Lecture 32 –Last lecture!! –Fatty acid biosynthesis.

Lecture 32

– Last lecture!!– Fatty acid biosynthesis

-oxidation• Strategy: create a carbonyl group

on the -C • First 3 reactions do that; fourth

cleaves the "-keto ester" in a reverse Claisen condensation

• Products: an acetyl-CoA and a fatty acid two carbons shorter

Acyl-CoA Dehydrogenase

• Oxidation of the C-Cbond

• 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.

Acyl-CoA Dehydrogenase

Net: 2 ATP/2 e- transferred

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1. Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD).

2. Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA

3. NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA.

4. C-C bond cleavage by -ketoacyl-CoA thiolase (KT)

Enoyl-CoA Hydratase• aka crotonases • Adds water across the double bond • Uses substrates with trans-2-and

cis 2 double bonds (impt in b-oxidation of unsaturated FAs)

• With trans-2 substrate forms L-isomer, with cis 2 substrate forms D-isomer.

• Normal reaction converts trans-enoyl-CoA to L--hydroxyacyl-CoA

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1. Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD).

2. Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA

3. NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA.

4. C-C bond cleavage by -ketoacyl-CoA thiolase (KT)

Hydroxyacyl-CoA Dehydrogenase

• Oxidizes the -Hydroxyl Group to keto group

• This enzyme is completely specific for L-hydroxyacyl-CoA

• D-hydroxylacyl-isomers are handled differently

• Produces one NADH

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1. Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD).

2. Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA

3. NAD+-dependent dehydrogenation of -hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA.

4. C-C bond cleavage by -ketoacyl-CoA thiolase (KT)

Thiolase

• Nucleophillic sulfhydryl group of CoA-SH attacks the -carbonyl carbon of the 3-keto-acyl-CoA.

• Results in the cleavage of the C-C bond.

• Acetyl-CoA and an acyl-CoA (-) 2 carbons are formed

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Figure 25-15 Mechanism of action of -ketoacyl-CoA thiolase.

1. An active site thiol is added to the substrate -keto group.

2. C-C bond cleavage forms an acetyl-CoA carbanion intermediate (Claisen ester cleavage)

3. The acetyl-CoA intermediate is protonated by an enzyme acid group (acetyl-CoA released)

4. CoA binds to the enzyme-thioester intermediate

5. Acyl-CoA is released.

Net reaction reduces fatty acid by 2C and

acyl-CoA group is free to pass through the cyle again.

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1. Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD).

2. Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA

3. NAD+-dependent dehydrogenation of -hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA.

4. C-C bond cleavage by -ketoacyl-CoA thiolase (KT)

-oxidation• Each round of -oxidation produces 1 NADH, 1 FADH2 and 1 acetyl-

CoA. -oxidation of palmitate (C16:0) yields 129 molecules of ATP

• C 16:0-CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA 8 acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+

• Acetyl-CoA = 8 GTP, 24 NADH, 8 FADH2 • Total = 31 NADH = 93 ATPs + 15 FADH2 = 30 ATPs

• 2 ATP equivalents (ATP AMP + PPi, PPi 2 Pi) consumed during activation of palmitate to acyl-CoA

• Net yield = 129 ATPs

Beta-oxidation of unsaturated fatty acids• Nearly all fatty acids of biological origin have cis double bonds between C9

and C10 (9 or 9-double bond).

• Additional double bonds occur at 3-carbon intervals (never conjugated).

• Examples: oleic acid and linoleic acid.

• In linoleic acid one of the double bonds is at an even-numbered carbon and the other double bond is at an odd-numbered carbon atom.

• 4 additional enzymes are necessary to deal with these problems.

• Need to make cis into trans double bonds

Figure 25-17Problems in the

oxidation of unsaturated fatty acids and their

solutions.

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-oxidation of unsaturated fatty acids

• -oxidation occurs normally for 3 rounds until a cis-3-enoyl-CoA is formed.

• Acyl-CoA dehydrogenase can not add double bond between the and carbons.

• Enoyl-CoA isomerase converts this to trans- 2 enoyl-CoA

• Now the -oxidation can continue on w/ the hydration of the trans-2-enoyl-CoA

• Odd numbered double bonds handled by isomerase

-oxidation of fatty acids with even numbered double bonds

-oxidation of odd chain fatty acids

• Odd chain fatty acids are less common• Formed by some bacteria in the stomachs of

ruminants and the human colon.• -oxidation occurs pretty much as w/ even

chain fatty acids until the final thiolase cleavage which results in a 3 carbon acyl-CoA (propionyl-CoA)

• Special set of 3 enzymes are required to further oxidize propionyl-CoA

• Final Product succinyl-CoA enters TCA cycle

Propionyl-CoA Carboxylase• The first reaction• Tetrameric enzyme that has a biotin prosthetic group• Reactions occur at 2 sites in the enzyme.

1. Carboxylation of biotin at the N1’ by bicarbonate ion (same as pyruvate carboxylase). Driven by hydrolysis of ATP to ADP and Pi-activates carboxyl group for transfer

2. Stereospecific transfer of the activated carboxyl group from carboxybiotin to propionyl-CoA to form (S)-methylmalonyl-CoA. Occurs via nucleophillic attack on the carboxybiotin by a carbanion at C2 of propionyl-CoA

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Methylmalonyl-CoA Racemase• 2nd reaction for odd

chain fatty acid oxidation

• Transforms (S)-methylmalonyl-CoA to (R)-methylmalonyl-CoA

• Takes place through a resonance stablized carbanion intermediate (p. 923)

Methylmalonyl-CoA mutase• 3rd reaction of the pathway: converts (R)-methylmalonyl-CoA to

succinyl-CoA• Utilizes 5’-deoxyadenosylcobalamin (AdoCbl) - coenzyme B12.• AdoCbl has a reactive C-Co bond that is used for 2 types of reactions:

1. Rearrangements in which a hydrogen atom is directly transferred between 2 adjacent C atoms.

2. Methyl group transfers between molecules.

-C1-C2-XH

-C1-C2-

X H

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Figure 25-21Structure of 5-deoxyadenosylcobalamin

(coenzyme B12).

Co is coordinated by the corrin ring’s 4 pyrrole N atoms, a N from the dimethylbenzimadazole (DMB), and C5’ from the deoxyribose unit.

One of only 2 known C-metal bonds in biology.

Figure 25-20The rearrangement catalyzed by methylmalonyl-CoA mutase.

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Methylmalonyl-CoA mutase• Mechanism begins with homolytic cleavage of the C-Co(III) bond. • The AdoCbl is a free radical generator• C-Co(III) bond is weak and it is broken and the radical is stabilized

favoring the formation of the adenosyl radical.• Rearrangement to form succinyl-CoA from a cyclopropyloxy radical• Abstraction of a hydrogen atom from 5’deoxyadenosine to regenerate

the adenosyl radical• Release of succinyl-CoA

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Odd chain fatty acids• Transform odd chain length FAs to succinyl-CoA• 3 enzymes• Propionyl-CoA carboxylase (biotin cofactor): activates bicarbonate

and transfers to propionyl-CoA to form S-methylmalonyl-CoA.• Methylmalonyl-CoA racemase: Transforms (S)-methylmalonyl-

CoA to (R)-methylmalonyl-CoA through a resonance-stabilized intermediate.

• Methylmalonyl-CoA mutase (B12 cofactor(AdoCbl)): Transforms (R)-methylmalonyl-CoA to succinyl-CoA by generating a radical.

• Succinyl-CoA enters TCA cycle

Combination of fatty acid activation, transport into mitochondrial matrix

and oxidation

• Resulting acetyl CoA enters citric acid cycle.

• Production of NADH, FADH2, oxidized by respiratory chain.

Fatty Acid Breakdown Summary• Even numbered fatty acids are broken down into acetyl-

CoA by 4 enzymes: acyl-CoA dehydrogenase (AD), enoyl-CoA hydratase (EH), 3-L-hydroxyacyl-CoA dehydrogenase (HAD) and -ketoacyl-CoA thiolase (KT).

• The breakdown of unsaturated fatty acids (cis double bonds) requires 4 additional enzymes in mammals: enoyl-CoA isomerase, 2,4 dienoyl-CoA reductase, 3,2-enoyl-CoA isomerase, and 3,5-2,4-dienoyl-CoA isomerase. In bacteria, they only need enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase.

• Have to convert cis double bonds to trans double bonds.

• Unsaturated fatty acids -oxidation results in the production of acetyl-CoA.

Fatty Acid Breakdown Summary• Odd numbered fatty acids are broken down into

propionyl-CoA.

• Propionyl-CoA is converted to S-Methylmalonyl-CoA by propionyl-CoA carboxylase with ATP and CO2. Uses a carboxybiotynyl cofactor for the mechanism.

• S-Methylmalonyl-CoA is converted to R-Methylmalonyl-CoA by methylmalonyl-CoA racemase.

• R-Methylmalonyl-CoA is converted to Succinyl-CoA by methylmalonyl-CoA mutase. Uses a 5’-deoxyadenosylcobalimin (AdoCbl aka coenzyme B12) cofactor for the mechanism.

Fatty Acid Synthesis• Fatty acid biosynthesis occurs through condensation

of C2 units (reverse of -oxidation)

• Acetyl-CoA is the precursor molecule; converted to malonyl-CoA

• In mammals fatty acid synthesis occurs primarily in the liver and adipose tissues

• Also occurs in mammary glands during lactation.• Fatty acid synthesis and degradation go by different

routes • There are four major differences between fatty acid

breakdown and biosynthesis

The differences between fatty acid biosynthesis and breakdown

• Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as compared to -SH groups of CoA)

• Synthesis in cytosol; breakdown in mitochondria• Enzymes of synthesis are one polypeptide in

eukaryotes.• Dissociated in bacteria• Biosynthesis uses NADPH/NADP+; breakdown

uses NADH/NAD+

ACP vs. Coenzyme A

•Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (ACP) as compared to -SH groups of CoA

Figure 25-28A comparison of fatty acid oxidation and fatty acid biosynthesis.

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Fatty Acid Synthesis Occurs in the Cytosol

• Must have source of acetyl-CoA• Most acetyl-CoA in mitochondria• Citrate-malate-pyruvate shuttle provides cytosolic

acetate units and reducing equivalents for fatty acid synthesis

Citrate synthaseCitrate Lyase

Malate dehydrogenase

Malate EnzymePyruvate

carboxylase

Fatty Acid Synthesis• Fatty acids are built from 2-C units derived

from acetyl-CoA• Acetate units are activated for transfer to

growing FA chain by conversion to malonyl-CoA

• Decarboxylation of malonyl-CoA and reducing power of NADPH drive chain growth

• Chain grows to 16-carbons (eight acetyl-CoAs)• Other enzymes add double bonds and more

carbons

Acetyl-CoA Carboxylase

• The "ACC enzyme" commits acetate to fatty acid synthesis

• Carboxylation of acetyl-CoA to form malonyl-CoA is the irreversible, committed step in fatty acid biosynthesis

Acetyl-CoA + HCO3- + ATP malonyl-CoA + ADP

Acetyl-CoACarboxylase

Regulation of Acetyl-CoA Carboxylase (ACCase)

• ACCase forms long, active filamentous polymers from inactive protomers

• Accumulation of palmitoyl-CoA (product) leads to the formation of inactive polymers

• Accumulation of citrate leads to the formation of the active polymeric form

• Phosphorylation modulates citrate activation and palmitoyl-CoA inhibition

Figure 25-30Association of acetyl-CoA carboxylase protomers.

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• Unphosphorylated ACCase has low Km for citrate and is active at low citrate

• Unphosphorylated ACCase has high Ki for palmitoyl-CoA and needs high palmitoyl-CoA to inhibit

• Phosphorylated E has high Km for citrate and needs high citrate to activate

• Phosphorylated E has low Ki for palmitoyl-CoA and is inhibited at low palmitoyl-CoA

Regulation of Acetyl-CoA Carboxylase (ACCase)

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1. Acetyl-CoA is converted by MAT to Acetyl ACP

2. Acetyl-ACP is attached to KS (condensation reaction).

3. Malonyl ACP is formed by MAT.

4. Acetyl-group is coupled to beta carbon of malonyl-ACP with release of CO2 to form acetoacetyl-ACP(2b) by KS.

5. Reduction of acetoacetyl-ACP with NADPH to form D--hydroxybutyrl-ACP by DH

6. Dehydration of D--hydroxybutyrl-ACP by ER to form a,b-trans-butenoyl-ACP

7. Reduction of the double bond to form butyryl-ACP

8. Repeat until Palmitoyl-ACP (C16) is formed.

9. ACP is cleaved by TE releasing free fatty acid.

Fatty Acid Synthesis

• Step 1: Loading – transferring acetyl- and malonyl- groups from CoA to ACP

• Step 2: Condensation – transferring 2 carbon unit from malonyl-ACP to acetyl-ACP to form 2 carbon keto-acyl-ACP

• Step 3: Reduction – conversion of keto-acyl-ACP to hydroxyacyl-ACP (uses NADPH)

• Step 4: Dehydration – Elimination of H2O to form Enoyl-ACP

• Step 5: Reduction – Reduce double bond to form 4 carbon fully saturated acyl-ACP

Step 1: Loading Reactions

H3C C

O

S CoA C C

O

S CoACO

O

H

H HS-ACPHS-ACP

HS-CoAHS-CoA

H3C C

O

S ACP C C

O

S ACPCO

O

H

H

acetyl-CoA

acetyl-ACP

malonyl-CoA

malonyl-ACP

acetyl-CoA:ACPtrans acylas e

malonyl-CoA:ACPtrans acylas eMAT

Step 2: Condensation Rxn

H3C C

O

S ACP

HS-Ketoacyl-ACP Synthase

HS-ACP

H3C C

O

S ketoacyl-ACP SynthaseC C

O

S ACPCO

O

H

H

CO2

C C

O

S ACPC

H

H

O

H3C

acetyl-ACP

malonyl-ACP

+

keto-ACP s ynthas e

acetoacetyl-ACP

-ketoacyl-ACP synthase (KS)

Step 3: Reduction

C C

O

S ACPC

H

H

O

H3C

NADP+

C C

O

S ACPC

H

H

OH

H3C

H

acetoacetyl-ACP

-hydroxybutyryl-ACP

NADPH+H+

Ketoacyl-ACPReductase

KR

Step 4: Dehydration

C C

O

S ACPC

H

trans-enoyl-ACP

H3C

H

H20

-hydroxyacyl-ACPdehydrase

C C

O

S ACPC

H

H -hydroxyacyl-ACP

OH

H3C

H

DH

Step 5: Reduction

C C

O

S ACPC

H

H3C

H

NADP+

C C

O

S ACPC

H

H3C

H

H

H

trans-enoyl-ACP

enoyl-ACP reductase

NADPH + H+

trans-enoyl-ACP

ER

Step 6: next condensation

C C

O

S ACPC

H

H

H3C

H

HHS-Ketoacyl-ACP Synthase

HS-ACP

C C

O

S KASC

H

H

H3C

H

H

C C

O

S ACPCO

O

H

H

CO2

C C

O

S ACP

H

H

C C

O

C

H

H

H3C

H

H

butyryl-ACP

malonyl-ACP

+

keto-ACP s ynthas e

ketoacyl-ACP

KS

Termination of Fatty Acid

Synthesis

C C

O

S ACPH3C

H

H

HS-ACP

C C

O

OH3C

H

H

AMP + PPi

C C

O

SH3C

H

H

CoA

14

Palmitoyl-ACP

14

Palmitic Acid

14

Thioesterase

ATP + HS-CoA

Palmitoyl-CoA

Acyl-CoA synthetase

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1. Acetyl-CoA is converted by MAT to Acetyl ACP

2. Acetyl-ACP is attached to KS (condensation reaction).

3. Malonyl ACP is formed by MAT.

4. Acetyl-group is coupled to beta carbon of malonyl-ACP with release of CO2 to form acetoacetyl-ACP(2b) by KS.

5. Reduction of acetoacetyl-ACP with NADPH to form D--hydroxybutyrl-ACP by DH

6. Dehydration of D--hydroxybutyrl-ACP by ER to form a,b-trans-butenoyl-ACP

7. Reduction of the double bond to form butyryl-ACP

8. Repeat until Palmitoyl-ACP (C16) is formed.

9. ACP is cleaved by TE releasing free fatty acid.

Organization of Fatty Acid Synthesis Enzymes

• In bacteria and plants, the fatty acid synthesis reactions are catalyzed individual soluble enzymes.

• In animals, the fatty acid synthesis reactions are all present on multifunctional polypeptide.

• The animal fatty acid synthase is a homodimer of two identical 250 kD polypeptides.

Animal Fatty Acid Synthase

Regulation of FA Synthesis• Allosteric modifiers, phosphorylation and

hormones• Malonyl-CoA blocks the carnitine

acyltransferase and thus inhibits beta-oxidation

• Citrate activates acetyl-CoA carboxylase• Fatty acyl-CoAs inhibit acetyl-CoA

carboxylase• Hormones regulate ACC• Glucagon activates lipases/inhibits ACC• Insulin inhibits lipases/activates ACC

Allosteric regulation of fatty acid synthesis occurs at ACCase and the carnitine acyltransferase

Glucagon inhibits fatty acid synthesis while increasing lipid breakdown and fatty acid -oxidation

Insulin prevents action of glucagon

Regulation• Pancreatic and cells directly sense the dietary and energy

state of the organism through [glucose] in the blood.

cells respond to low blood glucose by secreting glucagon.

cells respond to the high blood glucose by secreting insulin.

• Both involved in glycogen metabolism.

• These hormones determine whether fatty acids will be oxidized or synthesized.

• Target the flux-generating regulatory enzymes of fatty acid synthesis (acetyl-CoA carboxylase).

• Short-term regulation

• ACCase inhibited by cAMP-dependent phosphorylation (glucagon).

• Activated by insulin-dependent dephosphorylation.

Regulation• ACCase inhibitied by palmitoyl-CoA.

• Activated by citrate.

• Long-term regulation: control the amount of enzyme present over hours or days.

• Polyunsaturated fatty acids decreases the lipid biosynthesis enzymes.

• Adipose tissue lipoprotein lipase-enzyme that inititates fats for storage is increased by insulin and decreased by starvation.

• Starvation and/or regular exercise decreases blood glucose-changes hormone balance.

• Results in long-term changes in gene expression that increase the levels of fatty acid oxidation enzymes and decrease those of lipid biosynthesis.

Regulation• Fatty acid oxidation regulated by concentrations of fatty acids in

blood.

• Controlled by hydrolysis rates of triacylglycerols in adipose tissue by hormone-sensitive triacylgycerol lipase.

• Regulated by phosphorylation(active)/dephosphorylation (less active) in response to cAMP.

• Epinephrine and norepinephrine act to increase adipose tissue cAMP concentrations -> lead to protein kinase A phosphorylation, increase phosphorylation of enzymes.

• Stimulates lipolysis in adipose tissue raising blood fatty acid levels and activates -oxidation in liver and muscles.

Regulation• AMP-dependent protein kinase (AMPK) phosphorylates

ACCase (inactive) -inhibits fatty acid biosynthesis.

• AMPK measures energy levels of the cell. Activated by AMP and inhibited by ATP.

• Insulin has opposite effect of glucagon and epinephrine: stimulates glycogen and triacylglycerol formation.

• Decreases cAMP levels.

• Stimulates dephosphorylation of ACCase.

• Ratio of glucagon/insulin important for rate and direction of fatty acid metabolism.

• Carnitine palmitoyltransferase I is inhibited by malonyl-CoA.

• Keeps new fatty acids from getting into the mitochondria.

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