BIOC 303: Lipid Metabolism

Fatty Acid Transport into Mitochondria: Background

• the enzymes of fatty acid oxidation in animal cells are located in the mitochondria matrix 

• short and medium chain fatty acids (12 or less C’s) enter the mitochondria without the help of membrane transporter

• long chain fatty fatty acids (14 or more C’s) cannot pass directly thru the mitochondrial membranes, they must be transported thru the carnitine shuttle

  • majority of FFAs obtained in the diet or released from adipose tissue

Fatty Acyl-CoA Synthetase

• catalyzes the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl-CoA

  • activates the fatty acid

  • costs 2 ATP

• coupled to the cleavage of ATP to AMP and PP_i

• an isozyme specific for long-chain fatty acids and present in the outer mitochondrial membrane

Fatty Acyl-CoAs

• high energy compounds

• their hydrolysis to FFAs and CoA has a large negative standard free-energy change

• formation of fatty acyl-CoA is made more favorable by the hydrolysis of 2 high-energy bonds in ATP; the pyrophosphate is immediately hydrolyzed by inorganic pyrphosphatase

• fatty acyl-CoA ester formed on the cystolic side of the OMM can be transported into the mitochondrion and oxidized to produce ATP, or they can be used in the cytosol to synthesize membrane lipids

  • those destined for mitochondrial oxidation must be attached to carnitine to be shuttled across the IMM

The Oxidation of Long Chain FAs to Acetyl-CoA

• serves as a central energy-yielding pathway in many organisms and tissues

  • provides as much as 80% of the energetic needs in mammalian heart and liver

  • provides >40% of the daily energy requirement

• electrons removed from fatty acids during oxidation pass thru the respiratory chain, driving ATP synthesis

• acetyl-coA produced from the fatty acids may be completely oxidized to CO₂ in the citric acid cycle

Carnitine

Compound that transports fatty acyl-CoAs destined for mitochondrial oxidation across the inner mitochondrial mebrane

• small

• modified amino acid

• charged

• cells can easily recognize this

Carnitine Acyltransferase 1 (CAT1)

• catalyzes a transesterification reaction to transiently attach a fatty acyl-CoA to the hydroxyl group of carnitine to form fatty acyl-carnitine

• inhibitied by malonyl-coA, the first intermediate in fatty acid synthesis

  • prevents the simultaneous synthesis and degradation of fatty acids

• exchanges the S-CoA on the fatty acyl-CoA for carnitine in the OMM

  • S-CoA is a good leaving group

Acyl-Carnitine/Carnitine Cotransporter

• allows the passive transport of the fatty acyl-carnitine ester

• located in the IMM

• moves one molecule of carnitine from the matrix to the intermembrane space as one molecule of fatty acyl-carnitine moves into the matrix

Carnitine Acyltransferase 2 (CAT2)

• transfers the fatty acyl group from carnitine back to coenzyme A to regenerate fatty acyl-coA and free carnitine

• located on inner face of IMM

• the carnitine is now available to be transferred back thru the acyl carnitine/carnitine cotransporter to be used to shuttle the next fatty acid acros

Two Pools of Coenzyme A

• one pool in the cytosol, on in the mitochondria

• coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids and some amino acids

• cytosolic coenzyme A is used in the biosynthesis of fatty acids

Two Pools of Fatty Acyl-CoA

• one pool in the cytosol, on in the mitochondria

• fatty acyl-coA in the cytosolic pool can be used for membrane lipid synthesis of can be moved into the mitochondrial matrix for oxidation and ATP production

  • conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate

• mitochondrial fatty acyl-coA undergoes β-oxidation

Carnitine Shuttle is a Major Control Point

• carnitine-mediated entry is the rate-limiting step for oxidation of fatty acids in mitochondria

β-Oxidation: Details

• fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-coA, starting from the carboxyl end of the fatty acyl chain

• # of passes: n Carbons / 2 -1

  • -1 because the last two carbons will remain as acetyl-CoA

  • works for saturated (even C’s) and unsaturated fatty acids

• # of passes for odd numbered fatty acids: ( n Carbons - 3) / 2

• formation of each acetyl-coA requires removal of 4 hydrogen atoms (two pairs of electrons and four H⁺) from the fatty acyl moiety by dehydrogenases

• each acetyl-CoA can turn the TCA cycle once, producing 10 ATP equivalents

  • in total 14 ATP generated with β oxidation

β-Oxidation: Step 1

• oxidation (dehydrogenation) reaction of fatty acyl-CoA forms a double bond between the ⍺ and β carbons (C2 and C3)

  • yields trans-∆²-enoyl-coA

• catalyzed by acyl-coA dehydrogenase

• the new double bond has the trans configuration, whereas the double bonds in naturally occurring unsaturated fatty acids are normally in the cis configuration

• analagous to succinate dehydrogenation in the citric acid cycle

β-Oxidation: Acyl-CoA Dehydrogenase

• flavoprotein with tightly bound FAD

  • FAD gets reduced in this reaction, and FADH₂ can pass these two electrons to the ETC to generate energy in the form of 1.5 ATP

• 3 types of this isozyme, specific for a range of fatty acyl chain lengths:

  • very long chain acyl dehydrogenase (VLCAD): 12-18 carbons

  • medium chain (MCAD): 4-14 carbons

  • short chain (SCAD): 4-8 carbons

β-Oxidation: Step 2

• water is added to the double bond of the trans-∆²-enoyl-CoA to form the L-stereoisomer of β-hydroxyacyl-CoA (3-hydroxyacyl-CoA) 

• catalyzed by enoyl-CoA hydratase and it only works on trans double bonds

• analogous to fumarase reaction in the citric acid cycle

β-Oxidation: Step 3

• L-β-hydroxyacyl-CoA is dehydrogenated to form β-ketoacyl-CoA

• catalyzed by β-hydroxylacyl-CoA dehydrogenase and specific for the L-isomer

• NAD⁺ is the electron acceptor, and the NADH formed in the reaction donates its electrons to the ETC, leading to the formation of 2.5 ATP

• analogous to the malate dehydrogenase reaction in TCA cycle

β-Oxidation: Step 4

• catalyzed by acyl-CoA acetyltransferase (thiolase)

• reaction of β-ketoacyl-CoA with a free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA

• the other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms

• reverse claisen condensation reaction

Trifunctional Protein (TFP)

• a multienzyme complex associated with the inner mitochondrial membrane that catalyzes steps 2-4 of β-oxidation for fatty acyl chains of 12+ carbons (long chains)

• heterooctamer of ⍺₄β₄ subunits

  • each ⍺ subunit contains 2 activities, the enoyl-coA hydratas and β-hydroxylacyl-CoA dehydrogenase

  • β subunits contain the thiolase activity

• the tight association of these enzymes alllows efficient substrate channeling from one active site to the next, without diffusion of the intermediates away from the enzyme surface

• when TFP has shortened the fatty acyl chain to 12 or fewer carbons, further oxidations are catalyzed by medium/short chain enzymes

The Chemical Logic of the β-Oxidation Sequence

• the first 3 reactions of β-oxidation create a much less stable C-C bond

  • the ⍺ carbon (C2) is bonded to two carbonyl carbons (the β-ketoacyl-CoA intermediate)

• the ketone function on the β carbon (C3) makes it a good target for nucleophilic attack by the –SH of coenzyme A, catalyzed by thiolase 

• the acidity of the ⍺-hydrogen and the resonance stabilization of the carbanion generated by the departure of this hydrogen makes the terminal –CH₂–CO–S-CoA a good leaving group

  • facilitating breakage of the ⍺-β bond

One Pass of β-Oxidation

Fatty acyl-CoA + CoA + FAD + NAD⁺ + H₂O → 

shortened fatty acyl-CoA + acetyl-CoA + FADH₂ + NADH + H⁺

• 1 acetyl-CoA, 2 pairs of electrons, 4 protons are removed from the long-chain fatty acyl-CoA

• 4 molecules of ATP are formed for each 2-carbon unit removed in one pass thru the sequence

Repetition of β-Oxidation

• the shortened fatty acyl-CoA reenters the β-oxidation sequence for removal of another, and then another, acetyl-CoA

Genetic Defects in Fatty Acyl-CoA dehydrogenases

• inability to oxidize fatty acids from TAGs has serious health consequences

• individuals with 2 mutant MCAD alleles cannot oxidize fatty acids of 6-12 carbons

• lead to build up of medium chain fatty acids; long chains that have been shortened and transferred to MCAD will also not be able to do processes with MCAD

• symptoms include fatty liver, high blood levels of octanoic acid (8:0), coma and death

Water in β-Oxidation

• each pair of electrons transferred from NADH or FADH₂ to O₂ yields one H₂O (“metabolic water”)

• 2 H₂O produced per cycle

Further Oxidation of Acetyl-CoA

• the acetyl-CoA produced from β-oxidation of fatty acids can be oxidized to CO₂ and H₂O by the TCA cycle

• the second stage of fatty acid oxidation

n/2 Acetyl-coA + n O₂ + 10n ADP + 10n P_i → n CO₂ + 10n ATP + n H₂O

• n = number of carbons in the original fatty acyl-CoA chain

• From β-oxidation to end of TCA for even, saturated chains

  • -2 ATP from this equation because activating a fatty acid takes 2 ATP

fatty acyl-CoA (C_n) + (3n/2-1) O₂ + (7n - 4) ADP + (7n - 4) P_i → CoA + (7n - 4) ATP + n CO₂ + (3n/2 -1) H₂O

Oxidation of Unsaturated Fatty Acids

• most naturally occurring fatty acids have cis double bonds, and cannot be acted upon by enoyl-CoA hydratase

• requires two additional enzymes to transform into substrates for β-oxidation:

  • enoyl-CoA isomerase: converts cis double bonds to trans

    • only this is needed for monosaturated fats

  • 2,4-dienoyl-CoA reductase: reduces cis double bonds

    • required for PUFAs

Enoyl-CoA Isomerase Figure

2-4-Dienoyl-CoA Reductase

Oxidation of Odd-Number Fatty Acids

• oxidized in the same pathway as even-number fatty acids, beginning at the carboxyl chain

• the substrate for the last pass thru the β-oxidation sequence is a fatty acyl-CoA with a five carbon fatty acid

  • when oxidized and cleaved, the products are acetyl-CoA and propionyl-CoA

Propionate

• 3 carbon compounds formed by cattle and other ruminant animals during carbohydrate fermentation

• the propionate is absorbed into the blood and oxidized by the liver and other tissues

• CH₃–CH₂–COO^-

Oxidation of Propionyl-CoA: Step 1

• catalyzed by proprionyl-CoA carboxylase, which contains cofactor biotin 

• propionyl-CoA is carboxylated to form D-methylmalonyl-CoA 

• bicarbonate ion (HCO₃^-) is activated by attachment to biotin before its transfer to the propionate moiety

  • formation of the carboxybiotine intermediate requires ATP

Absence of Functional Propionyl-CoA Carboxylase

• leads to an accumulation of propionyl-CoA in mitochondria, depleting the available supply of coenzyme A for continuing β-oxidation

• propionyl-CoA is esterified to carnitine, transported out the mitochondria via the carnitine shuttle and released to blood as propionate

  • this severely acidifies blood and urine

Oxidation of Propionyl-CoA: Step 2

• catalyzed by methylmalonyl-CoA epimerase

• D-methylmalonyl-CoA is epimerized to its L stereoisomer 

Oxidation of Propionyl-CoA: Step 3

• catalyzed by methylmalonyl-CoA mutase

  • requires coenzyme B₁₂ 

• L-methylmalonyl-CoA undergoes intramolcular rearrangement to form succinyl-CoA, which can enter the TCA cycle

• net gain of oxidation of priopionyl-CoA = 4 (1 GTP, 1 FADH2 = 1.5 ATP, 1 NADH = 2.5 ATP)

  • was 5, but -1 from step 1

Fatty Acid Oxidation is Tightly Regulated

• oxidation of fatty acids consumes valuable energy stores, and is regulated to only occur when organism’s require energy

• in the liver, fatty acyl-CoA formed in the cytosol has 2 major pathways to it:

  • β oxidation by enzymes in mitochondria

  • conversion into triacylglycerols and phospholipids by enzymes in the cytosol

    • the pathway taken depends on the rate of transfer of long-chain fatty acyl-CoA into mitochondria

• the carnitine by which fatty acyl groups are carried into the mitochondrial matrix as fatty acyl carnitine is the rate limiting step for fatty acid oxidation

  • important point of regulation

  • once fatty acyl groups have entered the mitochondria, they are committed to oxidation to acetyl-CoA

Peroxisomes

• organelles found in plants and animals

• the major site of β oxidation in plant cells

β Oxidation in Peroxisomes

• the intermediates for β oxidation of fatty acids are coenzyme A derivatives

• process consists of 4 steps like in mitochondrial β oxidation: 1) dehydrogenation, 2) hydration, 3) oxidation of the β-hydroxyacyl-CoA to a ketone, 4) thiolytic cleavage by coenzyme A

• high concentrations of fats in the diet result in increased synthesis of the enzymes of peroxisomal β oxidation in the liver

• liver peroxisomes do not contain the enzymes of the citric acid cycle and cannot catalyze the oxidation of acetyl-CoA to CO₂

  • long chain/branched fatty acids are catabolized in peroxisomes to shorten chain producst, which are exported to mitochondria and completely oxidized there

Differences between Peroxisomal and Mitochondrial Pathways

Chemistry of the first step

• in peroxisomes, the flavoprotein acyl-CoA oxidase that introduces the double bond passes electron directly to O₂, producing H₂O₂

• this oxidant is immediately cleaved to H₂O and O₂ by catalase

• in peroxisomes, the energy released in step 1 is not conserved as ATP, but dissipated as heat 

• in mitochondria, the electrons removed in step 1 pass thru the ETC to O₂, producing water and ATP

Specificity of Chain Length

• the peroxisomal system is more active on very-long chain, and branched fatty acids

Genetic Defects in Peroxisomal Oxidation

• Zellweger syndrome: unable to make peroxisomes, and therefore lack all metabolism related to peroxisomes

• X-linked adrenoleukodystrophy (XALD): peroxisomes fail to oxidize VLCFA, due to the lack of function of the ABCD1 transporter in the peroxisomal membrane

Both defects lead to accumulation in the blood of VLCFA, and is a diagnostic sign of these disorders

• leads to neurological disorders including demyelination → motor impairment, memory loss and seizures

Treatments: bone marrow transplants, lentiviral therapies, Lorenzo’s oil

Malonyl-CoA Formation from Acetly-CoA: Details

• irreversible 3 step process that occurs in the cytoplasm

• catalyzed by acetyl-CoA carboxylase

  • contains a biotin prosthetic group covalently bound in amide linkage to the 𝞢-amino group of a Lys residue

Malonyl-CoA Formation from Acetly-CoA: Step 1

• a carboxyl group from HCO₃^- is transferred to biotin in an ATP dependent reaction

Malonyl-CoA Formation from Acetly-CoA: Step 2 + 3

• the carboxyl group is carried by the biotin to a different active site, where the CO₂ is transferred to acetyl-CoA in the third and final step to yield malonyl-CoA

• this carboxylation step renders the next step (condensation in FAS) more favorable thermodynamically

Why is bicarbonate used to activate Acetyl-CoA

• good leaving group once attached

• it’s around, it forms spontaneously when CO₂ dissolves in water

• biology is complex and doesn’t always use the most straightforward pathways

Fatty Acid Synthase (FAS1)

• catalyzes assembly of long carbon chains of fatty acids in the cytosol thru a repeating 4-step sequence

• begins with malonyl-CoA and acetyl-CoA

• each passage thru the cycle elongates chain by 2 carbons

• the product of each cycle feeds back into the cycle as the starting material for the next condensation step with malonyl-CoA

• 7 active sites to catalyze the 4 step cycle

FAS1 Reactions

• a condensation reaction is followed by a reduction-dehydration-reducation sequence to convert the C3 carbonyl to a methylene

• the last 3 steps are the chemical reverse of the OHO sequence in β oxidation of fatty acids

• the electron-cofactor and activating groups differ from β oxidation

  • the reducing agent in FAS is NADPH and the activating groups are two difference enzyme-bound –SH groups

• fatty acid synthesis leads to a single product

FAS Active Sties

• the active site for each enzyme is found in a separate domain within the larger polypeptide (FAS1)

• throughout FAS, the intermediates remain covalently attached as thioesters to one of two thiol groups

  • –SH group of a Cys residue in β-ketoacyl-ACP synthase

  • –SH group of ACP

• hydrolysis of thioesters is highly exergonic and the energy released helps make steps 1 and 5 thermodynamically favorable

Acyl Carrier Protein (ACP)

• as it goes thru the FAS cycle, the acyl group is covalently linked to ACP, which shuttles it from one active site to another in sequence

• part of FAS1 polypeptide

• no intermediates are released

• when the chain length reaches 16 carbons, the product, palmitate (16:0) leaves the cycle (7 cycles)

• C16 and C15 of palmitate are derived from acetyl-CoA, the rest of the carbon atoms in the chain are derived from acetyl-CoA via malonyl-CoA

4’-phosphopantetheine

• ACP is the shuttle that holds the system together, containing the prosthetic group 4’-phosphopantetheine, also found in coenzyme A

• serves as a flexible arm, tethering the growing fatty acyl chain to the surface of the fatty acid synthase complex while carrying the reaction intermediates from one enzyme active site to the next

Fatty Acid Synthase Receives the Acetyl and Malonyl Groups (before step 1)

• before the condesation rxns of FAS can begin, the two thiol groups on the enzyme complex must be charged with the correct acyl groups 

• the acetyl group of acetyl-CoA is transferred to ACP in a rxn catalyzed by the malonyl/acetyl-CoA ACP transferase (MAT) domain of the multifunctional polypeptide

  • the acetyl group is then transferred to the Cys–SH group of the β-ketoacyl-ACP synthase (KS)

• transfer of the malonyl group from malonyl-CoA to the –SH group of ACP

  • also catalyzed by MAT

FAS: Step 1 Condensation

• Claisen condesation of activated acetyl and malonyl groups to form acetylacetyl-ACP, molecule of CO₂ simultaneously produced

• catalyzed by β-ketoacyl-ACP synthase

• the acetyl group is transferred from the Cys-SH group of the enzyme to the malonyl group on the –SH of ACP, becoming the methyl terminal of the new acetoacetyl group

• the C atom of CO₂ produced is the same one that was introduced into malonyl-CoA from HCO₃^- in the acetyl-CoA carboxylase reaction 

  • therefore, CO₂ is only transiently in covalent linkage during FAS

  • its removed as each two-carbon unit is added 

Why add CO₂ to make malonyl group only to lose it during FAS?

• the use of activated malonyl groups rather than acetyl group makes the condensation reactions thermodynamically favorable

• the methylene carbon of malonyl group is sandwiched between carbonyl and carboxyl carbons, which forms a good nucleophile

• decarboxylation of the malonyl group facilitates nucleophilic attack of the methylene carbon on the thioester linking the acetyl group to β-ketoacyl-synthase, displacing the enzyme’s SH group

FAS: Step 2 Reduction

• reduction of the carbonyl group

• acetoacetyl-ACP undergoes reduction of the carbonyl group at C3 to form D-β-hydroxybutyryl-ACP

  • note different stereoisomer than the intermediate in fatty acid oxidation (L-β-hydroxyacyl)

• catalyzed by β-ketoacyl ACP reductase

• electron donor: NADPH

FAS: Step 3 Dehydration

• the elements of water are removed from C2 and C3 of D-β-hydroxybutyryl-ACP to yield a double bond in trans-∆²-butenoyl-ACP

• catalyzed by β-hydroxyacyl-ACP dehydratase 

FAS: Step 4 Reduction

• the double bond of trans-∆²-butenoyl-ACP is reduced (saturated) to form butyryl-ACP 

• catalyzed by enoyl-ACP reductase

• electron donor: NADPH

FAS: Step 5 Translocation

• production of the 4-carbon, saturated fatty acyl-ACP marks completion of one pass thru the fatty acid synthase complex

• the butyryl group is transferred from teh phosphopantetheine–SH group of ACP to the Cys–SH group of β-ketoacyl-ACP synthase (which initially bore the acetyl group)

FAS: Step 6 Repeat

• to start the next cycle of 4 rxns that lengthens the chain by two more carbons, another malonyl group is linked to the now unoccupied phosphopantetheine–SH group of ACP

• condesation occurs as the butyryl group, acting like the acetyl group in the first cycle, is linked to two carbons of the malonyl-ACP group with concurrent loss of CO₂

Overall Reaction of FAS (Synthesis of Palmitate)

Formation of 7 malonyl-CoA molecules

7Acetyl-CoA + 7CO₂ + 7ATP → 7 malonyl-CoA +7ADP +7P_i

7 cycles of condensation and reduction

Acetyl-CoA + 7malonyl-CoA+14NADPH +14H⁺ → palmitate + 7CO₂ + 8CoA +14NADP⁺ + 6H₂O

Overall process:

8 Acetyl-CoA + 7ATP +14NADPH +14H⁺ → palmitate +8CoA+7ADP +7P_i +14NADP⁺ + 6H₂O

Citrate Transporter: Moving Acetyl Units out of Matrix

• intramitochondrial acetyl-CoA reacts with oxaloacetate to form citrate, catalyzed by citrate synthase (also an enzyme in TCA cycle)

  • acetyl-CoA made from pyruvate dehydrogenase or β-oxidation

• citrate then passes thru the IMM on the citrate transporter

• in the cytosol, citrate cleavage by citrate lyase regenerates acetyl-CoA and oxaloacetate in an ATP dependent reaction (for FA synthesis)

  • oxalocaetate can’t return to the matrix, as there’s no oxaloacetate transporter

Citrate Transporter: Malate Fates

Matrix Fate

• cytosolic malate dehydrogenase reduces the oxaloacetate to malate, which can return to the mitochondrial matrix on the malate-⍺-ketoglutarate transporter, in exchange for citrate

  • in the matrix, malate is reoxidized to oxaloacetate to complete the shuttle

OR

Cytosol Fate

• be converted in pyruvate by malic enyme which is transported into the matrix by the pyruvate transporter and converted into oxaloacetate

  • this pathway produces NADPH, which is required for FA synthesis and is one of the major ways in which the cell generates this electron acceptor

Pyruvate Transporter

Transports pyruvate into the matrix where it is converted to oxaloacetate by pyruvate carboxylase

Citrate Malate Shuttle Figure

• in the resulting cycle, two ATP molecules are consumed (by citrate lyase and pyruvate carboxylase) for every molecule of acetyl-CoA delivered to FA synthesis

  • citrate lyase (cytosol): cleaves citrate → acetyl-CoA + oxaloacetate (1ATP)

  • pyruvate carboxylase (matrix): regenerates oxaloacetate from pyruvate after the cycle runs (1ATP)

Acetyl-CoA Carboxylase (ACC) Regulation

• we want to synthesize fatty acids when there is an abundance of energy and Acetyl-CoA available

  • we want to reduce/restrict synthesis when there is not

• ACC, which synthesizes malonyl-CoA (substrate of FA synthesis), is inhibited by glucagon and by high levels of palmitoyl-CoA 

  • glucagon: secreted when blood glucose levels are low in the fasting state

  • palmitoyl-CoA: ultimate product of FAS, negative feedback regulator

• the rxn catalyzed by ACC is the rate-limiting step in FA synthesis

• when concentration of mitochondrial acetyl-CoA and ATP increase, citrate is transported out of the mitochondria

  • it then becomes the precursor of cytosolic acetyl-CoA and an allosteric signal of the activation of ACC

Acetyl-CoA Carboxylase (ACC): Phosphorylation Regulation

• Dephosphorylation: ON

• Phosphorylation: OFF

ACC and CAT1 Regulation

• if FAS and β oxidation proceeded simultaneously, energy would be wasted

  • because ATP and NADPH are spent to make fatty acids only to immediately oxidize them back into acetyl-CoA.

• high blood glucose dephosphorylates ACC, making it more active

• the product, malonyl-CoA inhibits CAT1, restricting the amount of fatty acyl-CoA that can enter the mitochondria for oxidative breakdown (β oxidation)

• in the fasting state, ACC is inactive and malonyl-CoA is not being synthesized, β-oxidation is favored