Lecture 31 –Quiz on Wed (today): fatty acid synthesis Final on Monday morning 8-10AM.

Lecture 31

– Quiz on Wed (today): fatty acid synthesis Final on Monday morning 8-10AM

• Anchor proteins to the exterior of the eukaryotic membrane.

• Alternative to transmembrane polypeptides

• Proteins destined to be anchored to the surface of the membrane are synthesized with membrane spanning C-terminal sequences which are removed after GPI addition.

Glycosylphosphatidylinositol (GPI)

Figure 11–20 Plasma membrane proteins have a variety of functions.

Figure 9.19 Passive transport of solute molecules through a permeable membrane

Figure 9.20 Three types of membrane transport

Figure 9.23 Glucose permease of erythrocyte membrane

Passive transport system - intracellular [glucose] =< plasma [glucose] concentration.

Also transports epimers of glucose - mannose, galactose at slower

rates (20%)

Figure 9.23 Transport of sodium and potassium ions by Na+-K+ ATPase transporter (pump)

Three Na+ ions are transported out of the cell for every two K+ that move inside

• How are lipids accessed for energy production?

• Know the differences between the triacylglycerol lipase and phospholipase A2 mechanisms.

• Triacylglycerol lipase uses a catalytic triad similar to Ser proteases (Asp, His, Ser)

• Phospholipase A2 uses a catalytic triad but substitutes water for Ser.

Lipases

Figure 25-3aSubstrate binding to phospholipase A2. (a) A hypothetical model of phospholipase A2 in complex

with a micelle of lysophosphatidylethanolamine.

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Figure 25-4aThe X-ray structure of porcine phospholipase A2 (lavender) in complex with the

tetrahedral intermediate mimic MJ33.

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Figure 25-4bThe catalytic mechanism of

phospholipase A2.

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What other mechanism does this look like?

What are the differences?

• Fatty acids form complexes with intestinal fatty acid-binding protein (I-FABP) which makes them more soluble.

• Chylomicrons-transport exogenous (dietary) triacylglycerols and chloestorl packaged into lipoprotein molecules from the intestine to the tissues.

• Chylomicrons are released into the bloodstream via transport proteins named for their density.

• VLDL (very low density lipoproteins), LDL (low density lipoproteins) - transport endogenous (internally produced) triacylglycerols and cholesterols from the liver to tissues “Bad”

• HDL (high density lipoproteins), - transport endogenous cholesterol from the tissues to liver - “Good”

Fatty acid binding proteins

Table 12-6 Characteristics of the Major Classes of Lipoproteins in Human Plasma.

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• Hydrolyzes triacylglycerol components fo chylomicrons and VLDL to free fatty acids and glycerol.

• Fatty acids taken up by tissues.

• Glycerol is returned to liver or kidneys to convert to DHAP

Lipoprotein lipase

• Once fatty acids are taken into the cell they undergo a series of oxidations to yield energy.

• In eukaryotes, occurs in the matrix of mitochondria (same place as TCA cycle)

• Triglycerides found in fat cells (adipocytes) or in cytoplasm.

• Was found by Knoop back in the day (1904) through the following experiment that the oxidation of the carbon atom to the carboxyl-group is involved in fatty acid breakdown.

Fatty acid oxidation

Figure 25-8 Franz Knoop’s classic experiment indicating that fatty acids are metabolically oxidized at

their -carbon atom.

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Beta Oxidation of Fatty Acids

• Process by which fatty acids are degraded by removal of 2-C units

• -oxidation occurs in the mitochondria matrix

• The 2-C units are released as acetyl-CoA, not free acetate

• The process begins with oxidation of the carbon that is "beta" to the carboxyl carbon, so the process is called"beta-oxidation"

• Triglycerides are broken down into free fatty acids in the cytoplasm.

• Beta-oxidation takes place in the mitochondrial matrix.

• Fatty acids must be imported into the matrix

• Requires activation of fatty acids in the cytosol (fatty acids converted to acyl-CoA form)

• Activated fatty acids (acyl-CoA form) are imported into the mitochondrion.

Fatty acid oxidation

Fatty acids must first be activated by formation of acyl-CoA

• Acyl-CoA synthetase condenses fatty acids with CoA, with simultaneous hydrolysis of ATP to AMP and PPi

• Formation of a CoA ester is expensive energetically

• Reaction just barely breaks even with ATP hydrolysis Go’ATP hydroysis = -32.3 kJ/mol, Go’ Acyl-CoA synthesis +31.5 kJ/mol.

• But subsequent hydrolysis of PPi drives the reaction strongly forward (Go’ –33.6 kJ/mol)

Fatty acid + CoA + ATP acyl-CoA + AMP + PPi

Import of acyl-CoA into mitochondria• -oxidation occurs in the mitochondria, requires import of

long chain acyl-CoAs

• Acyl-CoAs are converted to acyl-carnitines by carnitine acyltransferases.

• A translocator then imports Acyl carnitine into the matrix while simultaneously exporting free carnitine to the cytosol

• Acyl-carnitine is then converted back to acyl-CoA in the matrix

Figure 25-10 Acylation of carnitine catalyzed by carnitine palmitoyltransferase.

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Figure 25-11 Transport of fatty acids into the mitochondrion.

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Import of acyl-CoA into mitochondria• The acyl group of cytosolic acyl-CoA is transferred to

carnitine-releases CoA to cytosol.

• Acyl-carnitine is transported into the matrix by the carnitine carrier protein

• The acyl group is transferred to a CoA molecule from the mitochondrion.

• Carnitine is returned to the cytosol.

Deficiencies of carnitine or carnitine transferase or translocator activity are

related to disease state

• Symptons include muscle cramping during exercise, severe weakness and death.

• Affects muscles, kidney, and heart tissues.• Muscle weakness related to importance of fatty acids as

long term energy source• People with this disease supplement diet with medium chain

fatty acids that do not require carnitine shuttle to enter mitochondria.

Activation of fatty acids for -oxidation

• Activation of fatty acid to acyl-CoA form by acyl-CoA synthetase-requires CoASH and ATP (converted to AMP + PPi ) in the cytosol.

• Acyl-CoA is converted to acyl-carnitine by carnitine acyltransferase (carnitine palmitoyl transferase I) in the cytosol for transport into the mitochondrion.

• Acyl-carnitine is transported across the membrane by the carnitine carrier protein.

• Acyl-carnitine is converted to acyl-CoA by carnitine palmitoyl transferase II in the mitochondrial matrix.

• The fatty acyl-CoA is ready for the reactions of the oxidation pathway

-oxidation• The first 3 steps resemble the citric acid reactions that convert succinate to

oxaloacetate.-2OC-CH2-CH2-CO2

-

Succinate dehydrogenase

Succinate

Fumarate

FAD

FADH2

-2OC-C=C-CO2

-

H

H

-2OC-CH2-C-CO2

-

OH

Fumarase

H2O

NAD+

NADH + H+

-2OC-CH2-C-CO2

-

Malate dehydrogenase

Malate

OOxaloacetate

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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)

-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

• Mitochondria have four acyl-CoA dehydrogenases• Specificities for short (C4 to C6), medium (C6 to C10), long

(C8-C12), very long (C12 to C18) chain fatty acyl-CoAs.• Reoxidized via the Electron Transport Chain.

Figure 25-13Ribbon diagram of the active site region in a subunit of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria in complex with octanoyl-

CoA.

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FAD = greenOctonoyl=blueCoA =whiteGlu376 -redGeneral base

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

Enoyl hydratase

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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 b-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 dimehylbenzimadazole (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• Unusual barrel enzyme. Most barrel enzymes have active

sites at the C-terminus, but the methlymalonyl-CoA mutase AdoCbl group is located at the N-terminus.

• The Co atom is coordinated by His610 instead of the N from the 5,6 dimehylbenzimadazole (DMB)

• Has a narrow tunnel through the center of the barrel for the substrate and provides the only access to the active site, protecting the free radical intermediates that are produced from the side reactions.

Figure 25-22a X-Ray structure of P. shermanii methylmalonyl-CoA mutase in complex with 2-carboxypropyl-CoA and AdoCbl. (a) The catalytically

active subunit.

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2-carboxypropyl-CoA

AdoCb

N-term

C-term

Figure 25-22b The arrangement of AdoCbl and 2-

carboxypropyl-CoA molecules in the barrel of P. shermanii 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.

Ketone Bodies• A special source of fuel and energy for certain tissues

• Produced when acetyl-CoA levels exceed the capacity of the TCA cycle (depends on OAA levels)

• Under starvation conditions no carbos to produced anpleorotic intermediates

• Some of the acetyl-CoA produced by fatty acid oxidation in liver mitochondria is converted to acetone, acetoacetate and -hydroxybutyrate

• These are called "ketone bodies"

• Source of fuel for brain, heart and muscle

• Major energy source for brain during starvation

• They are transportable forms of fatty acids!

Ketone bodies• Acetyl-CoA can be

converted through ketogenesis to acetoacetate, D--hydroxybutyrate, and acetone.

• Acetoacetate and D--hydroxybutyrate are carried in the bloodstream to other tissues where they are converted to acetyl-CoA.

Ketone bodies• Acetoacetate and D--

hydroxybutyrate are carried in the bloodstream to other tissues where they are converted to acetyl-CoA.

• Catalyzed by three enzymes: -hydroxybutyrate dehydrogenase, 3-ketoacyl-CoA transferase, thiolase

Formation of ketone bodies

Re-utilization of

ketone bodies

Ketone Bodies and Diabetes• Lack of insulin related to uncontrolled fat breakdown in

adipose tissues• Excess -oxidation of fatty acids results in ketone body

formation.• Can often smell acetone on the breath of diabetics.• High levels of ketone bodies leads to condition known as

diabetic ketoacidosis.• Because ketone bodies are acids, accumulation can lower

blood pH.