carnitines via CACT. β-Oxidation in the matrix produces acetyl-CoA, which is also formed from glycolytic pyruvate through the action of pyruvate dehydrogenase. Hence, acetyl-CoA links both glycolysis and fatty acid oxidation. The resulting acetyl-CoA can enter the TCA cycle when energy is needed.

The mitochondrial carnitine/acylcarnitine carrier protein helps transport acylcarnitines of different lengths across the mitochondrial inner membrane for β-oxidation into the mitochondrial matrix. Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the human mitochondrial carnitine/acylcarnitine carrier protein AlphaFold model (O43772)

Figure \(\PageIndex{6}\): Mitochondrial carnitine/acylcarnitine carrier protein AlphaFold model (O43772). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...DdDNu9xVJGg2i9

The transmembrane helices are shown in gray. The N- (Met 1) and C-terminal (Leu 301) amino acids are shown in spacefill color CPK.

Malonyl-CoA, produced in the first committed step in fatty acid synthesis, inhibits CPT1. This should make biological sense, as fatty acid oxidation should not occur when fatty acids are being synthesized. Palmitoyltransferase II (CPT II), which converts acylcarnitine to fatty acyl-CoA, traps the molecules within the mitochondrial matrix.

In contrast to this regulated transport mechanism, very long-chain fatty acids (VLCFAs) and branched-chain fatty acids are transported into peroxisomes by the ABCD1-3 transporters through an ATP-dependent process.

The mitochondrial β-oxidation of fatty acids has four steps in the mitochondrial matrix. In those steps, a 16:0 fatty acid (for example) is converted to a (14):0 fatty acid and the 2C molecule acetyl-CoA. The (14):0 fatty acid undergoes six more rounds of the β-oxidation cycle until the entire 16:0 fatty acid is fully converted to 8 acetyl-CoAs.

Step 1: Acyl-CoA dehydrogenase

There are long-chain acyl-CoA dehydrogenases (LCAD), medium-chain acyl-CoA dehydrogenases (MCAD), and short-chain acyl-CoA dehydrogenases (SCAD), which catalyze the first oxidative step in the β-oxidation pathway. These enzymes catalyze the formation of a trans double bond between the α and β carbons (C2 and C3) on the acyl-CoA substrates. A stronger oxidizing agent than NAD⁺ is required to form an alkene between the two methylene groups, so FAD is used. Eventually, the reduced FADH₂ produced will produce 1.5 equivalents of ATP in the mitochondrial electron transport chain/oxidative phosphorylation.

Figure \(\PageIndex{7}\) shows the key oxidative step in the mechanism of acyl-CoA dehydrogenase to 2-enoyl-CoA by acyl-CoA dehydrogenases

Figure \(\PageIndex{7}\): Mechanism for conversion of acyl-CoA to enoyl-CoA by acyl-CoA dehydrogenase. (after Ghisla and Thorpe. Eur. J. Biochem. 271, 494–508 (2004) doi:10.1046/j.1432-1033.2003.03946.x)

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with octanoyl-CoA, a substrate (3MDE)

Figure \(\PageIndex{8}\): Medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with octanoyl-CoA substrate (3MDE). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SiAZExwXVV2TG6

Two subunits of the biologically active tetramer are shown (dark gray and cyan). FAD is shown in each subunit (sticks, CPK colors, labeled). A bound substrate, octanoyl-CoA (spacefill, CPK colors, labeled CO8) is also shown in each subunit. The catalytic base, Glu 376, is depicted in sticks, with CPK colors and labeled.

The structures of the unliganded and acyl-CoA forms of the enzymes are very similar, so there are no large conformational changes on binding octanoyl-CoA. The ligand binds to the enzyme at the FAD's rectus (re) face with the acyl chain buried. The fatty acyl chain of the thioester substrate is buried inside the polypeptide, and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The carbonyl oxygen of octanoyl-CoA interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. Glu-376 acts as a general base, removing the alpha proton in the reaction.

Step 2. Enoyl CoA hydratase

This enzyme catalyzes a hydration step of the double bond between the α and β carbons (C2 and C3), adding an OH group to the β carbon, in a reaction that poses a low energy barrier. Figure \(\PageIndex{9}\) shows a likely mechanism for enoyl-CoA hydratase.

Figure \(\PageIndex{9}\): Mechanism for enoyl-CoA hydratase

Figure \(\PageIndex{10}\) shows an interactive iCn3D model of the rat enoyl-CoA hydratase in complex with hexadienoyl-CoA (1MJ3)

Figure \(\PageIndex{10}\): Rat enoyl-CoA hydratase in complex with hexadienoyl-CoA (1MJ3). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...byx3eEKewEeEz9

For clarity, only one subunit of the biological hexamer is shown. Two glutamic acids (141 and 164) appear to activate a water molecule for the hydration reaction. Alanine 98 and Gly 141 are also in the oxyanion hole, stabilizing the transition state and intermediate.

Water addition is syn since the proton and the OH group are added to the same side of the double bond. The glycine amide NH forms a strong hydrogen bond to the carbonyl of the substrate, hexadienoyl-CoA, thereby polarizing the ene-one. The substrate trans-2-crotonyl-CoA is converted to the 3(S) alcohol rather than the 3(R) alcohol by a large factor. The cis and trans isomers of a substrate analog (hexadienoyl-CoA) can bind to the enzyme, but only the cis isomer is polarized. Since the transition state is polarized as well, the bound cis isomer is also strained and destabilized, suggesting that its binding is an example of transition-state binding catalysis.

Step 3. Beta-hydroxyl acyl CoA dehydrogenase

After the addition of the OH on C3 (beta) OH during the hydration reaction, the resulting ROH is oxidized to a ketone, β-ketoacyl-CoA, by the oxidizing agent NAD⁺ using the enzyme β-hydroxyl acyl CoA dehydrogenase. The resulting NADH is reoxidized to NAD⁺ through the mitochondrial electron transport chain, ultimately leading to 2.5 molecules of ATP for each NADH. Figure \(\PageIndex{11}\) shows a plausible mechanism for the beta-hydroxyl acyl CoA dehydrogenase-catalyzed reaction.

Figure \(\PageIndex{11}\): Mechanism for production of β-ketoacyl-CoASH by beta-hydroxyl acyl CoA dehydrogenase

His 158 acts as a general base, and Glu 170 increases its basicity. The other group is involved in H-bond and electronic stabilization interactions.

Step 4. Acetyl-CoA acetyltransferase, mitochondrial - ACAT1 (also called 3-ketoacyl-CoA thiolase)

The final step in the beta-oxidation pathway involves cleavage of the bond between the alpha and beta carbon by CoASH. This step is catalyzed by beta-keto thiolase and is a thiolytic reaction (as opposed to cleavage by water, a hydrolysis reaction). The reaction produces one molecule of acetyl-CoA and a fatty acyl-CoA that is two carbons shorter. The process repeats until the even-chain fatty acid is completely converted into acetyl-CoA. The enzyme's activity is reversible, and it can also catalyze the Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA, as we will see in the next chapter section on the synthesis of ketone bodies.

The reaction begins with the acylation of the nucleophilic Cys 89 by the carbonyl group of the 3-oxoacyl-CoA, accompanied by the concomitant release of acetyl-CoA. This forms a Cys 89-acyl covalent intermediate. In the next step, Cys 378 acts as a general base to facilitate the nucleophilic attack of free CoASH on the acyl-intermediate. His 348 acts as a general acid, protonating the thiolate leaving group. The amino acids (Cys89, Cys378, and His348) are generally conserved in thiolases (Bhaskar et al. 2020). Kinetically, this mechanism is a ping-pong reaction. A reaction mechanism is shown in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Reaction mechanism for mitochodrial acetyl-CoA acetyltransferase (ACAT1, thiolase T1)

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of Human Mitochondrial 3-Ketoacyl-Coa Thiolase (T1) (4C2J)

Figure \(\PageIndex{13}\): Human Mitochondrial 3-Ketoacyl-Coa Thiolase (T1) (4C2J). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...dzAHKEY77WpNi7

Two subunits in the biological function dimer are shown (cyan and gray). The active site is shown in the gray subunit as CPK-colored sticks and labeled. The numbers differ slightly from those shown in the mechanistic figure. CoASH is shown in each subunit as sticks. The fatty-acyl tail appears to bind in a tunnel.

A few more enzymes are needed.

Steps 1 through 4 outlined above apply to the beta-oxidation of a saturated fatty acid with an even-numbered carbon skeleton. Unsaturated fatty acids, such as oleate (18:1) and linoleate (18:2), contain cis double bonds that must be isomerized to the trans configuration by the enzyme enoyl CoA isomerase or reduced by the 2,4-dienoyl CoA reductase (24DCR), using NADPH.

Enoyl CoA isomerase

This enzyme catalyzes the isomerization of cis double bonds to the trans form, which mimics those formed by acyl-CoA dehydrogenase using FAD in step 1. Figure \(\PageIndex{14}\) shows a plausible mechanism for converting cis double bonds to their trans isomer.

Figure \(\PageIndex{14}\): Cis to trans isomerization by enoyl-CoA isomerase (https://www.ebi.ac.uk/thornton-srv/m-csa/entry/341/)

Glu 136 acts as a general base, while the amide Hs of Leu 66 and Gly 111 stabilize the intermediate oxyanion and, hence, the developing charge in the transition state. They are optimally situated in the oxyanion hole.

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of the Human mitochondrial Δ³-Δ²-enoyl-CoA isomerase (1SG4)

Figure \(\PageIndex{15}\): Human mitochondrial Δ³-Δ²-enoyl-CoA isomerase (1SG4) (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...H6LxEzBKajq8b7

The three subunits are shown in different colors. The substrate analog octanoyl-CoA is shown in spacefill CPK colors bound to the gray subunit. The catalytic residues are shown in sticks with CPK colors and labeled. The distal omega end binds in a hydrophobic tunnel.

2,4-dienoyl CoA reductase or 24DCR

An alternative way to deal with the cis double bond is to reduce it, in this case, with NADPH. This enzyme acts on all C=C bonds at even-numbered positions and on more at odd-numbered positions. A mechanism for the reduction is shown in Figure \(\PageIndex{16}\).

Figure \(\PageIndex{16}\): a mechanism for the reduction of double bonds in unsaturated fatty acids (after Fillgrove and Anderson, Biochemistry (2001) https://doi-org.ezproxy.csbsju.edu/10.1021/bi0111606

Figure \(\PageIndex{17}\) shows an interactive iCn3D model of a monomer of the homotetrameric human mitochondrial 2,4-dienoyl-Coa reductase (1W6U)

Figure \(\PageIndex{17}\): Monomer of the homotetrameric human Mitochondrial 2,4-Dienoyl-Coa Reductase (1W6U) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...pQueKc84sFqzt7

The model shows bound NADP⁺ and the substrate trans-2,trans-4-dienoyl-CoA. The active site is sufficiently open to accommodate fatty acids of varying lengths. Tyr-199 and Asn-148 stabilize the enolate and the oxidized nicotinamide.

For odd-number chain fatty acids, propionyl-CoA to succinyl-CoA

Although most fatty acids of biological origin have even numbers of carbons, not all do. The oxidation of fatty acids with odd numbers of carbons ultimately produces an intermediate with three carbons, propionyl-CoA, which cannot be oxidized further in the beta-oxidation pathway. These additional steps are necessary:

1. carboxylation to make (S)-methylmalonyl-CoA;
2. isomerization to (R)-methylmalonyl-CoA;
3. rearrangement to form succinyl-CoA. The last step of the process utilizes the enzyme methylmalonyl-CoA mutase, which uses the B12 coenzyme in its catalytic cycle. Succinyl-CoA can then be metabolized in the citric acid cycle.

Figure \(\PageIndex{18}\) shows the pathway for converting propionyl-CoA to succinyl-CoA.

 

Figure \(\PageIndex{18}\): Conversion of propionyl-CoA to succinyl-CoA

---

Mechanisms for conversion of propionyl-CoA to succinyl-CoA.

We will look at each enzyme in turn.

Propionyl-CoA carboxylase

The net reaction for this enzyme is shown in Figure \(\PageIndex{19}\) below.

Propionyl-CoA + ATP + HCO₃^- →  (S)-methylmalonyl-CoA + ADP + P_i + H⁺

Figure \(\PageIndex{19}\): Propionyl-CoA carboxylase reaction

The enzyme has three subunits.  The alpha subunit contains a biotin carboxyl carrier protein domain and a biotin carboxylase domain.  The beta subunit has carboxytransferase activity.  The Streptomyces coelicolor propionyl-CoA carboxylase (PCC) mechanism is shown in two parts below.   

In Part 1, biotin is carboxylated by HCO₃^- to form an activated bicarbonate derivative (similar to an anhydride) that is high energy in comparison to its hydrolysis product, as shown in Figure \(\PageIndex{20}\) below. This reaction takes place in the alpha subunit.

Figure \(\PageIndex{20}\): Formation of carboxylated biotin by propionyl-CoA carboxylase - Part 1.   M-CSA.  Gemma L. Holliday, Daniel E. Almonacid, Jonathan T. W. Ng.  Creative Commons Attribution 4.0 International (CC BY 4.0) License

In Part 2, shown in  Figure \(\PageIndex{21}\) below, the carboxyl group on biotin is transferred to propionyl-CoA to form methymalonyl-CoA. This reaction occurs in the beta subunit.

Figure \(\PageIndex{21}\): Part 2 - Formation of (S)-methylmalonyl-CoA by activated CO₂ transfer from carboxybiotin by propionyl-CoA carboxylase. Holliday et al. ibid.

The roles of the main chain atoms of Ala 182 and Gly 183 in the alpha subunit are to stabilize the propionyl-CoA, while the backbone atoms of Gly 429 and Ala 430 in the beta subunit are to stabilize the oxyanion of carboxylated biotin.

Figure \(\PageIndex{22}\) shows an interactive iCn3D model of biotin and propionyl-CoA bound to Acyl-CoA Carboxylase Beta Subunit from S. coelicolor (1XNY)

Figure \(\PageIndex{22}\): Monomer of the biotin and propionyl-CoA bound to Acyl-CoA Carboxylase Beta Subunit from S. coelicolor (1XNY). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...K46r5EdZezF4o6

Biotin (labeled BTN) and propionyl-CoA are shown in spacefill in CPK colors.

Methylmalonyl-CoA Epimerase

The net reaction is shown in Figure \(\PageIndex{23}\) below.

Figure \(\PageIndex{23}\): Methylmalonyl-CoA Epimerase reaction

This reaction proceeds via an enolate intermediate after abstraction of a proton from the chiral center of methylmalonyl-CoA, as shown in the reaction mechanism for the enzyme from Propionibacterium freudenreichii subsp. shermanii is shown Figure \(\PageIndex{24}\) below. The reaction is readily reversible.

Figure \(\PageIndex{24}\):  Mechanism for methylmalonyl-CoA Epimerase.  Gemma L. Holliday et al. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/33/.    Creative Commons Attribution 4.0 International (CC BY 4.0) License

Note the presence of a Co²⁺  ion in the active site. 

Figure \(\PageIndex{25}\) shows an interactive iCn3D model of Methylmalonyl-CoA epimerase in complex with 2-nitronate-propionyl-CoA from S. coelicolor (6WFI)

Figure \(\PageIndex{25}\): Methylmalonyl-CoA epimerase in complex with 2-nitronate-propionyl-CoA from S. coelicolor (6WFI). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Sqc98CCjAvzkp7

The enzyme is a dimer with two identical subunits and is catalytic. One of the subunits is shown with a transparent blue surface that contains the bound 2-nitronate-propionyl-CoA inhibitor. The other is shown in a gray cartoon, with the key side chains involved in substrate binding and catalysis shown as color sticks and labeled.

 

Methylmalonyl-CoA Mutase

In this reaction, a methyl group is removed from  (R)-methylmalonyl-CoA to form succinyl-CoA, as shown in Figure \(\PageIndex{26}\) below.

Figure \(\PageIndex{26}\): Methylmalonyl-CoA reaction

This reaction, going in the opposite direction, is an example of a methyltransferase.  Another similar enzyme (which we will see in Chapter 18.4 - Amino Acid Degradation) catalyzes a methyl transfer from homocysteine to a new cofactor, cobalamin, which then transfers it to cysteine to form methionine, a reaction catalyzed by methionine synthase.  Cobalamin and its methylated form are derivatives of Vitamin B12.  We will leave the details of cobalamin biochemistry to the next chapter and present a mechanism for methylmalonyl-CoA mutase here.    

We present the reaction mechanism for the reverse reaction, the conversion of succinyl-CoA to (R)-methylmalonyl-CoA, catalyzed by the enzyme from Propionibacterium freudenreichii subsp. shermanii in two parts below.  The enzyme is a heterodimer composed of an alpha and a beta subunit and requires the cofactor adenosylcobalamin (coenzyme B12). In contrast, the human mutase, a homodimer, is similar to the alpha subunit.

First, a free radical is formed from the adenosyl group on the cofactor.  This promotes a free-radical rearrangement of succinyl-CoA to (R)-methylmalonyl-CoA (or the reverse for the pathway of interest here).  The reaction is shown in two parts for optimal viewing.

Figure \(\PageIndex{27}\) below shows the first part of the reaction for the conversion of succinyl-CoA to (R)-methylmalonyl-CoA by the mutase from Propionibacterium freudenreichii subsp. shermanii. 

Figure \(\PageIndex{27}\): Part 1 of the conversion of succinyl-CoA to (R)-methylmalonyl-CoA by methylmalonyl-CoA mutase.  Gemma L. Holliday, Gail J. Bartlett, Daniel E.  Almonacid.  M-CSA.  https://www.ebi.ac.uk/thornton-srv/m-csa/entry/62/.  Creative Commons Attribution 4.0 International (CC BY 4.0) License

Figure \(\PageIndex{28}\) below shows the rest of the reaction for the conversion of succinyl-CoA to (R)-methylmalonyl-CoA. 

 

Figure \(\PageIndex{28}\): Part 2 of the conversion of succinyl-CoA to (R)-methylmalonyl-CoA by methylmalonyl-CoA mutase. Gemma L. Holliday et al. ibid.  

The last product from the bottom-left reaction, the reformed active adenosylcobalamin cofactor, is not shown. The enzyme facilitates the hemolytic cleavage of the Co-C bond. A free-radical rearrangement follows this. 

Figure \(\PageIndex{29}\) shows an interactive iCn3D model of the methylmalonyl-coenzyme A mutase from Propionibacterium freudenreichii subsp. shermanii (1REQ)

Figure \(\PageIndex{29}\): Methylmalonyl-coenzyme A mutase from Propionibacterium freudenreichii subsp. shermanii (1REQ). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...qPYm2sXjraCuN8

An analog of CoASH, desulfo-CoA (DCA), is shown in spacefill, CPK colors, and labeled.  Adenosylcobalamin is shown in colored sticks and labeled B12.  The alpha subunit is gray, with the key amino acids from the enzyme mechanism presented above shown as colored sticks and labeled.  The beta subunit is shown in blue.

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Propionyl-CoA is also produced as a product of methionine, valine, isoleucine, and threonine oxidation. (See the amino acid metabolism chapter for more details on mechanisms.)

Very long chain oxidation

In contrast to the oxidation of short- and medium-chain fatty acids, which, under beta-oxidation, require four distinct enzymes, the oxidation of very long-chain fatty acids (VLCFs) is carried out by two proteins, with the second protein expressing three enzyme activities. The first step, analogous to step 1 in beta-oxidation described above, is catalyzed by very long-chain acyl-CoA dehydrogenase (VLCAD). A single trifunctional protein (TFP) with two subunits carries out the next three reactions. The α-subunit carries out the hydration (2-enoyl-CoA hydratase, ECH) and next oxidation step (3-hydroxyl-CoA dehydrogenase (HAD), while the β-subunit has 3-ketothiolase (KT) activity. Deficiencies of TFP can cause significant disease and even death.

TFP is a heterotetramer of two α and two β subunits, with the beta subunits forming a central homodimer. The two alpha units bind at each end, and the whole complex forms an arc. A "tunnel" appears to allow substrate transfer after each step. This minimizes premature intermediate release. Figure \(\PageIndex{30}\) shows an interactive iCn3D model of the human mitochondrial trifunctional protein, a fatty acid beta-oxidation metabolon (6DV2)

Figure \(\PageIndex{30}\): Human mitochondrial trifunctional protein fatty acid beta-oxidation metabolon (6DV2). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...XiUXru4vkRVh87

• Grays: two thiolase subunits: reversible thiolytic cleavage of 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA, a 2-step reaction involving a covalent intermediate formed with a catalytic cysteine. The catalytic residues (C138, C458, and H428) are shown as sticks with CPK colors.
• Cyan and magenta subunits: enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)
• The red dots represent the inner leaflet of the inner mitochondrial membrane, where proteins reside in the mitochondrial matrix.

Regulation of beta-oxidation

We observed that the metabolic decision to utilize carbohydrate energy reserves (glycogen) is highly regulated. Glycogen breakdown occurs during fasting and periods of high energy need. Fatty acids, our largest energy stores, are released from triglyceride reserves in adipose cells through extracellular epinephrine and glucagon activation of pathways that activate intracellular hormone-sensitive lipase. Released fatty acids are bound to the serum protein albumin, which transports them to the tissues. Additionally, as mentioned above, malonyl-CoA inhibits fatty acid transport into mitochondria. Malonyl-CoA is the first committed product of fatty acid biosynthesis. Each acyl-CoA product of the four enzymes engages in product inhibition for the enzyme that produced it. 3-ketoacyl-CoA also inhibits enoyl-CoA hydratase and acyl-CoA dehydrogenase [17]. The NADH/NAD⁺ and acetyl-CoA/CoA ratios also influence the beta-oxidation pathway through allosteric regulation.   For example, the acetyl-CoA/CoA ratio affects the activity of ketoacyl-CoA thiolase. 

Fatty acids also bind to the transcription factors called peroxisome proliferator-activated receptors (PPARs) and coactivator PGC-1α, which regulate the transcription of enzymes in the beta-oxidation pathways. PPARs and the retinoid X receptor form heterodimers that bind to the PPAR response element in key promoter sites involved in fatty acid degradation. These include CPT1, long-chain acyl-CoA dehydrogenase (LCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and acyl-CoA synthetase (ACS). The PPARs have tissue specificity. We will discuss PPARs in more detail in the chapter on fatty acid synthesis.

Peroxisomal α-Oxidation

Alpha oxidation of fatty acids occurs in the peroxisome, where the metabolism of phytanic acid (3,7,11,15-tetramethyl hexadecanoic acid) in dairy products, animal fat, and some fish occurs. Phytanic acid is produced in ruminants on the degradation of plant material and derives from phytol, an isoprenoid alcohol esterified to chlorophyll. Phytol is first converted to phytanic acid.

Fatty acid β-oxidation can also occur in peroxisomes. In animals, peroxisomes are believed to be important in the initial breakdown of very long-chain fatty acids and methyl-branched fatty acids [11]. The enzymes involved in fatty acid oxidation in peroxisomes differ from those in mitochondria. An important difference is acyl-CoA oxidase, the first enzyme in peroxisomal β-oxidation, which transfers the hydrogen to oxygen, producing H₂O₂ rather than FADH₂. Catalase breaks down H₂O₂ to water. The fatty acyl-CoA intermediates formed during β-oxidation are the same in peroxisomes and mitochondria. Peroxisomes also contain the enzymes for α-oxidation, which are necessary to oxidize some fatty acids with methyl branches.

Branched-chain fatty acids also require additional enzymatic modification to enter the alpha-oxidation pathway within peroxisomes. Phytanic acid (3,7,11,14-tetramethylhexadecanoic acid) requires additional peroxisomal enzymes to undergo beta-oxidation. Phytanic acid initially forms phytanyl CoA, which is then hydroxylated at the alpha carbon by phytanyl CoA hydroxylase (alpha-hydroxylase), encoded by the PHYH gene. The alpha-carbon-hydroxyl bond then undergoes two successive rounds of oxidation to form pristanic acid. Pristanic acid undergoes beta-oxidation, which produces acetyl-CoA and propionyl-CoA in alternative rounds. As with peroxisomal beta-oxidation of VLCFAs, this process generally ends when the carbon chain length reaches 6-8 carbons.  At this point, the molecule is shuttled to the mitochondria by carnitine for complete oxidation to carbon dioxide and water. Figure \(\PageIndex{31}\) shows the steps in the catabolism of phytanic acid (3,7,11,14-tetramethylhexadecanoic acid).

Figure \(\PageIndex{31}\): Alpha oxidation pathway of catabolism of phytanic acid (3,7,11,14-tetramethylhexadecanoic acid)

Omega-oxidation

The omega-oxidation pathway occurs in the endoplasmic reticulum and metabolizes larger fatty acids, which, due to their hydrophobicity, can damage cells at high concentrations. In this pathway, fatty acids are metabolized to dicarboxylic acids, increasing their water solubility for excretion in the urine. The first step utilizes cytochrome P450 enzymes, which also modify xenobiotic compounds with dioxygen, thereby increasing their solubility. Figure \(\PageIndex{32}\) shows the omega oxidation pathway.

Figure \(\PageIndex{32}\): The ER omega oxidation pathways for catabolism of fatty acids

Three subfamily members of the cytochrome P450s (CYPs) show a preference for the hydroxylation of short-chain fatty acids (C7-C10, CYP4B), medium-chain (C10-C16, CYP4A), and long-chain (C16-C26, CYP4F) fatty acids, which can be saturated, unsaturated, and branched. Figure \(\PageIndex{33}\) shows a summary of the alpha, beta, and omega oxidation pathways

Figure \(\PageIndex{33}\): https://febs.onlinelibrary.wiley.com...8.2010.07947.x

Diseases of fatty acid metabolism

A few select diseases listed below directly involve defective fatty acid metabolism through intrinsic enzyme deficiencies, or indirectly prevent its proper functioning through extrinsic enzyme deficiencies. Many, but not all, deficiencies of enzymes involved in fatty acid oxidation result in abnormal neurological development and/or function early in life; a brief list of signs and symptoms is provided under the selected diseases.

MCAD Deficiency

Medium-chain acyl dehydrogenase is the most common inherited defect of fatty acid oxidation in people.  As expected, medium-chain, 6-8 carbon molecules accumulate in this disease. Clinical manifestations of MCAD deficiency primarily present during fasting conditions and include lethargy, weakness, sweating, and hypoglycemia, most commonly in children under the age of 5. Serum measurements of octanoyl carnitine are usually elevated in these patients and can aid in the diagnosis. These abundant molecules are then oxidized by the cytochrome P450 system, which is involved in omega-oxidation, leading to dicarboxylic acidemia and dicarboxylic aciduria.

Zellweger Syndrome

Zellweger syndrome results from autosomal recessive mutations in the PEX genes, which encode peroxin proteins essential for peroxisome assembly. Almost 70% of all peroxisomal biogenesis disorders (PBDs) result from a PEX1 gene mutation. Many different fatty acid compounds, including VLCFAs and phytanic acid, can accumulate without the oxidative machinery of peroxisomes. Manifestations of this disease generally include the brain, kidneys, and skeleton.

X-Linked Adrenoleukodystrophy (X-ALD)

X-ALD is a genetic deficiency of the ABCD transporters in the membrane of peroxisomes, as mentioned previously. It results in the pathological accumulation of phytanic acid and VLCFAs within cells and is most clinically significant when the ABCD1 transporter is absent. The disease presents with neurodegenerative and adrenal abnormalities.

Refsum Disease

Refsum disease results from a genetic deficiency of the enzyme phytanyl CoA 2-hydroxylase, which, as previously mentioned, is involved in the alpha-oxidation of phytanic acid, a breakdown product of chlorophyll. Notable clinical manifestations of Refsum disease include cardiac malfunction and defective functioning of the olfactory and auditory nerves due to the accumulation of phytanic acid.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter describes the oxidative catabolism of fatty acids, tracing the biochemical pathways by which highly reduced acyl chains are systematically cleaved and oxidized to produce acetyl-CoA, NADH, and FADH₂—the fuels for the citric acid cycle and oxidative phosphorylation.

Fatty acid oxidation occurs in three distinct subcellular compartments, each handling different classes of fatty acids. The mitochondria process the majority of cellular fatty acids through β-oxidation, generating acetyl-CoA that enters the TCA cycle and reduced electron carriers that fuel ATP synthesis. Peroxisomes handle very-long-chain fatty acids (VLCFAs, C24–C26) and branched-chain fatty acids that cannot be initially processed in the mitochondria; they also perform α-oxidation. A key difference is that the first peroxisomal oxidative step uses FAD with O₂ as the terminal electron acceptor, producing H₂O₂ rather than FADH₂ — the H₂O₂ is immediately detoxified by catalase. The endoplasmic reticulum performs ω-oxidation using cytochrome P450 enzymes to hydroxylate the terminal methyl carbon of fatty acids, producing dicarboxylic acids that are excreted in urine; this pathway handles medium- and long-chain fatty acids that would otherwise accumulate to toxic levels, as seen in MCAD deficiency.

Entry of long-chain fatty acids into the mitochondrial matrix requires the carnitine shuttle. At the outer mitochondrial membrane, CPT-1 transfers the acyl group from CoASH to carnitine; the acylcarnitine is then translocated across the inner membrane by CACT; and CPT-2 transfers the acyl group back to matrix CoASH. Malonyl-CoA — the committed intermediate of fatty acid synthesis — inhibits CPT-1, ensuring that fatty acid synthesis and oxidation are not simultaneously active. Very-long-chain fatty acids, by contrast, enter peroxisomes via ATP-binding cassette transporters (ABCD1-3).

The four-step mitochondrial β-oxidation cycle removes two carbons per turn as acetyl-CoA. Step 1: acyl-CoA dehydrogenase (LCAD, MCAD, or SCAD depending on chain length) introduces a trans double bond between C2 and C3, using FAD as the oxidant — the more positive reduction potential of FAD compared to NAD⁺ is required for this thermodynamically demanding dehydrogenation of a saturated C–C bond. Glu376 serves as the general base abstracting the α-proton. Step 2: enoyl-CoA hydratase adds water across the trans double bond in a syn addition, producing the (S)-3-hydroxyacyl-CoA; two conserved glutamate residues activate the water molecule, and oxyanion hole residues stabilize the transition state. Step 3: β-hydroxyacyl-CoA dehydrogenase oxidizes the β-hydroxyl to a ketone using NAD⁺, with His158 as the general base and Glu170 enhancing its basicity. Step 4: thiolase (ACAT1) cleaves the C2–C3 bond by thiolysis — a nucleophilic attack by CoASH rather than water — through a ping-pong mechanism involving a covalent Cys89-acyl intermediate, releasing acetyl-CoA and a shortened acyl-CoA ready for the next cycle. For palmitate (16:0), seven cycles produce eight acetyl-CoAs, seven FADH₂, and seven NADH, ultimately yielding approximately 106 ATP.

Unsaturated fatty acids require auxiliary enzymes: enoyl-CoA isomerase converts cis double bonds to the trans configuration needed by hydratase, while 2,4-dienoyl-CoA reductase uses NADPH to reduce double bonds at even-numbered positions in polyunsaturated fatty acids. For odd-chain fatty acids — generated in ruminants from propionate and in humans from some amino acid catabolism — the final three-carbon product, propionyl-CoA, is converted to succinyl-CoA in three steps: biotin-dependent carboxylation by propionyl-CoA carboxylase (mechanistically identical to pyruvate carboxylase), epimerization by methylmalonyl-CoA epimerase through an enolate intermediate with a Co²⁺ cofactor, and radical rearrangement by methylmalonyl-CoA mutase using the adenosylcobalamin (vitamin B12) cofactor through homolytic cleavage of the Co–C bond. Succinyl-CoA then enters the citric acid cycle directly.

For very-long-chain fatty acids, a single trifunctional protein (TFP) — an α₂β₂ heterotetramer — performs steps 2, 3, and 4 of β-oxidation, with a substrate-channeling tunnel between active sites minimizing intermediate release. The α-subunit contains enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, while the β-subunit contributes thiolase activity.

The peroxisomal α-oxidation pathway handles methyl-branched fatty acids such as phytanic acid (from chlorophyll degradation in ruminants) that cannot undergo β-oxidation because the methyl group at C3 blocks thiolase. Phytanyl-CoA hydroxylase (encoded by PHYH) hydroxylates the α-carbon, and subsequent oxidative steps release one carbon as CO₂ to generate pristanic acid, which can then undergo β-oxidation. The resulting medium-chain products are exported to mitochondria for complete oxidation.

Defects in fatty acid oxidation produce characteristic clinical syndromes. MCAD deficiency — the most common inherited defect of fatty acid oxidation — impairs medium-chain fatty acid catabolism, causing hypoglycemia, lethargy, and accumulation of octanoylcarnitine during fasting, with overflow to ω-oxidation producing dicarboxylic aciduria. Zellweger syndrome results from PEX gene mutations that prevent peroxisome assembly, causing accumulation of VLCFAs and phytanic acid with severe neurological, renal, and skeletal consequences. X-linked adrenoleukodystrophy (X-ALD) results from a deficiency of the ABCD1 transporter, which prevents VLCFA import into peroxisomes and causes neurodegenerative and adrenal disease. Refsum disease arises from phytanyl-CoA hydroxylase deficiency, leading to phytanic acid accumulation that causes cardiac arrhythmias and sensory neuropathy.