Bio98 Lecture 16 Fatty Acid Catabolism & Biosynthesis

Fatty Acid Oxidation requires…

1) activation

2) transport

3) beta-oxidation

Catabolism of FAs (Fatty Acid Oxidation summary)

-lipids have the highest yield of energy per gram (9cal) vs. protein and carbohydrates (4.5 cal)

-1) FA activates using ATP for energy → makes a high energy Acyladenylate

-2) Energy from acyladenylate → makes Acyl-CoA

-3) Acyl-CoA → Acylcarnitine (to enable transport to mitochondria)

• transports to intermembrane space and then mitochondrial matrix

-4) Acylcarnitine → converted back to Acyl-CoA (in mitochondria)

-5) Acyl-CoA is broken down piece by piece into multiple Acetyl-CoA

Fatty Acid Oxidation: 1) Activation

Net: fatty acid + ATP ➔ acyl-CoA + AMP + PPi (pyrophosphate)

(steps 1-3)

-FA is activated by 2 ATP (AMP + 2 Pi released) for energy to make a high energy ACYLADENYLATE
—> done by ACYL-CoA SYNTHETASE
—> happening in the CYTOSOL

- HS-CoA attached to ACYLADENYLATE and the energy is used to make ACYL-CoA
—> done by ACYL-CoA SYNTHETASE again

- ACYL-CoA is converted to ACYLCARNITINE so that it can enter the MITOCHONDRIA
—> done by CARNITINE ACYLTRANSFERASE (CAT1)
—> carried out at the OUTER MITOCHONDRIAL MEMBRANE

Fatty Acid Oxidation: 2) Transport

(steps 3-4)

- THE CARNITINE CYCLE
—> RATE-CONTROLLING STEP for fatty acid oxidation b/c ACYL-CoA (fatty acyl-CoA) CANNOT enter the MITOCHONDRIA MATRIX directly

- ACYLCARNITINE is transported into the INTERMEMBRANE SPACE and then into the MATRIX

- ACYLCARNITINE is then converted back to ACYL-CoA
—> done by CARNITINE ACYLTRANSFERASE (CATII)

Fatty Acid Oxidation: 3) beta-oxidation

(final: step 5)

- ACYL-CoA is broken down into multiple ACETYL-CoAs
—> 2 CARBONS of the fat are REMOVED each time —> attached to the CoA as ACETYL-CoA
—> last 2 CARBONS are just the ACETYL-CoA

- acyl-CoA gets 2 CARBONS SHORTER each time acetyl-CoA is produced

Beta-Oxidation Cycle

- 4 rxns per cycle including:

• 2 oxidations (4e-) involving NADH + FADH2

• 1 H2O addition

• Making Acetyl-CoA

Beta-Oxidation Cycle: Step 1

1) Fatty acyl-CoA → oxidized into trans-Δ²-enoyl-CoA

-carried out by acyl-CoA dehydrogenase (AD)

-resulted in a double bond between the alpha and beta carbons

-FAD → generates FADH2 bc electrons got captured; contribute to ETC

Beta-Oxidation Cycle: Step 2

2) trans-Δ²-enoyl-CoA → 3-L-Hydroxylacyl-CoA

-carried out by Enoyl-CoA hydratase (EH)

-doubled bond of the enoyl-CoA is hydrated (broken) to yield/hold a hydroxyl group (H2O)

Beta-Oxidation: Step 3

3) 3-L-Hydroxylacyl-CoA → Beta-ketoacyl-CoA

-carried out by 3-L-Hydroxylacyl-CoA dehydrogenase (HAD)

-generates NADH from NAD+

-hydroxyl group is oxidized to a carbonyl, yielding a B-keto acyl group.

Beta-Oxidation: Step 4

4) Beta-ketoacyl-CoA → fatty acyl-CoA (2 carbons shorter) → acetyl-CoA!

-carried out by Beta-ketoacyl-CoA thiolase (KT)

-the remaining fatty acyl-CoA undergoes further rounds of Beta-oxidation

-Acetyl-CoA is further metabolized by the citric acid cycle

Beta-Oxidation Yield

108 ATP, 15 FADH2, 21 NADH

Unsaturated fatty acids for B-oxidation

Unsaturated (C=C) fats enter the β-oxidation cycle after first undergoing a separate oxidation to remove the double bond (costs 1x FADH2)

Uneven-numbered fatty acids

-Uneven number of carbons will end oxidation with a C3 propionyl-CoA, not C2 acetyl-CoA

-C3 PROPIONYL-CoA is converted to SUCCINYL-CoA
—> needs ATP
—> carried out by PROPIONYL-CoA CARBOXYLASE (biotin)

- SUCCINYL-CoA then goes 1 round of the TCA cycle

Fatty Acid ATP calculation

- ODD # CHAIN - # of cycles: [((# of C - 1) / 2) - 1]

- EVEN # CHAIN - # of cycles: [(# of C) / 2)) - 1]

- 1 ACETYL-CoA = 10 ATP

- 1 PROPIONYL-CoA = 19 ATP
—> converting propionyl-CoA to succinyl-CoA costs 1 ATP
—> succinyl-CoA goes for 2 TCA ROUNDS = 10 ATP x 2
—> 20 ATP - 1 ATP = 19 ATP

Fatty Acid Calculation: Calculate the total ATP generated for a 21-carbon fatty acid with one double bond ➔ 21 C = 9 x acetyl-CoA + 1 x propionyl-CoA

1) ODD # CHAIN - # of cycles: [((# of C - 1) / 2) - 1]. = (21-1/2)-1 → 9 cycles

• producing 9 acetyl-CoA + 9 NADH + 8 FADH2 (minus one FADH2 for one double bond)

2) 1 acetyl-CoA = 10 ATP & 1 propionyl-CoA= 19 ATP

• Propionyl CoA → succinyl CoA (costs 1 ATP)

• Succinyl CoA undergoes 2 rounds of TCA cycle: 10 ATP x 2 = 20 ATP

• 1 propionyl CoA = 20-1 = 19 ATP

• REMIND: NADH = 2.5 ATP, FADH2= 1.5 ATP

3) (9×10)+(1×19)+(9×2.5)+(8×1.5) -2 = 141.5 ATP

• 9 acetyl coA + 1 propionyl CoA + 9 NADH + 8 FADH2

—> 2 ATP SUBTRACTED b/c 2 ATP were used to activate the fatty acid during STEP 1

Fatty Acid Biosynthesis

IN CYTOSOL/CYTOPLASM

- Acetyl-CoA carboxylase (ACC)

• Builds malonyl-CoA

• Malonyl-CoA is used by FAS

- Fatty acid synthase (FAS)

• Catalyzes 7 different reactions

• 4 are similar to beta-oxidation

• 3 are completely different

• Builds acetyl-CoA and malonyl-CoA into growing FA chains

Fatty Acid Biosynthesis Step 0: Malonyl-CoA Biosynthesis by ACC

1) Biotin-carrier + Biotin carboxylase + transcarboxylase = acetyl-CoA carboxylase (ACC)

2) ACC → Malonyl-CoA through transcarboxylase

- uses 1 ATP to convert ACETYl-CoA (2C) into MALONYL-CoA (3C) + cofactor

Subsequent steps: Catalyzed by Fatty Acid Synthase (FAS)

-There are 7 domains; each one catalyzes one reaction

-all rxns happen while the intermediates are connected to x y z.

-it’s just a handoff rxn at the ACP domain

ACP

-acyl carrier protein, is the domain of fatty acid synthase

-has a bound cofactor, where it’s essentially its own cofactor

-phosphopantathione or PPT cofactor with the reactive sulfur group on the end like CoA

• only difference it has from CoA is that it’s fused covalently directly to the protein through an active site serine residue; NOT DERIVED FROM ATP OR ADENOSINE

-the purpose of these fused or covalently bound cofactors is to act as a swing arm to coordinate substrate transfer or product transfer to different domains of these enzyme complexes, or in the case of fatty acid synthase, between domains of a single enzyme.

Coenzyme A

-freely diffusible cofactor; comes in and out from different enzymes that it binds to.

-has phosphopantathione or PPT cofactor with the reactive sulfur group on the end

The Swing Arm: Nature’s Machinery

- PURPOSE: to transfer reactive intermediates between enzyme subunits

ex. LIPOIC ACID in PDH (E2), BIOTIN or ACC (step 0 FA Biosynthesis), PPT + ACP domain in FAS

FAS

-fatty acid synthase; FAS is all one protein

-consists of 7 domains: each one catalyzes a different rxn

-all reactions are completed while the intermediates are connected to ACP (acyl-carrier protein)

-2 REDUCTION rxns occur —> NADPH turns into NADP+ (x2)
-1 DEHYDRATION rxn occurs —> H2O produced

Fatty Acid Oxidation vs Biosynthesis: Opposing Processes

OXIDATION:

• Location: mitochondrion

• e- carrier: NAD/FAD

• Acetyl-carrier: CoA

• Acetyl-donor: Acetyl CoA (C2)

• Cycle enzymes: many; separate

• Cycle intermediates: L-OH acyl CoA

• Metabolic timing: Low EC starvation

BIOSYNTHESIS:

• Location: cytoplasm

• e- carrier: NADP/NADPH

• Acetyl-carrier: ACP

• Acetyl-donor: malonyl ACP (C3)

• Cycle enzymes: one protein chain

• Cycle intermediates: D-OH acyl ACP

• Metabolic timing: High EC well fed