Chapters 17,21 Fatty acid catabolism , Lipid biosynthesis

17| Fa'y Acid Catabolism

2013 W. H. Freeman and Company

21| Lipid Biosynthesis

Oxida=on of fa'y acids is a major energy source in many organisms

About one-third of our energy needs comes from dietary triacylglycerols

About 80% of energy needs of mammalian heart and liver are met by oxida

Fats provide efficient fuel storage

The advantage of fats over polysaccharides: Fa=y acids carry more energy per carbon because they are more reduced

Fa=y acids carry less water along because they are nonpolar (aggregate in lipid droplets and are unsolvated)

Glucose and glycogen are for short-term energy needs, quick delivery

Fats are for long-term (months) energy needs, good storage, slow delivery

Fat Storage in White Adipose Tissue

Nuclei

Squeezed

Dietary fa'y acids are absorbed in the vertebrate small intes=ne

Emulsification by biological detergents (bile)

Breakdown of TAG to DAG, MAG, FFA and glycerol

Uptake by intestinal cells

Chylomicrons (lipoproteins)

Bloodstream to target tissues

2nd breakdown of TAG

Used for energy (muscles) or reesterified for energy (adipose)

Remaining chylomicrons go to liver and enter by RME used for ketone bodies synthesis. When diet contains more f.a. than needed, liver converts them to TAG and packages them into VLDL to be transported to adipocytes

Lipids are transported in the blood as chylomicrons

Apoliporpotein + lipids particles = lipoprotein Lipoproteins range in density: VLDL to VHDL

Hormones trigger mobiliza=on of stored triacylglycerols

Hydrolysis of TAGs is catalyzed by lipases - can produce MAGs, DAGs, FFA and glycerol

Some lipases are regulated by hormones glucagon and epinephrine Recall: Epinephrine means: We need energy now Glucagon means: We are out of glucose

Hormones trigger mobiliza=on of stored triacylglycerols

Perilipins proteins that coat lipid droplets and restrict access to lipids to prevent premature mobiliza

Glycerol from fats enters glycolysis

Only 5% of biologically-ac

Fa'y Acid Transport into Mitochondria

Fats are degraded into fa=y acids and glycerol in the cytoplasm of adipocytes

Fa=y acids are transported to other

Conversion of a fa'y acid to a fa'y acylCoA (1)

Nucleophilic attack by f.a. anion

Phosphoester linkage between f.a. carboxyl and phosphate of ATP

Thioester linkage between f.a. carboxyl and thiol group of CoA-SH Hydrolysis of PPi to 2Pi is highly exergonic and pulls the first reaction forward

Acyl-Carni=ne/Carni=ne Transport

(2)

(3)

Transesterification to carnitine

Transesterification to CoA

2 separate pools of CoA: Matrix CoA used mostly in oxidative degradation (pyr, f.a., a.a.) Cytosolic CoA used in biosynthesis of f.a.

Carnitine-mediated entry is the rate limiting step for oxidation of f.a. in mito

Stages of Fa'y Acid Oxida=on

Stage 1 consists of oxida

Stages of Fa'y Acid Oxida=on

The -Oxida=on Pathway Each pass removes one acetyl moiety in the form of acetyl-CoA.

Palmitate (C16) undergoes seven passes through the oxidative sequence

Formation of each acetyl-CoA requires removal of 4 H atoms {2 e pairs and 4 H+})

Step 1: Dehydrogena=on of Alkane to Alkene

Catalyzed by isoforms of acyl- CoA dehydrogenase (AD) on the mitochondrial inner membrane Very-long-chain AD (VLCAD, 1218 carbons)

Medium-chain AD (MCAD, 414 carbons)

Short-chain AD (SCAD, 48 carbons) Results in trans double bond, different from naturally occurring

unsaturated fa=y acids, between and C

Analogous to succinate dehydrogenase reac

Step 2: Hydra=on of Alkene

Catalyzed by two isoforms of enoyl-CoA hydratase: Soluble short-chain hydratase (crotonase) Membrane-bound long-chain hydratase, part of trifunc

Step 3: Dehydrogena=on of Alcohol

Catalyzed by -hydroxyacyl-CoA dehydrogenase The enzyme uses NAD cofactor as the hydride acceptor Only L-isomers of hydroxyacyl CoA act as substrates Analogous to malate dehydrogenase reac

Step 4: Transfer of Fa'y Acid Chain

Catalyzed by acyl-CoA acetyltransferase (thiolase) via covalent mechanism The carbonyl carbon in -ketoacyl-CoA is electrophilic Ac

Trifunc=onal Protein (TFP)

Hetero-octamer Four subunits

enoyl-CoA hydratase ac

Similar mechanisms introduce carbonyls in other metabolic pathways

NADH and FADH2 serve as sources of ATP

Oxida=on of Unsaturated Fa'y Acids Naturally occurring Unsaturated Fa=y acids contain cis double bonds Are NOT a substrate for enoyl-CoA hydratase

Two addi

Oxida=on of Monounsaturated Fa'y Acids

Oleate (18:1 9) converted to oleoyl-CoA and imported into mito via carnitine shuttle

Oxida=on of Polyunsaturated

Fa'y Acids

Linoleate (9,12)

First double bond requires isomeriza=on

Second requires reduc=on/isomeriza=on

Oxida=on of odd-numbered fa'y acids

Most dietary fa=y acids are even-numbered Many plants and some marine organisms also synthesize odd-numbered fa=y acids

Propionyl-CoA forms from -oxida

Carboxyla=on of Propionyl-CoA

Isomeriza=on to Succinyl-CoA CAC

Isomeriza=on in propionate oxida=on requires coenzyme B12

Complex Cobalt-Containing Compound: Coenzyme B12

Very unstable bond Breaks to yield CH2

. and Co3+ Used to transfer the hydrogen

atom to a different C in the molecule (isomerization)

No mixing of the transferred H atom with the hydrogen of the solvent (H2O)

The formation of this complex cofactor occurs in one of two known reactions that cleaves a triphosphate from ATP

Regula=on of Fa'y Acid Synthesis and Breakdown

Cytosol

Occurs only when need for energy requires it 2 pathways for f.a.CoA in liver: TAG synthesis in cytosol or f.a. oxida

Gene=c defects in fa'y acyl-CoA dehydrogenases

Inability to oxidize fats for energy has serious effects on health

More than 20 human gene

-Oxida=on in Mitochondria vs. Peroxisomes

Differ in the first step: - passes es directly to O2 forming H2O2 which is quickly removed by the ac

Forma=on of Ketone Bodies

Entry of acetyl-CoA into citric acid cycle requires oxaloacetate

When oxaloacetate is depleted, acetyl-CoA is converted into ketone bodies (acetone, acetoacetate and D--hydroxybutyrate) Frees Coenzyme A for con

Release of Free Coenzyme A

Another condensation with acetyl-CoA yields HMG-CoA

Forma=on of Ketone Bodies

Cleaved into acetoacetate and acetyl-CoA

Specific for the D-isomer; dont confuse it with L--hydroxyacyl-CoA DH of oxidation

Untreated diabetes [acetoacetate] is high more acetone produced exhaled (odor)

Ketone Bodies as fuel In extrahepatic tissues: Ketone bodies can be used as fuels in all tissues except the liver The liver is a producer, not a consumer, of ketone bodies

CAC Found in all tissues except the liver

Lipids fulfill a variety of biological func=ons

Energy storage Cons

Catabolism and anabolism of fa'y acids proceed via different pathways

Catabolism of fa=y acids (excergonic and oxida=ve) produces acetyl-CoA produces reducing power (NADH and FADH2) ac

Subcellular localiza=on of lipid metabolism

Overview of Fa'y Acid Synthesis

Fa=y acids are built in several passes, processing one acetate unit at a

Malonyl-CoA is formed from acetyl-CoA and bicarbonate

The reac

Synthesis of fa'y acids is catalyzed by fa'y acid synthase (FAS)

FAS system: Catalyzes a repea

FAS I vs. FAS II

FAS I Single polypep

Fa'y Acid Synthesis Overall goal: a=ach two-C acetate unit from malonyl-CoA to a

growing chain and then reduce it Reac

The General Four-Step Fa'y Acid Synthase I Reac=on in Mammals (1)

Prep: Malonyl CoA and acetyl CoA (or longer fa=y acyl chain) are bound to FAS I

- bind via thioester terminus of a Cys of the FAS - ac

Step 1 of FAS I: Elonga=on

Steps 2-4 of the FAS I rxn

Overall Palmitate Synthesis

Acyl Carrier Protein (ACP) serves as a shu'le in fa'y acid synthesis

Contains a covalently a=ached prosthe

Charging ACP and FAS I with acyl groups ac=vates them

Two thiols must be charged with the correct acyl groups before condensa

Charging, Ac=va=on with ACP, and the Four-Step Sequence of Mammalian Fa'y

Acid Synthesis

Carbonyl at C-3 is reduced to form D--hydroxybutyryl-ACP NADPH is e donor

Catalyzed by -ketoacyl-ACP reductase (KR)

OH and H removed from C-2 and C-3 of -hydroxybutyryl-ACP to form trans-2-butenoyl-ACP

Catalyzed by -hydroxyacyl-ACP dehydratase (DH)

NADPH is the electron donor to reduce double bond of trans-2-butenoyl-ACP to form butyryl-ACP

Catalyzed by enoyl-ACP reductase (ER)

Enzymes in Fa'y Acid Synthase

Condensa

The Transferase and FAS rxns are repeated in new rounds

Product of first round is butyryl-ACP (bound to phosphopantetheine-SH group of ACP)

Butyrul gp is transferred to the Cys of -ketoacyl-ACP synthase In the first round, acetyl-CoA was bound here

New malonyl-CoA binds to ACP Aoer new round of four steps, six-C product is made (bound to ACP)

Beginning of the Second Round of Fa'y Acid Synthesis

Stoichiometry of Synthesis of Palmitate (16:0)

1) 7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs using ATP

7 AcCoA + 7 CO2 + 7 ATP 7 malCoA + 7 ADP + 7 Pi

2) Seven cycles of condensa

Acetyl-CoA is transported into the cytosol for fa'y acid synthesis

In nonphotosynthe

Fa'y acid synthesis occurs in cell compartments where NADPH levels are high

Cytosol for animals, yeast Chloroplast for plants

Sources of NADPH: In adipocytes: pentose phosphate pathway and malic enzyme

NADPH is made as malate converts to pyruvate + CO2 In hepatocytes and mammary gland: pentose phosphate pathway NADPH is made as glucose-6-phosphate converts to ribulose 6-phosphate

In plants: photosynthesis

Pathways for NADPH Produc=on

Acetyl-CoA, generated in the mitochondria, is shu'led to the cytosol as citrate

In most eukaryotes, the acetyl-CoA for lipid synthesis is made in the mitochondria But lipid synthesis occurs in the cytosol

And there is no way for acetyl-CoA to cross mitochondrial inner membrane to the cytosol

So acetyl-CoA is converted to citrate Acetyl-CoA + oxaloacetate citrate

Same rxn as occurs in CAC Catalyzed by citrate synthase Citrate passes through citrate transporter

Citrate is cleaved to regenerate acetyl-CoA

Citrate (now in cytosol) is cleaved by citrate lyase Regenerates acetyl-CoA and oxaloacetate Rxn requires ATP Acetyl-CoA can now be used for lipid synthesis

What happens to the oxaloacetate because there is no oxaloacetate transporter either?

Oxaloacetatecyt is converted to malate

Malate dehydrogenase in cytosol reduces oxaloacetate to malate

Two poten

Shu'le for Transfer of Acetyl Groups from Mitochondria to Cytosol

Fa'y acid synthesis is =ghtly regulated via ACC

Acetyl CoA carboxylase (ACC) catalyzes the rate-limi

Importance of Citrate to Regula=on of Fa'y Acid Synthesis

In animals, citrate s

ACC is also regulated by covalent modifica=on

Inhibited when energy is needed Glucagon and epinephrine:

reduce sensi

Regula=on of Fa'y Acid Synthesis in Vertebrates

Addi=onal Modes of Regula=on in Fa'y Acid Synthesis

Changes in gene expression Example: Fa=y acids (and eicosanoids) bind to transcrip

Palmitate can be lengthened to longer-chain fa'y acids

Elonga

Palmitate and stearate can be desaturated

Palmitate(16:0)palmitoleate(16:1; 9) Stearate (18:0)oleate (18:1; 9)

Catalyzed by fa=y acyl-CoA desaturase in animals Also known as the fa=y acid desaturases Requires NADPH; enzyme uses cytochrome b5 and cytochrome b5 reductase

Note that this is a 9-desaturase! It reduces the bond between C-9 and C-10.

Vertebrate fa'y acyl desaturase is a non-heme, iron-containing, mixed func=on

oxidase

O2 accepts four electrons from two substrates Two electrons come from saturated fa=y acid Two electrons come from ferrous state of cytochrome

b5

Desatura=on of a Fa'y Acid by Fa'y Acyl-CoA Desaturase

Plants can desaturate posi=ons beyond C-9

Humans have 4, 5, 6, and 9 desaturases but cannot desaturate beyond 9

Plants can produce: linoleate 18:2(9,12) -linolenate 18:3 (9,12,15)

These fa=y acids are essen=al to humans Polyunsaturated fa=y acids (PUFAs) help control membrane fluidity

PUFAs are precursors to eicosanoids Implica

Oxidases, Monooxygenases, and Dioxygenases

Many enzymes use oxygen as an e acceptor, but not all of them incorporate oxygen into the product.

Oxidases do not incorporate oxygen into the product Oxygen atoms usually end up in H2O2

Oxygenases do incorporate oxygen into the product Monooxygenases incorporate one of the oxygen atoms into the product

Dioxygenases incorporate both oxygen atoms into the product

Monooxygenases incorporate one oxygen into the product

AH + BH2 + O-O A-OH + B + H2O Product is ooen hydroxylated, so also called hydroxylases or mixed-func=on oxygenases

Example: Phenylanine hydroxylase hydroxylates phenylalanine to form tyrosine

Deficiency causes phenylketonuria (PKU)

Cytochrome P450s are monooxygenases

Important in drug metabolism Hydroxylate nonpolar molecules

usually inac

Dioxygenases incorporate two oxygens in the product

Usually metalloproteins Ac

Eicosanoids are potent short-range hormones made from arachidonate

Eicosanoids are paracrine signaling molecules They include prostaglandins, leukotrienes, thromboxanes

Created from arachidonic acid, 20:4 (5,8,11,14) Arachidonate is incorporated into the phospholipids of membranes

In response to s

Prostaglandins are made by prostaglandin H2 synthase (cyclooxygenase, COX)

COX (aka PGH2 synthase) is a bifunc

Conversion of Arachidonate to Prostaglandins and Other Eicosanoids

Thromboxane synthase present in thrombocytes converts PGH2 to thromboxane A2

Induce the constric

PGH2 synthase has two isoforms

COX-1 catalyzes synthesis of prostaglandins that regulate gastric mucin secreEon

COX-2 catalyzes synthesis of prostaglandins that mediate pain, inflammaEon, and fever

NSAIDs inhibit cyclooxygenase ac=vity

Aspirin (Acetylsalicylate) is an irreversible inhibitor Acetylates a Ser in the ac

A Few NSAIDs that Inhibit PGH2

Arachidonate (substrate)

Advil, motrin Aleve

COX-2-specific inhibitors have a checkered history

Developed to inhibit prostaglandin forma

Leukotriene synthesis also begins with arachidonate

O2 is added to arachidonate via lipoxygenases Creates species that differ in the posi

Biosynthesis of Triacylglycerols Synthesized or ingested fa=y acids are either stored for energy or used in membranes depending on the needs of the organism

Animals and plants store fat for fuel

Plants: in seeds, nuts Typical 70-kg human has ~15 kg fat

Enough to last 12 wks Compare with 12 hrs worth glycogen in liver and muscle

Animals and plants and bacteria make phospholipids for cell membranes

The precursor for the backbone of fat and phospholipids is glycerol 3-phosphate

Both pathways start by the forma

Acyl transferases a'ach two fa'y acids to glycerol 3-phosphate

Phospha

To make TAG, phospha=dic acid is dephosphorylated and acylated

Phospha

Conversion of Phospha=dic Acid into Triacylglycerol

Regula=on of Triacylglycerol Synthesis by Insulin

Insulin results in s

Regula=on of Fat Metabolism by Glucagon and Epinephrine

Glucagon and epinephrine result in s

Triacylglycerol breakdown and re-synthesis create a fu=le cycle

Seventy-five percent of free fa=y acids (FFA) released by lipolysis are reesterified to form TAGs rather than be used for fuel Some recycling occurs in adipose

Benefits of this fu=le cycle?

Recycling con

What is the source of the glycerol 3-phosphate needed for fa'y acid

reesterifica=on?

During lipolysis (s

Glyceroneogenesis makes DHAP for glycerol 3-phosphate genera=on

Glyceroneogenesis contains some of the same steps of gluconeogenesis Converts pyruvate DHAP Basically, a shortened version of gluconeogenesis in the liver and adipose

Glyceroneogenesis

Regula=on of PEPCK expression is =ssue-dependent

Cor

Regula=on of Glyceroneogenesis via Glucocor=coid Hormones

Cor=sol and glucagon can elevate blood sugar

1) PEPCK expression in liver gluconeogenesis (so [glucose])

2) PEPCK expression in adipose

Thiazolidinedione drugs target insulin resistance by increasing glyceroneogenesis

Elevated FFA levels seem to promote insulin resistance

Thiazolidinediones upregulate PEPCK in adipose

Thiazolidinediones/Glitazones

Have this group in common

Avandia (Rosiglitazone) removed from market due to associa

Regula=on of Glyceroneogenesis via Thiazolidinediones

Biosynthesis of Membrane Phospholipds

Begin with phospha

Further Details on A'aching the Head Group

Either one of the alcohols is ac

Synthesis of Phospha=dylethanolamine and Phospha=dylcholine in Yeast

Phospha

Phospholipid Synthesis in Mammals Phospha7dylserine isnt synthesized from CDP-diacylglycerol as it is in yeast and bacteria

Made backwards from PE or PC via head group exchange rxns Catalyzed by specific synthases Pathway salvages the choline

Sphingolipids are made in four steps

1) Synthesis of sphinganine from palmitoyl-CoA and serine

2) A=achment of fa'y acid via amide linkage 3) Desatura=on of N-acylsphinganine

(dihydroceramide) Yields N-acylsphingosine (ceramide)

4) A=achment of head group Can yield a cerebroside or ganglioside

ER

Golgi

Phospholipids must be transported from the ER to membranes

Phospholipids are: synthesized in the smooth ER transported to Golgi complex for addi

Four Steps of Cholesterol Synthesis

1) Three acetates condense to form 5-C mevalonate

2) Mevalonate converts to phosphorylated 5-C isoprene

3) Six isoprenes polymerize to form the 30-C linear squalene

4) Squalene cyclizes to form the four rings that are modified to produce cholesterol

Step 1: Forma=on of Mevalonate from Acetyl-CoA

2 Acetyl-CoAs Acetoacetyl-CoA Catalyzed by acetyl-CoA acyl transferase

(thiolase)

Acetyl-CoA + Acetoacetyl-CoA -hydroxyl--methylglutaryl-CoA (HMG-CoA) Catalyzed by HMG-CoA synthase

NOT the mitochondrial HMG-CoA synthase used in ketone body forma

Sta=n drugs inhibit HMG-CoA reductase to lower cholesterol

Sta

Step 2: Conversion of Mevalonate to Two Ac=vated Isoprenes

3 PO43 transferred stepwise from ATP to mevalonate

Decarboxyla

Step 3: Six Ac=vated Isoprene Units Condense to Form Squalene

The two isoprenes join head -to-tail, displacing one set of diphosphates forms10-C geranyl

pyrophopshate

Geranyl pyrophosphate joins to another isopentenyl pyrophosphate forms 15-C farnesyl

pyrophosphate

Two farnesyl pyrophosphates join head-to-head to form phosphate-free squalene

Step 4: Conversion of Squalene to Four-Ring Steroid Nucleus

Squalene monooxygenase adds one oxygen to the end of the squalene chain forms squalene 2,3-epoxide

Here pathways diverse in animal cells vs. plant cells

The cycliza

Conversion of Squalene to Cholesterol

Fates of Cholesterol Aner Synthesis

In vertebrates, most cholesterol synthesized in the liver, then exported: - As bile acids, biliary cholesterol or cholesteryl esters

Other

Bile Acids Assist in Emulsifica=on of Fats

Bile is stored in the gall bladder, secreted into small intes

Cholsteryl esters are more nonpolar than cholesterol

Contain a fa=y acid esterified to the oxygen Comes from a fa=y acyl-CoA Makes the cholesterol more hydrophobic, unable to enter membranes

Transported in lipoproteins to other

Cholesterol and other lipids are carried on lipoprotein par=cles

Lipids are carried through plasma on spherical par

Four Major Classes of Lipoprotein Par=cles

Named based on posi

Electron Microscope Pictures of Lipoproteins

Apolipoproteins in Lipoproteins

Apo for without So apolipoprotein refers to the protein part of a lipoprotein par

Chylomicrons carry fa'y acids to =ssues

Have more TAG and less protein hence, least dense.

Have ApoB-48, ApoE, and ApoC-II

ApoC-II ac

Chylomicron remnants deposit their cholesterol in the liver

When chylomicrons are depleted of their TAG, remnants go to liver

ApoE receptors in liver bind the remnants, take them up by endocytosis

Remnants release their cholesterol in the liver

VLDLs transport endogenous lipids

Cholesteryl esters and TAGs from excess FA and cholesterol are packed into very low-density lipoproteins (VLDL)

Excess carbohydrate in the diet can also be made into TAG in the liver and packed into VLDL

Contain apoB-100, apoC-I, apoC-II, apoC-III, and apoE

VLDLs take TAGs to adipose =ssue and muscle

Again, ApoC-II ac

VLDL remnants become LDL

Removal of TAG from VLDL produces LDL Because TAG removed, LDL is enriched in cholesterol/chloesteryl esters

ApoB-100 is the major apolipoprotein

LDLs carry cholesterol from liver to muscle and adipose =ssue

Muscle and adipose

Cholesterol Uptake by Receptor-Mediated Endocytosis

Familial hypercholesterolemia is associated with LDL receptor muta=ons

Muta

HDL carries out reverse cholesterol transport

HDLs contain a lot of protein Including ApoA-I and lecithin-cholesterol acyl transferase (LCAT) Catalyzes the forma

Five Modes of Regula=on of Cholesterol Synthesis and Transport

1) Covalent modifica

Regula=on of Cholesterol Metabolism

HMG-CoA reductase is most ac=ve when dephosphorylated

1) AMP-dependent protein kinase - when AMP rises, kinase phosphorylates the enzyme ac

Longer-term Regula=on of HMG-CoA Reductase through Transcrip=onal Control

Sterol regulatory element-binding proteins (SREBPs) When sterol levels are high, SREBP is in ER membrane with other proteins

When sterol levels decline, complex is cleaved, moves to the nucleus

SREBP ac

Regula=on of Cholesterol Synthesis by SREBP

Regula=on of HMG-CoA Reductase by Proteoly=c Degrada=on

Insig (insulin-induced gene protein) senses cholesterol levels. Binds to HMG-Co-A reductase, Triggers ubiquina

Cardiovascular disease (CVD) is mul=-factorial

Very high LDL-cholesterol levels tend to correlate with atherosclerosis Although many heart a=ack vic

How Plaques Form

LDL with partly oxidized fa=y acyl groups s

How Plaques Form (cont.)

Foam cells undergo apoptosis Remnants accumulate, along with scar

Familial Hypercholesterolemia

Due to gene

Reverse cholesterol transport by HDL explains why HDL is cardioprotec=ve

HDL picks up cholesterol from non-liver

Reverse Cholesterol Transport

Ques=on 7 (Take home exam) Due: NEXT WEEK (js=ban@birzeit.edu)

Please solve ques=ons: 1. 6 (uncouplers) 2. 17 (ATP turnover) 3. 22 (alanine) 4. 24 (diabetes) For wri[en answers, I prefer to have them typed in Word. I can accept the assignment in one file sent to my email. For answers that require solving mathemaEcally, you can either type them or write them down and scan them.