Fatty Acid Catabolism and Digestion

Fatty Acid Catabolism

• Fatty acids are catabolized, and their inherent energy is captured by organisms.
• Fatty acids are utilized by organisms for energy storage.
• Carbon in fatty acids is mostly -CH2- and reduced, allowing its oxidation to yield the most energy possible.
• Fatty acids are not hydrated like mono- and polysaccharides, which allows them to pack more closely in storage tissues.

Fats Mobilization

• Most "fats" in diet and adipose tissue are triglycerides.
• Triglycerides provide major energy input in the modern American diet.
• Triglycerides are the major form of stored energy in the body.
• Hormones like glucagon, epinephrine, and ACTH trigger the release of fatty acids from adipose tissue.
• Triglycerides occupy most of the volume of adipose cells.

Fat Digestion

• Pancreatic lipases and PLA2 break down TAGs.
• After absorption, they are reconstituted.

Fatty Acids Breakdown

• Knoop showed that fatty acids must be degraded by removing 2-C units.
• Albert Lehninger showed that this process occurs in the mitochondria.
• F. Lynen and E. Reichart showed that the 2-C unit released is acetyl-CoA, not free acetate.
• The process begins with oxidation of the carbon that is "β" to the carboxyl carbon, hence it is called "β-oxidation".
• Fatty acids are degraded by repeated cycles of oxidation at the β-carbon, with cleavage of the Cα-Cβ bond to yield acetate units in the form of acetyl-CoA.

CoA Activation

• Acyl-CoA synthetase condenses fatty acids with CoA, with simultaneous hydrolysis of ATP to AMP and PPi.
• Formation of a CoA ester is energetically expensive.
• Free energy change barely breaks even with ATP hydrolysis.
• Subsequent pyrophosphate (PPi) hydrolysis drives the reaction forward.
• The acyl-adenylate intermediate is present in the mechanism.
• Acyl-CoA synthetase activates fatty acids for β-oxidation.
• The reaction is driven by ATP hydrolysis to AMP and pyrophosphate, and by subsequent pyrophosphate hydrolysis.
• The acyl-CoA synthetase reaction involves fatty acid carboxylate attack on ATP, forming an acyl-adenylate intermediate.
• The fatty acyl-CoA thioester product is formed by CoA attack on this intermediate.

Carnitine as a Carrier

• Carnitine carries fatty acyl groups across mitochondrial membranes.
• Short-chain fatty acids are carried directly into the mitochondrial matrix.
• Long-chain fatty acids cannot be directly transported into the matrix.
• Long-chain FAs are converted to acyl-carnitines, and transported into the mitochondria.
• Acyl-CoA esters are formed inside the inner mitochondrial membrane from transported acylcarnitines.

β-Oxidation Reactions

• It is a repeated sequence of 4 reactions.
• The strategy is to create a carbonyl group at the β-C.
• The first 3 reactions create a carbonyl group at the β-C, the fourth cleaves the β-keto ester in a reverse Claisen condensation.
• Products are an acetyl-CoA and a fatty acid two carbons shorter.
• The first three reactions are crucial and classic, appearing again in other pathways.
• The β-oxidation of saturated fatty acids involves a cycle of four enzyme-catalyzed reactions.

Acyl-CoA Dehydrogenases

• Acyl-CoA dehydrogenases are a family of membrane-bound and soluble matrix enzymes.
• As a fatty acyl chain is shortened in successive β-oxidation cycles, it moves from the membrane-bound complex to the family of soluble matrix enzymes.
• Acyl-CoA dehydrogenase mechanism involves proton abstraction, followed by double-bond formation and hydride removal by FAD.
• Electrons are passed to an electron transfer flavoprotein (ETF), and then to the electron transport chain.
• Very long-chain fatty acids proceed through several β-oxidation cycles via membrane-bound enzymes in mitochondria.
• They proceed before becoming substrates for the separate soluble enzymes of β-oxidation.
• Two electrons are removed in the acyl-CoA dehydrogenase reaction are delivered to the electron transport chain in the form of reduced coenzyme Q (UQH2).
• The mechanism of acyl-CoA dehydrogenase is the removal of a proton from the α-carbon, followed by hydride transfer from the β-carbon to FAD.

Enoyl-CoA Hydratase

• Enoyl-CoA Hydratase adds water across the double bond.
• The addition of the elements of H2O across the new double bond is stereospecific, yielding corresponding L-hydoxyacyl-CoA.
• The reaction is catalyzed by enoyl-CoA hydratase, also called crotonase.
• These enzymes convert trans-enoyl-CoA derivatives to L-β-hydroxyacyl-CoA.

Hydroxyacyl-CoA Dehydrogenase

• L-Hydroxyacyl-CoA Dehydrogenase oxidizes the β-Hydroxyl Group.
• The third reaction of this cycle is the oxidation of the hydroxyl group at the β-position to produce a β-ketoacyl-CoA derivative.
• L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD+ as a coenzyme catalyzes
• NADH produced in this reaction represents metabolic energy.
• Each NADH produced by this reaction in mammalian mitochondria drives the synthesis of 2.7 ATP.

Thiolase

• Aka β-ketothiolase.
• Cysteine thiolate on the enzyme attacks the β-carbonyl group, leading to acetyl-CoA release.
• Thiol group of a new CoA attacks the shortened chain, forming a new acyl-CoA.
• This is the reverse of a Claisen condensation through attack of the enolate of acetyl-CoA on a thioester.
• The reaction is favorable and drives the other three reactions even though it forms a new thioester.
• Cys thiolate group attack at the β-carbonyl carbon produces a tetrahedral intermediate, decomposes with departure of acetyl-CoA, enzyme thioester intermediate.
• Attack by the thiol group of a second CoA yields a new acyl-CoA.

Trifunctional Protein Complex

• Oligomeric associations of constituent enzymes facilitate efficiencies for metabolic pathways.
• A trifunctional enzyme (TFE) is an important component of the β-oxidation pathway.
• Bacterial and human forms of this complex are similar with two types of subunits.
• The α-subunit contains the enoyl-CoA hydratase (ECH) and hydroxyacyl-CoA dehydratase (HCAD) activities
• The β-subunit contains the ketoacyl thiolase (KACT) activity.
• A substrate channel on the surface of the complex facilitates substrate channeling.

β-Oxidation Summary and Palmitic Acid Oxidation

• Repetition of the cycle gives a succession of acetate units.
• Palmitic acid yields eight acetyl-CoAs.
• Complete β-oxidation of one palmitic acid yields 106 molecules of ATP.
• Large energy yield results from highly reduced state of the carbon in fatty acids.
• Highly reduced state and low density makes fatty acids the fuel of choice for migratory birds and many other animals.
• Reduced Coenzymes drive synthesis of ATP in oxidative phosphorylation.
• Complete oxidation of palmitoyl-CoA yields a net of 106 ATP.
• 1 AcCo gives 10 ATP (in TCA, ETC).
• 1 NADH gives 2.5 ATP.
• 1 FADH2 gives 1.5 ATP.
• For an even FA with n carbons, n/2-1 beta oxidations, n/2-1(NADH + FADH2) and n/2 AcCoA.
• C16 gives 8AcCoA, 7(NADH +FADH2) – 2ATP invested to make initial CoAester.

Migratory Birds and Fatty Acid Oxidation

• Because they represent the most highly concentrated form of stored biological energy, fatty acids are the metabolic fuel of choice for sustaining the long flights of migratory birds.
• American golden plovers fly from Alaska to Hawaii nonstop – 3300 km in 35 hours – more than 250,000 wing beats.
• The ruby-throated hummingbird flies nonstop across the Gulf of Mexico
• These prodigious feats are accomplished by storing large amounts of triacylglycerols prior to flight.
• These birds are often 70% fat by weight when migration begins.

Metabolic Water and Fatty Acid Oxidation

• Large amounts of metabolic water are generated by β-oxidation.
• The oxidation of stored fatty acids can be a significant source of dietary water for certain animals, including desert animals (such as gerbils), killer whales (which do not drink seawater), and camels (whose hump is a large fat deposit).
• Metabolism of fatty acids from stores provides needed water and metabolic energy when drinking water is not available.

Odd-Carbon Fatty Acid Oxidation

• β-Oxidation of odd-carbon fatty acids yields propionyl-CoA.
• Odd-carbon fatty acids are metabolized normally, until a 3-C fragment - propionyl-CoA is reached.
• Three reactions convert propionyl-CoA to succinyl-CoA.
• Biotin and vitamin B12 involvement.
• Catalytic power of the epimerase reaction considered.
• Succinyl-CoA pathway for net oxidation considered.
• The conversion of propionyl-CoA (formed from β-oxidation of odd-carbon fatty acids) to succinyl-CoA is carried out by a trio of enzymes.
• Succinyl-CoA can enter the TCA cycle.

L-Methylmalonyl-CoA and B12 Catalysis

• L-Methylmalonyl-CoA catalyzed by B12 yields Succinyl-CoA.
• The methylmalonyl-CoA epimerase mechanism involves a resonance-stabilized carbanion at the α-position.

Unsaturated Fatty Acids Oxidation

• Consider monounsaturated fatty acids like oleic acid and palmitoleic acid.
• Proceed with normal β-oxidation for three cycles.
• Cis-Δ3 acyl-CoA cannot be utilized by acyl-CoA dehydrogenase.
• Enoyl-CoA isomerase converts this to trans-Δ2 acyl CoA.
• β-oxidation continues from this point.
• β-Oxidation of unsaturated fatty acids-in the case of oleoyl-CoA, three β-oxidation cycles produce three molecules of acetyl-CoA and leave cis-Δ3-dodecenoyl-CoA
• Enoyl-CoA isomerase rearranges this, giving the trans-Δ2 species, which then proceeds normally through the β-oxidation pathway.

Polyunsaturated Fatty Acids

• Degradation of polyunsaturated fatty acids is slightly more complicated.
• The process proceeds the same as for oleic acid, through 3 cycles of β-oxidation.
• Enoyl-CoA isomerase then converts the cis-Δ3 double bond to a trans-Δ2 double bond.
• Permits 1 more round of β-oxidation.
• The resulting trans-Δ2, cis-Δ4 structure is a problem,
• 2,4-Dienoyl-CoA reductase solves this problem.
•  Polyunsaturated FA use beta oxidations until the first C=C bond is reached, where enoyl-CoA-isomerase makes the trans delta 2 unsaturated ester and beta oxidation continues until the second double bond is reached
•  2,4-Acyl-CoA dehydrogenase uses NADPH to reduce the ends to a single C=C.

Peroxisomal β-Oxidation

• Peroxisomes are organelles with flavin-dependent oxidations, with oxidized flavins regenerating by reaction with O2 to produce H2O2.
• Similar to mitochondrial β-oxidation, but initial double bond formation is by acyl-CoA oxidase.
• Electrons go directly to O2 from the flavoprotein, and there is no electron transport.
• There are fewer ATPs.
• Requires FAD dependent acyl-CoA oxidase.
• Captured electrons used to make HOOH, needed for degradative processes (not available to ATP).
• Acyl-CoA requires FAD-dependent acyl-CoA oxidase.

Branched-Chain Fatty Acids and α-Oxidation

• α-Oxidation is required as an alternative to β-oxidation
• Branched chain FA with branches with odd-number carbons are not good substrates for β oxidation and are alternatives in this instance.
• Phytanic acid α-oxidase decarboxylates with oxidation at the alpha position.
•  β-oxidation past branch.
• Branched-chain FA are oxidized by α-oxidation- product is suitable for normal β-oxidation Isoputiryl-CoA and propionyl-CoA can both be converted to Succinyl-CoA, and can enter the CAC.
• Refsum’s disease results from the mitochondrial α-oxidizing Enzyme.

Refsum’s disease

• Rare inherited disorder-patients lack mitochondrial α-oxidizing e.
• Consequence large quantities phytanic acid in tissue and serum
• Cerebellar ataxia (lack of muscle control), retinitis pigmentosa (genetic disorder eyes), nerve deafness peripheral neuropathy
• Restriction dairy products, ruminant meat alleviate symptoms.

ω-Oxidation

• Yields small amounts dicarboxylic acids.
• Dicarboxylic acids are a form by oxidation of the omega carbon of fatty acids (Cytochrome P-450 dependent reaction).

Ketone Bodies

• Special source to tissues.
• Acetyl-CoA from fatty acid oxidation -> Acetone Acetoacetate, α-hydroxybutyrate.
• Source fuel brain heart muscle.
• Transportable Fa.

Ketone Metabolism, Synthesis

• Synthesis ketone bodies only mitochondrial matrix
• Figure 23.26 reverse Thiolase
• Second reaction HMG-CoA
• Mitochondrial analogs of first two Cholesterol Synthesis Third HMG-CoA lyase same Citrate Synthase.

Ketone Bodies & Diabetes

• "Starvation" Cell plenty glucose abound blood, uptakes cells low
• Cells metabolically starved turns gluconeogenesis fat protein catabolism
• Diabetic OAA (oxaloacetic acid) - Low excess gluconeogenesis acetyl-CoA (catabolism/protein) does not go into CAC = Ketone production
• Acetone detected breathe

Ketone Bodies & Acidosis

• Relativley strong acids (Pka ~3.5) lower the pH
• Impairs ability hemoglobin to bind oxygen.