Cell Metabolism 2 (TCA & β-oxidation)

What is the TCA cycle?

The tricarboxylic acid (TCA) cycle, also called the Krebs cycle, is an aerobic mitochondrial pathway that oxidises acetyl-CoA to generate NADH, FADH2, GTP and CO2.

Where does the TCA cycle occur?

The TCA cycle occurs in the mitochondrial matrix. Most enzymes are soluble proteins in the mitochondrial matrix.

Why is the TCA cycle considered aerobic?

The TCA cycle depends on re-oxidation of NADH and FADH2 by oxidative phosphorylation, which requires oxygen.

What enters the TCA cycle?

Acetyl-CoA combines with oxaloacetate to begin the TCA cycle.

What does one turn of the TCA cycle produce?

3 NADH, 1 FADH2, 1 GTP and 2 CO2.

How many ATP equivalents are produced from one acetyl-CoA in the TCA cycle?

Approximately 12 ATP equivalents: 3 NADH × 3 ATP + 1 FADH2 × 2 ATP + 1 GTP × 1 ATP.

Why is oxaloacetate important?

Oxaloacetate is required for acetyl-CoA to enter the TCA cycle.

What happens to the carbon atoms of acetyl-CoA in the TCA cycle?

The two carbons entering as acetyl-CoA are ultimately released as CO2.

What is the main purpose of the TCA cycle?

To generate reduced cofactors (NADH and FADH2) that drive ATP production through oxidative phosphorylation.

What are glucogenic amino acids?

Amino acids whose carbon skeletons can be converted into glucose.

What are ketogenic amino acids?

Amino acids whose carbon skeletons can be converted into ketone bodies.

General strategy of amino acid degradation

The amino group is removed and eventually excreted as urea, while the carbon skeleton enters gluconeogenesis or the TCA cycle.

What seven molecules result from degradation of all 20 amino acids?

Pyruvate, acetyl-CoA, acetoacetyl-CoA, α-ketoglutarate, succinyl-CoA, fumarate and oxaloacetate.

What is transamination?

A group transfer reaction in which an amino group is transferred from an amino acid to a keto acid, generating a new amino acid and a new keto acid.

What type of reaction is transamination?

Group transfer.

Alanine transamination

Alanine + α-ketoglutarate → Pyruvate + Glutamate via alanine aminotransferase.

What enzyme catalyses alanine transamination?

Alanine aminotransferase (ALT).

What happens to glutamate after transamination?

Glutamate can be converted back to α-ketoglutarate by glutamate dehydrogenase, releasing NH4+ for urea synthesis.

Why is NAD+ regeneration important?

Without regeneration of NAD+, glycolysis rapidly stops because NAD+ is required for glyceraldehyde-3-phosphate dehydrogenase.

What is the purpose of NADH shuttles?

To transfer high-energy electrons from cytosolic NADH into mitochondria for oxidative phosphorylation.

What are the two major NADH shuttles?

The glycerol phosphate shuttle and the malate-aspartate shuttle.

Which tissues primarily use the glycerol phosphate shuttle?

Skeletal muscle and brain.

Which tissues primarily use the malate-aspartate shuttle?

Liver, kidney and heart.

How does the glycerol phosphate shuttle work?

Cytosolic glycerol-3-phosphate dehydrogenase transfers electrons from NADH to DHAP, producing glycerol-3-phosphate. A mitochondrial form transfers the electrons to FAD and then coenzyme Q.

What is transferred across the mitochondrial membrane by NADH shuttles?

Electrons, not NADH itself.

What reactions occur in the malate-aspartate shuttle?

Redox reactions and transamination reactions.

What enzyme does MDH stand for?

Malate dehydrogenase.

What enzyme does AT stand for?

Aspartate transaminase.

What is the biological significance of glycolysis and the TCA cycle beyond ATP production?

They provide building blocks for biosynthesis of amino acids, nucleotides, lipids, sugars and other cellular molecules.

What is the Warburg effect?

The preferential generation of lactate from glucose despite adequate oxygen availability.

Which TCA cycle enzymes are commonly mutated in cancer?

Succinate dehydrogenase, fumarase and isocitrate dehydrogenase.

How might TCA cycle defects contribute to cancer?

They decrease TCA cycle activity and enhance aerobic glycolysis (the Warburg effect).

What is β-oxidation?

The mitochondrial pathway that degrades fatty acids to generate acetyl-CoA, NADH and FADH2.

Why are fats a rich energy source?

On a weight basis, fatty acids yield approximately twice as much energy as carbohydrates.

When does fatty acid oxidation become particularly important?

During prolonged fasting and energy deprivation.

First step of β-oxidation

Fatty acids are activated by conversion into acyl-CoA.

Enzyme for fatty acid activation

Acyl-CoA synthetase.

Fatty acid activation reaction

Fatty acid + CoA + ATP → Acyl-CoA + AMP + PPi.

How many high-energy phosphate bonds are consumed during fatty acid activation?

Two high-energy phosphate bonds because ATP is converted to AMP.

Why is the carnitine shuttle required?

Long-chain fatty acyl-CoA molecules cannot directly cross the inner mitochondrial membrane.

What is the function of the carnitine shuttle?

To transport activated fatty acids from the cytosol into the mitochondrial matrix.

How does the carnitine shuttle work?

Acyl-CoA is converted to acyl-carnitine, transported across the membrane by a translocase and then reconverted to acyl-CoA inside the matrix.

What is primary carnitine deficiency?

An autosomal recessive disorder caused by mutations affecting the carnitine transporter, reducing cellular uptake of carnitine.

What gene is mutated in primary carnitine deficiency?

SLC22A5.

Symptoms of primary carnitine deficiency

Encephalopathy, cardiomyopathy, muscle weakness and hypoglycaemia.

Treatment of primary carnitine deficiency

Carnitine (levocarnitine) supplementation.

What four reactions make up β-oxidation?

Oxidation, hydration, oxidation and thiolysis.

What is produced during one cycle of β-oxidation?

One acetyl-CoA, one NADH, one FADH2 and an acyl-CoA shortened by two carbons.

What is thiolysis?

The cleavage reaction in β-oxidation that releases acetyl-CoA using CoA.

What happens during β-oxidation of palmitoyl-CoA?

Seven cycles of β-oxidation generate eight acetyl-CoA molecules, seven NADH and seven FADH2.

How many acetyl-CoA molecules does palmitate generate?

Eight acetyl-CoA molecules.

How many β-oxidation cycles are needed for palmitate?

Seven cycles.

Overall β-oxidation products of palmitoyl-CoA

8 acetyl-CoA + 7 NADH + 7 FADH2.

Net ATP yield from complete oxidation of palmitate

Approximately 129 ATP after accounting for activation costs.

Why does β-oxidation yield so much ATP?

It generates large amounts of NADH and FADH2, and multiple acetyl-CoA molecules enter the TCA cycle.

What are ketone bodies?

Acetoacetate, D-3-hydroxybutyrate and acetone.

When are ketone bodies formed?

When fat breakdown predominates, such as during prolonged fasting.

Why might acetyl-CoA form ketone bodies instead of entering the TCA cycle?

Because oxaloacetate is diverted for gluconeogenesis and is unavailable for acetyl-CoA entry into the TCA cycle.

Meaning of the phrase "Fat burns in the flame of carbohydrate"

Acetyl-CoA can enter the TCA cycle only when sufficient oxaloacetate from carbohydrate metabolism is available.

What is lipogenesis?

The biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA.

How many major enzymes drive lipogenesis?

Two: acetyl-CoA carboxylase and fatty acid synthase.

What are the two major enzymes of lipogenesis?

Acetyl-CoA carboxylase and fatty acid synthase.

What substrates are used in lipogenesis?

Acetyl-CoA and malonyl-CoA.

How are fatty acids built during lipogenesis?

By sequential decarboxylative condensation reactions that add two carbons at a time.

What enzyme activities occur after each cycle of fatty acid elongation?

Ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER).

What carrier protein is used during fatty acid synthesis?

Acyl carrier protein (ACP).

Three distinctions between lipogenesis and β-oxidation

1. Carrier: ACP in lipogenesis vs CoA in β-oxidation. 2. Reducing power: NADPH in lipogenesis vs FAD and NAD+ in β-oxidation. 3. Location: Cytoplasm in lipogenesis vs mitochondrial matrix in β-oxidation.

Where does lipogenesis occur?

Mainly in the cytoplasm.

Where does β-oxidation occur?

In the mitochondrial matrix.

Which cofactor provides reducing power for lipogenesis?

NADPH.

Which cofactors are used in β-oxidation?

FAD and NAD+.

What carrier is used in lipogenesis?

Acyl carrier protein (ACP).

What carrier is used in β-oxidation?

CoA.

Overall lipogenesis reaction

Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ → Palmitate + 7 CO2 + 6 H2O + 8 CoA-SH + 14 NADP+.

What is the main fatty acid product of lipogenesis?

Palmitate (16 carbons).

What is elongation of fatty acids?

The extension of fatty acids beyond 16 carbons after palmitate synthesis.

Where does elongation of fatty acids occur?

In the mitochondria and endoplasmic reticulum.

What is desaturation of fatty acids?

The introduction of double bonds by fatty acyl-CoA desaturases.

What is Δ9 desaturase?

The enzyme that introduces a double bond nine carbons from the carboxyl group, producing oleic acid from stearate and palmitoleic acid from palmitate.

Where is de novo fatty acid synthesis normally found in adults?

Mainly in the liver, adipose tissue and lactating breast.

Why is fatty acid synthesis important in cancer?

Many cancer cells reactivate fatty acid synthesis pathways to support growth and survival.

What enzyme is a major target in cancer fatty acid metabolism?

Fatty acid synthase (FASN).

What evidence links FASN to cancer?

Inhibition of FASN by cerulenin has been shown to reduce ovarian tumour growth.

Why might FASN be a useful cancer therapy target?

Some cancers depend on fatty acid synthesis, so blocking FASN may inhibit tumour growth.

What are acyl-CoA dehydrogenases?

A family of enzymes that catalyse the initial oxidation step of β-oxidation for fatty acids of different chain lengths.

Why are multiple acyl-CoA dehydrogenases needed?

Fatty acids become progressively shorter during β-oxidation and different enzymes recognise different chain lengths.

What is MCADD?

Medium-chain acyl-CoA dehydrogenase deficiency, a disorder of fatty acid β-oxidation.

Inheritance pattern of MCADD

Autosomal recessive.

What enzyme is deficient in MCADD?

Medium-chain acyl-CoA dehydrogenase.

Why can MCADD be dangerous?

Affected individuals cannot effectively generate energy from medium-chain fatty acids, especially during fasting.

How is MCADD diagnosed?

Newborn heel-prick screening.

How common is MCADD in the UK?

Approximately 1 in 10,000 live births.

Relationship between MCADD and SIDS

If undiagnosed, MCADD is thought to account for around 1 in 100 sudden infant death syndrome cases.

Lifestyle advice for patients with MCADD

Avoid prolonged fasting, follow a high-carbohydrate diet and seek glucose supplementation during illness.

Why might intravenous glucose be needed in MCADD?

To prevent reliance on fatty acid oxidation during illness, vomiting or poor oral intake.

Key summary of lipogenesis

Lipogenesis is essentially the reverse of β-oxidation, uses ACP and NADPH, occurs in the cytoplasm, is driven mainly by acetyl-CoA carboxylase and fatty acid synthase, and produces palmitate as the primary fatty acid product.

What are three examples of ketone bodies produced during fasting?

Acetoacetate, beta-hydroxybutyrate, and acetone.

What is the net result of each “turn” of the Kreb’s cycle?

Each turn of the Kreb's cycle produces 3 NADH, 1 FADH2, 1 GTP (or ATP), and releases 2 CO2.

Which family of enzymes catalyses the first oxidation step in the β-oxidation of fatty acids?

The acyl-CoA dehydrogenase family of enzymes catalyzes the first oxidation step in the β-oxidation of fatty acids, converting acyl-CoA to trans-Enoyl-CoA while reducing FAD to FADH2.

Why is the TCA cycle described as an amphibolic pathway?

It is both catabolic (oxidising acetyl-CoA to generate energy) and anabolic (providing intermediates for biosynthesis).