Fatty ACid Catabolism of Unsaturated and Odd chain fatty acids

Beta-oxidation of monosaturated fatty acid

• Cis oleoyl-CoA (18:1 delta 9), goes through 3 beta-oxidation cycles creating a 12-Carbon cis-delta3,4-fatty acyl-CoA

  • Cis 3,4-CoA double bond cannot be processed by the first two beta-oxidation enzymes without rearrangement, must get a cis 3,4 double bond to trans 2,3 double bond

• Enoyl-CoA isomerase converts the stalled cis-delta3-enoyl-CoA into the trans-delta2-enyol-CoA

• Can then enter standard beta-oxidation by enoyl-CoA hydratase

• It skips the first step as the double bond is already present in the substrate, FADH2 isn't produced at this cycle, leads to fewer ATP equivalents

• Pre-existing unsaturation bypasses the initial dehydrogenation and reduces the total ATP yield per carbon

Mechanism of enoyl-CoA isomerase repositions double bonds to sustain beta-oxidation of monounsaturated fats

Issues with MUFAs

• Beta-oxidation enzymes act only on trans-delta2-enoyl-CoA intermediates

• Oxidation of a double bond located at an odd carbon creates a delta3-enoyl-CoA intermediates

Solution

• Enoyl-CoA isomerase repositions the double bond to form the required trans-delta2-enoyl-CoA, allows beta-oxidation to proceed

• Catalyzes an intramolecular redox-neutral isomerization

Mechanism

• Proton abstraction and re-addition causes double bond migration from C3-C4 to C2-C3 configuring the bond to trans

• The active site uses a conserved glutamate to abstract the alpha proton (C2) and re-protonate at C4 to shift the pi bond

• No change in oxidation state of substrate, just positional isomerization

• Always yield trans-delta2-product with correct geometry for the next beta-oxidation step

Enzymatic Strategies in polyunsaturated fatty acid beta-oxidation

Start

• Undergoes regular beta-oxidation until it reaches the double bond

• Will use enoyl-CoA isomerase to shift a cis-delta3 double bond to the trans-delta2 form for continued beta-oxidation

Problem

• After 4th round of oxidation and isomerization, it generates a 2,4-dienoyl-CoA which has 2 conjugated double bonds

• Standard beta-oxidation enzymes only act on substrates with a single trans-delta2 doble bond

Solution

• 2,4-dienoyl-CoA reductase reduces the remaining conjugated dienoyl-CoA intermediates to trans-delta3-enoyl-CoA which is converted to trans-delta2enoyl-CoA by enoyl-CoA isomerase and processed by the standard beta-oxidation enzymes

2-4-dienoyl-CoA Reductase

Problem

• Beta-oxidation of PUFAs produces 2,4-dienoyl-CoA intermediates (conjugated 2,4 double bonds)

• Cannot be processed by standard beta-oxidation enzymes

Solution

• Enzyme reduces the conjugated system to a single trans-delta3-enoyl-CoA

• Isomerized to beta-oxidation compatible trans-delta2-enoyl-CoA by enoyl-CoA isomerase

Mechanism

• DECR catalyzes conjugated diene to monoene reduction (2 double bond to 1)

• NADPH transfers a hydride to C5, causes conjugated diene to be at C1 and C3 (oxygen takes electrons from C=O)

• C2 is protonated to converts the 1-2 double bond into a 1-2 single bond

• Only remaining doble bond is trans 3

• Tyr and Asn active site residues stabilize tetrahedral intermediate to facilitate proton transfer

Enoyl-CoA isomerase

• Cis delta 3 to trans delta 2

• Standard hydratase needs trans delta 2

• Glutamate abstracts alpha proton and reprotonates C4

• Turns cis double bond into trans

2,4-dienoyl-CoA reductase

• For conjugated 2,4-dienoyl-CoA to trans-delta3-enoyl-CoA

• Standard enzymes reject conjugated systems

• NADPH donates hydride to C5 and a proton to C2

• Tyr and ASN stabilize the tetrahedral intermediate

• Creates a trans double bond at C3

Final cycle of beta-oxidation, propionyl-CoA to TCA cycle

1. 5C FA turns into Acetyl-CoA and propionyl-CoA

2. Propionyl-CoA uses propionyl-CoA carboxylase to add a carboxyl group on the middle carbon can produces D-methylmalonyl-CoA

   1. Uses HCO3, ATP as substrates, biotin cofactor, ADP and Pi as product

3. Methylmalonyl-CoA epimerase converts D-methylmalonyl-CoA to L-methylmalonyl-CoA

   1. CoA is moved from Carboxyl on the send to the newly added one on the middle carbon

4. Methylmalonyl-CoA mutase uses B12 to isomerize it into succinyl-COA

   1. L-MM-CoA and succinyl-Coa are epimers (stereoisomers that differ at 1 chiral centre)

   2. Carboxyl with CoA switches with a hydrogen on the other carbon end

Regulation of fatty acid breakdown

Key points

1. Entry into mitochondria - Carnitine Palmitoyltransferase 1 (CPT-1)

   1. Rate limiting step of beta-oxidation

   2. Inhibited by malonyl-COA linking fatty acid synthesis to degradation

2. Hormonal regulation

   1. Glucagon and epinephrine stimulate beta-oxidation by increasing lipolysis and activating CPT-1 indirectly by reducing malonyl-CoA. MORE ENERGY

   2. Insulin suppresses fatty acid breakdown, promotes malonyl CoA, and inhibit lipolysis, Don't need energy

1. Energy charge control

   1. Buildup high NADH/NAD+ and acetyl-CoA slow beta-oxidation by mass-action (Q) and thermodynamic feedbac

Acetyl-CoA serves many purposes

• Energy production

  • Feeds TCA for ATP generation

• Biosynthesis

  • Building block for fatty acids, cholesterol, ketone bodies, isoprene's and some amino acids

• Regulation

  • Donates acetyl groups for protein acetylation, influencing enzyme activity and gene expression

• Integration of metabolism

  • Central node linking catabolic and anabolic pathways

  • Helps cell balance energy supply and biosynthetic demands

Condensation of acetyl-group enolates produces the branched intermediates beta-hydroxy-beta methylglutaryl-CoA (HMG-CoA)

• Mitochondrial matrix, HMG-COA is cleaved to ketone bodies for export as fuel

• In cytosol, converted into a 5 carbon isoprene unit, building block for terpenes, cholesterol and other complex lipids

  • Multiple copies of 5 carbon isoprene unit can be joined to form complex carbon skeletons in terpenes and steroids

HMG-CoA synthesis

1. 2 acetyl-CoA join together, thiolase displaced CoA from one acetyl-CoA to form acetoacetyl-CoA

   1. Reversible direction, depends on acetyl-CoA Concentration

   2. High concentration = condensation is favoured over splitting, drives synthesis of downstream products

2. HMG-CoA synthase joins another acetyl-S-CoA to HMG-S-Coa + HS-CoA (+H2O)

   1. Combines a third CoA tag, analogous to citrate synthase

   2. Incoming acetyl-Coa forms an enolate to attack the carbonyl of acetoacetyl-CoA

   3. Reaction is endergonic but driven forward by high acetyl-CoA levels and exergonic cleavage of acetyl-CoA thioester

3. Reaction is the same between mitochondrial HMG-CoA synthase for ketones and cytosolic HMG-COA synthase for isoprenoids

Ketogenesis, in the mitochondrial matrix

• HMG-lyase in mito matrix cleaves CoA-linked acetyl group on HMG to generate acetoacetate for export from mitochondrial

  • HMG-S-COA > acetoacetate + acetyl-CoA

• Acetoacetate unstable beta-keto carboxylate spontaneously decarboxylate to acetone (unusable fuel)

  • Uses acetoacetate decarboxylate to speed up reaction for ketone regulation

• Reduction of beta keto group in acetoacetate by beta-hydroxybutyrate dehydrogenase to produce D-beta-hydroxybutyrate (BHB)

  • Ruses NADH and H+ as substrate

• In humans most circulating ketone bodies are BHB, doesn't have a reactive ketone body

Ketolysis

• Keton body catabolism in peripheral tissue is to generate energy

• Requires bypass reaction as HMG-CoA synthase is absent in peripheral tissue

• Converts BGB to acetyl-CoA for energy

Ketolysis Steps

1. BHB is oxidized to acetoacetate using BHB dehydrogenase and generates NADH

   1. In all mitochondrial matrix of all tissue but the liver

2. ACA is activated by a CoA transfer from succinyl-CoA to from acetoacetyl-CoA which enters the TCA cycle as acetyl-CoA for ATP production

   1. Succinyl-CoA:acetoacetate CoA-transferase or thiophorase

   2. Transfers CoA from succinyl-CoA to acetoacetate forming acetoacetyl-CoA

   3. Liver lacks this enzyme and cannot use keton bodies as fuel

3. Acetoacetyl-CoA is cleaved by thiolase into 2 molecules of acetyl-CoA to enter TCA cycle for oxidation and ATP production

Why acetyl-CoA cannot be turned into glucose and why it uses acetyl-CoA to make ketone bodies instead

1. Some sources of carbon can become glucose and are glucogenic

   1. Intermediates of glycolysis, lactate and TCA 4 carbon intermediates can be used to make glucose

   2. Enter gluconeogenesis

2. Acetyl-CoA cannot become glucose

   1. Pyruvate dehydrogenase irreversibly converts pyruvate to acetyl-COA

   2. There is no enzyme that converts acetyl-CoA back into pyruvate

3. Carbon in acetyl-CoA is ketogenic

   1. Acetyl-CoA cannot contribute to net glucose production

   2. It is ketogenic carbon, form ketone bodies instead of glucose

   3. It doesn't increase pool of oxaloacetate, for every one acetyl-CoA it generates one oxaloacetate

Ketogenic exportable fuel

• Ketogenic carbon is stored as fatty acids in adipocytes

• Fasting or glucagon stim released fatty acids are oxidized by the liver to acetyl-CoA

• When Acetyl-CoA exceeds the liver's oxidative capacity, it diverts to ketogenesis forming acetoacetate, beta-hydroxybutyrate and acetone

When ketone bodies are formed

• Ketone bodies are exported from the liver and use as an immediate, water-soluble energy source by heart, skeletal muscle, renal cortex and brain when need

• Tissue converts acetoacetate and beta-hydroxybutyrate back to acetyl-CoA

• Acetyl-CoA enters the TCA to generate ATP

• Acetone is exhale and not metabolized

 

Ketone vs Fatty acids

• Ketone body are not a storage form of energy, it is a transportable intermediate

• Ketone body is produced by the liver during carbohydrate scarcity to distribute acetyl-CoA to tissue that need it

  • Acetyl-CoA is membrane impermeable and can't leave cells

• Liver synthesize but can't use ketone

  • Lack enzyme to convert back to acetyl-CoA

  • Peripheral tissue has thiophorase

4-carbon pool availability decides acetyl-CoA fate in liver

1. In a glucagon response, liver gets ATP from FA catabolism (glucose is for export not catabolism)

2. 4C dicarboxylate pool, OAA, is used for gluconeogenesis. During fasting liver's priority is to export glucose.

   1. OAA > PEP > gluconeogenesis > glucose

3. If glucagon signal is prolonged, OAA is depleted turning into glucose

   1. Lower OAA = less acetyl-CoA enter TCA cycle

   2. Acetyl-COA accumulates and is diverted to ketogenesis

• Less acetyl-CoA entering TCA = less ATP supply so then liver uses amino acids for energy

During fasting OAA is used to make glucose, leaving too little to run the TCA cycle, forces accumulate acetyl-CoA to be converted into ketone bodies

HMG-CoA: Branch point for ketones and isoprenoids

• Assembly for acetyl-group enolates produces a branched intermediates HMG-CoA

• This is where it differs

Mitochondrial matrix

• HMG-CoA serves as the precursor for ketone body synthesis

  • HMg-CoA > acetoacetate > acetone and reduces to BHB (D-betahydroxybutyrate) (ketogenesis)

  • BHB > acetoacetate > acetoacetyl-CoA (uses thiophorase)

  • Acetoacetate is able to transport to and from the blood

• Occurs in all tissue but the liver

In the cytosol

• HMG-CoA is diverted to produce 5-carbon isoprene units

• Building blocks for cholesterol and other isoprenoids

• Occurs in all tissues

Isoprenoid synthesis

Stage 1: Formation of mevalonate

• Mirror ketogenesis but occur in the cytosol using distinct isozymes

1. Formation of acetoacetyl-CoA

   1. 2 acetyl-CoA condense to form acetoacetyl-CoA

   2. thiolase

   3. Reversible and doesn't commit cell to isoprenoid synthesis

2. Formation of HMG-CoA

   1. Acetoacetyl-CoA condenses with a third acetoacetyl-CoA condenses with a third acetyl-CoA to generate HMG-COA

   2. HMG-CoA synthase

   3. Reaction is reversible

3. Key regulatory step

   1. HMG-CoA reductase

      1. Catalyze NADPH-dependent, 4 electron reduction of HMG-CoA to mevalonate, double reduction of thio-ester  turns into a primary alcohol and thiol

      2. HMG-CoA + 2NADPH + 2H+ > Mevalonate + 2NADP+ + CoA-SH

   2. This is the committing the pathway to cholesterol and isoprenoid synthesis

   3. Committed step and primary regulatory enzyme of cholesterol biosynthesis

   4. HMG-CoA reductase is found in the liver, in the smooth endoplasmic reticulum as a membrane protein

   5. Statins: reduce cholesterol by blocking HMG-CoA reductase

Stage 2: Mevalonate activation and formation of activated isoprenes (IPP and DMAPP)

• Mevalonate is sequentially phosphorylated three times, to create a highly activated intermediate

• First 2 carboxylation occurs at C5, third one is at C3

1. Mevalonate (mevalonate 5-phosphotransferase) + ATP > 5-phosphomevalonate

2. 5-phosphomevalonate (phosphomevalonate kinase) + ATP > 5-pyrophosphomevalonate

3. 5-pyrophosphomevalonate (pyrophospho-mevalonate decarboxylase) + ATP > 3-phospho-5-pyrophosphomevalonate

• The third phosphorylation at C3 primes the molecule for a decarboxylation reaction, eliminates phosphate and carboxyl group to form a double bond creating Isopentenyl pyrophosphate (IPP), first activated isoprene

• IPP undergo reversible isomerization to generate dimethylallyl pyrophosphate (DMAPP), second activated isoprene

  • Double changes from C2=C3 to C3=C4

• IPP and DMAPP serve as the fundamental five-carbon building blocks for downstream isoprenoid and sterol synthesis

 

IPP is the universal five-carbon building block feeding the entire isoprenoid family

• Includes rubber, quinones, carotenoids, retinoids, fat-soluble vitamins and all sterol

• Central launching point for vast structural and functional diversity in biology

• Multiple isoprene units polymerize to squalene which is completely hydrophobic

• Cyclization's make multi-ring steroid nucleus, 4 fused rings , 3 6c 1 5c rings

  • Used to create cholesterol, steroids, bile salts

Liver

• Creates bile acid for emulsifying lipids in digestive tract

• Taurocholate

Skin

• Produces vitamin D for calcium signaling and gene regulation

• The steroid nucleus in the presence of UV light creates cholecalciferol, Vitamin D3

Adrenal cortex

• Creates steroid hormones

• Glucocorticoids like cortisol

• Anti-inflammation signaling

Gonads

• Creates steroid hormones

• Sex hormones like androgens, estrogen and testosterone

Know HMG-CoA reductase is the committed and primary regulatory step in isoprene/cholesterol biosynthesis

1. HMG-CoA reductase

   1. Catalyze NADPH-dependent, 4 electron reduction of HMG-CoA to mevalonate, double reduction of thio-ester  turns into a primary alcohol and thiol

   2. HMG-CoA + 2NADPH + 2H+ > Mevalonate + 2NADP+ + CoA-SH

2. This is the committing the pathway to cholesterol and isoprenoid synthesis

3. Committed step and primary regulatory enzyme of cholesterol biosynthesis

4. HMG-CoA reductase is found in the liver, in the smooth endoplasmic reticulum as a membrane protein

5. Statins: reduce cholesterol by blocking HMG-CoA reductase