BIOC 3022 - Quarter 4

What triggers fatty acid synthesis?

High energy charge → NADH and citrate accumulate → citrate is exported to cytosol to initiate synthesis.

Which TCA intermediate contributes to lipid synthesis?

Citrate

How does citrate leave the mitochondria?

Via citrate transport protein across inner membrane; outer membrane is freely permeable.

What enzyme converts citrate into cytosolic acetyl-CoA?

ATP-citrate lyase

What happens to oxaloacetate in the cytosol?

Reduced to malate (uses NADH), then converted to pyruvate via malate enzyme.

What is produced when malate is converted to pyruvate?

NADPH + CO2.

Why is the OAA → malate → pyruvate pathway important during fatty acid synthesis?

It generates NADPH, which provides the reducing power required for the reduction steps in fatty acid synthesis.

What is the rate-limiting step of fatty acid synthesis? What enzyme catalyzes this step?

Conversion of acetyl-CoA to malonyl-CoA. Acetyl-CoA carboxylase (ACC).

What does Acetyl-CoA carboxylase (ACC) require?

ATP and biotin (carboxybiotin intermediate)

What is the function of malonyl-CoA?

Donor of 2-carbon units for fatty acid elongation.

What is another key role of malonyl-CoA?

Inhibits fatty acid transport into mitochondria (blocks β-oxidation).

How is ACC regulated allosterically?

Activated by citrate; inhibited by palmitoyl-CoA

How is ACC regulated by phosphorylation?

AMPK phosphorylates and inactivates ACC when AMP is high.

What hormone promotes ACC activation?

Insulin (via dephosphorylation).

What energy state activates AMPK?

Low energy (high AMP/ATP ratio).

How does citrate regulate glycolysis?

Inhibits PFK-1, slowing glycolysis.

What type of enzyme is fatty acid synthase?

Multi-enzyme complex (homodimer).

How many functional components does FAS have?

7 components (6 enzymes + ACP).

What is ACP?

Acyl Carrier Protein; carries the growing fatty acid chain (like CoA).

Where is the acyl chain temporarily stored in FAS?

ACP and cysteine residue in ketoacyl synthase.

List the 7 components of FAS:

— Transferase

— Ketoacyl synthase (KS)

— Ketoacyl reductase

— Dehydratase

— Enoyl reductase

— Thioesterase

— ACP

How is FAS loaded initially? what enzyme is involved?

Acetyl-CoA → cysteine in ketoacyl synthase; malonyl-CoA → ACP. Both use transacylase and cysteine and ACP are on fatty acid synthase

In what direction does elongation occur?

Inside out (new carbons added to interior).

What is the main product of FAS?

Palmitate (C16 fatty acid).

How many cycles are needed to make palmitate (C16)?

7 cycles. You start with 2 initially and then adding two each time for seven rounds yields 16 carbons

How much NADPH is required to synthesize one molecule of palmitate (C16)?

14 NADPH are required because each of the 7 elongation cycles uses 2 NADPH for reduction steps.

What is the ATP cost to synthesize one molecule of palmitate (C16)

7 ATP are required to convert 7 acetyl-CoA into 7 malonyl-CoA via acetyl-CoA carboxylase.

What is the total ATP equivalent cost of synthesizing palmitate (C16)

~42 ATP equivalents (7 ATP + energy equivalent of 14 NADPH ≈ 35 ATP).

Why does fatty acid synthesis require NADPH?

NADPH provides the reducing power needed to convert the growing fatty acid chain into a fully saturated hydrocarbon during each elongation cycle

Why is fatty acid synthesis still energetically favorable overall?

Even though synthesis costs ~42 ATP, oxidation of palmitate via β-oxidation produces significantly more ATP, resulting in a net energy gain.

How is the fatty acid chain released from fatty acid synthase (FAS)?

The enzyme thioesterase hydrolyzes the thioester bond, releasing free palmitate (C16 fatty acid).

Where does further elongation of fatty acids occur after FAS?

In the endoplasmic reticulum (ER) and mitochondria.

What carrier is used for fatty acid elongation instead of ACP?

Coenzyme A (CoA) is used instead of ACP.

What reactions occur during elongation outside FAS?

The same four steps: condensation, reduction, dehydration, and reduction.

What is the function of desaturase enzymes?

They introduce double bonds into fatty acid chains, producing unsaturated fatty acids.

What do desaturases require to function?

NADPH and oxygen; they remove hydrogen atoms and form double bonds.

What molecule provides the backbone for triacylglycerol (TAG) synthesis?

Glycerol-3-phosphate.

Where does glycerol-3-phosphate come from?

It is produced by reduction of dihydroxyacetone phosphate (DHAP), a glycolysis intermediate.

What enzyme converts DHAP to glycerol-3-phosphate?

Glycerol-3-phosphate dehydrogenase.

What do acyl transferase enzymes do in lipid synthesis?

They transfer fatty acid chains from acyl-CoA to glycerol-3-phosphate, forming ester bonds.

What is the product after the first fatty acid is added to glycerol-3-phosphate?

Lysophosphatidic acid (monoacylglycerol).

What is formed after the second fatty acid is added?

Phosphatidic acid.

What is the structure of phosphatidic acid?

C1: saturated fatty acid

C2: unsaturated fatty acid

C3: phosphate group

How is diacylglycerol (DAG) formed from phosphatidic acid?

The phosphate group is removed by phosphatidic acid phosphohydrolase (PAP).

What enzyme converts diacylglycerol (DAG) into triacylglycerol (TAG)?

Diacylglycerol acyltransferase (DGAT), which adds a third fatty acid.

Where are triacylglycerols (TAGs) stored in cells?

In lipid droplets, primarily in adipose tissue.

What surrounds lipid droplets?

A phospholipid monolayer and proteins such as perilipins.

What are the functions of lipid droplets?

Energy storage, supply of signaling molecules, structural roles, and protection from excess lipid toxicity.

What health issues are associated with excess TAG accumulation?

Obesity, insulin resistance, steatohepatitis, and cardiomyopathy.

When does ketogenesis occur?

During starvation or prolonged fasting when glucose is limited.

Where does ketogenesis occur?

In the liver.

Why are ketone bodies important?

They provide an alternative fuel source for the brain when glucose is unavailable.

What are ketone bodies?

Water-soluble molecules produced from fatty acid breakdown that can be used for energy.

What is the normal concentration of ketone bodies in blood?

Less than 3 mg/100 mL.

What are the starting molecules for ketone body synthesis?

Two acetyl-CoA molecules.

What enzyme combines two acetyl-CoA molecules in ketogenesis?

Thiolase makes acetoacetyl-CoA

What enzyme forms HMG-CoA in ketogenesis?

HMG-CoA synthase, which adds a third acetyl-CoA.

Why is HMG-CoA important in metabolism?

It is a key intermediate in both ketogenesis and cholesterol synthesis.

What is ketolysis?

The breakdown of ketone bodies into acetyl-CoA for energy production in peripheral tissues.

What pathway produces cholesterol?

The mevalonate pathway.

What key intermediate links ketogenesis and cholesterol synthesis?

HMG-CoA.

What enzyme converts HMG-CoA to mevalonate?

HMG-CoA reductase.

What cofactor is required by HMG-CoA reductase?

NADPH.

What type of drugs inhibit HMG-CoA reductase?

Statins.

What is SREBP and its function?

A transcription factor that regulates genes involved in cholesterol synthesis.

How is SREBP activated when cholesterol is low?

SCAP activates cleavage of SREBP, allowing it to enter the nucleus and increase gene expression.

What shuttle system allows acetyl-CoA to move from mitochondria to cytosol for fatty acid synthesis?

The citrate shuttle.

How are fatty acid synthesis and fatty acid oxidation reciprocally regulated?

Malonyl-CoA inhibits fatty acid oxidation, while AMPK inhibits fatty acid synthesis.

How does cytosolic acetyl-CoA get generated for fatty acid synthesis (step-by-step)?

1. Citrate accumulates in mitochondria (high energy)

2. Citrate transported to cytosol

3. ATP-citrate lyase cleaves citrate → acetyl-CoA + OAA

How is NADPH generated during fatty acid synthesis (mechanism)? —

1. OAA reduced to malate (uses NADH)

2. Malate converted to pyruvate via malic enzyme

3. NADP+ → NADPH (provides reducing power for synthesis)

How is malonyl-CoA formed (mechanism)?

1. Acetyl-CoA binds acetyl-CoA carboxylase (ACC)

2. Biotin carries activated CO2

3. ATP hydrolysis drives carboxylation

4. Malonyl-CoA (3C) is produced

What happens during the condensation step of fatty acid synthesis?

1. Malonyl-CoA is decarboxylated (releases CO2)

2. 2-carbon fragment attacks acetyl group

3. Forms 4-carbon acetoacetyl-ACP

What are the four repeating steps of fatty acid elongation (mechanism)?

1. Condensation (adds 2 carbons)

2. Reduction (NADPH)

3. Dehydration (removes H2O)

4. Reduction (NADPH)

What happens during the first reduction step in FAS?

β-keto group is reduced to hydroxyl using NADPH → forms hydroxybutryl-ACP using 3-ketoacyl-ACP reductase

What happens during the dehydration step in FAS?

Water is removed → forms a trans double bond between α and β carbons using 3-hydroxyacyl-ACP dehydrase, forming butenoyl-ACP

What happens during the second reduction step in FAS?

Double bond on butenoyl-ACP is reduced using NADPH → fully saturated acyl chain. Uses enoyl-ACP reductase and forms butyryl-ACP

How does the fatty acid chain continue to elongate (cycle mechanism)?

1. Growing acyl chain transferred from ACP → cysteine

2. New malonyl-CoA loaded onto ACP

3. Condensation occurs again

4. Cycle repeats, adding 2 carbons each round

How is palmitate (C16) produced (mechanism summary)?

1. 1 acetyl-CoA primes the system

2. 7 malonyl-CoA added sequentially

3. 7 cycles of elongation

4. Chain reaches 16 carbons

5. Thioesterase releases palmitate

How does fatty acid elongation occur outside FAS (mechanism)?

1. Acyl-CoA used instead of ACP

2. Malonyl-CoA provides 2C units

3. Same 4 steps: condensation → reduction → dehydration → reduction 4. occurs in ER or mitochondria

How do desaturase enzymes introduce double bonds (mechanism)?

Remove hydrogen atoms from adjacent carbons

Use O2 and NADPH

Form C=C double bond → unsaturated fatty acid

How are fatty acids attached to glycerol (mechanism)?

1. Acyl transferase catalyzes nucleophilic attack

2. Hydroxyl group of glycerol attacks acyl-CoA thioester

3. Ester bond forms + CoA released

How is phosphatidic acid formed (stepwise)

1. First FA added → lysophosphatidic acid

2. Second FA added → phosphatidic acid

How is triacylglycerol (TAG) formed (mechanism)?

1. Phosphatidic acid dephosphorylated by PAP → DAG

2. DGAT adds third fatty acid → TAG

How are ketone bodies synthesized from acetyl-CoA (step-by-step)?

1. 2 acetyl-CoA → acetoacetyl-CoA + H-SCoA (thiolase)

2. acetoacetyl-CoA +acetyl-CoA → HMG-CoA + H-SCoA (HMG-CoA synthase)

3. HMG-CoA → acetoacetate + acetyl-CoA(HMG-CoA lyase)

4. acetoacetate + NADH + H+ -> 3-hydroxybutyrate (3-hydroxybutyrate dehydrogenase)

Why are ketone bodies produced during fasting (mechanistic reasoning)?

1. β-oxidation ↑ → acetyl-CoA accumulates

2. TCA cycle limited (OAA used for gluconeogenesis)

3. Excess acetyl-CoA diverted → ketogenesis

How is HMG-CoA formed in cholesterol synthesis (mechanism)?

Same first steps as ketogenesis:

1. 2 acetyl-CoA → acetoacetyl-CoA

2. acetyl-CoA → HMG-CoA

How is mevalonate formed from HMG-CoA (mechanism)?

HMG-CoA reductase reduces HMG-CoA using NADPH → mevalonate

How does the mevalonate pathway lead to cholesterol (high-level mechanism)?

1. Mevalonate → activated isoprenes

2. Isoprenes → squalene

3. Squalene → cholestero

How does AMPK inhibit fatty acid synthesis (mechanism)?

1. Low energy → AMPK activated

2. AMPK phosphorylates ACC

3. ACC becomes inactive → ↓ malonyl-CoA → ↓ FA synthesis

How does malonyl-CoA inhibit fatty acid oxidation (mechanism)?

Malonyl-CoA inhibits fatty acyl transport into mitochondria (CPT1), preventing β-oxidation

How does citrate coordinate metabolism (mechanism)?

1. High citrate → activates ACC → FA synthesis

2. High citrate → inhibits PFK-1 → slows glycolysis

What are the 3 major purposes of amino acids?

1) Energy substrates 2) Protein synthesis 3) Synthesis of other products (heme, purines, pyrimidines, coenzymes, biogenic amines)

Why is nitrogen removal important during amino acid catabolism?

Too much nitrogen is toxic; amine groups must be removed and excreted as urea (excess) or uric acid (normal route)

What 4 carbon intermediates do amino acid carbon chains get converted to?

Pyruvate, Oxaloacetate, Acetyl-CoA, α-Ketoglutarate

What is the Glucose-Alanine Cycle?

A cycle that moves nitrogen from muscle to liver

What are the steps for the Glucose-alanine cycle, starting in the muscle?

muscle aminotransferase transfers amino group from glutamate to pyruvate → alanine → travels in blood to liver → liver converts alanine back to pyruvate (gluconeogenesis) + enters urea cycle

Under what conditions does the glucose-alanine cycle occur?

In cases of extreme starvation

What does the liver do with alanine received from muscle?

Converts it back to pyruvate via alanine aminotransferase; nitrogen enters the urea cycle; pyruvate used for gluconeogenesis → glucose released to blood

What do transaminases (aminotransferases) do?

Transfer α-amino groups from an amino acid onto an α-keto acid, producing a new α-keto acid and a new α-amino acid

What are the two key transamination reactions?

Glu: amino acid + α-ketoglutarate → α-keto acid + glutamate; Asp: amino acid + oxaloacetate → α-keto acid + aspartate