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Ch 17-19: Lipids1) Digestion, Absorption, Transport

2) Fatty Acid Oxidation (Breakdown)

3) Ketone Bodies

4) Fatty Acid Synthesis

5) Regulation

6) Membrane Lipids

7) Cholesterol Metabolism

Lipid Metabolism

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Marathon - Hitting the Wall muscle 1-2% glycogen by weight; liver cells nearly 10%

(a limited supply)

Depletion of glycogen (and glucose)shift in metabolism - ATP generated from fatty acid oxidation(much slower than glycolysis;

80% of energy in heart and liver)

1. Fatty Acids

saturated unsaturated

18:0 18:1 18:2 18:3

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1. Triacylglycerols (TAGs)

TAG or fat or triglyceride

90% of dietary lipids

the major form of energy storage in mammals

(how much energy comes from a palmitateor C16 fatty acid compared to glucose?)

1. Triacylglycerols (TAGs)

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1. Lipids As Energy

Energy yields:

1 g fatty acid = 9 kcal1 g sugar = 4 kcal

Hydrophobic vs. Hydrophilic consequences:

1 g of glycogen binds 2 g of water, so ...1.33 kcal/g weight

1 g of lipid is 9 kcal/g weight(6x the stored energy of carbohydrate)

1. Lipids As Energy

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1. Metabolic Orientation

1. DigestionTAGs cannotbe absorbedby theintestine.

Bile Acids:emulsification oflipids in the smallintestine; gallbladder production

IntestinalLipases:TAGs to free fattyacids andmonoglycerides

exogenous = dietendogenous = synthesized

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1. Bile Acids

Bile acids: amphipathic detergent like molecules - emulsifylipids; and essential for downstream processing

Gallbladder removal patients:

difficulty digesting lipids

Problem: TAGs are water insoluble and digestiveenzymes are water soluble

1. Bile AcidsAction of bile salts in emulsifying fats in the intestine:

bile acidionizes to its cognate bile salt

The hydrophobic surface of the bile salt associates with TAG. Complexes aggregate as a micelle

The polar surface of the bile salts faces outward -> associates with pancreatic lipase/colipase

Hydrolysis by lipase frees fatty acids -> smaller micelles for intestinal mucosa absorbtion

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How do you move a hydrophobic molecule into ahydrophilic one?

Lipaseforms a complex with co-lipase, and is only functionalat the water-fat interface.

1. Intestinal Lipase

protein-lipidinteractionopens the

lipase activesite

Co-lipase:preventsinhibition

resulting frombinding of bileacids to lipase

1. Interfacial Catalysis

notice the lidmovement

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1. Absorption

Bile acids and

fatty acidbindingproteins allowtransport ofFA micellesacross themucosa of theintestinal wall.

1. Chylomicrons

Mucosal cellsconvert FA toTAG andpackage theminto

chylomicrons.

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1. Transport - Lipoproteins

Lipoproteins are lipidproteincomplexes that allow movement ofapolar lipids through aqueousenvironments.

Generalized structure of a plasmalipoprotein:

The spherical particle, part of whichis shown, contains neutral lipids inthe interior and phospholipids,cholesterol, and protein at thesurface.

1. Transport - Lipoproteins

type of apolipoproteinvaries with lipoprotein

LDL cartoon

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1. ApolipoproteinsCoating the surface of lipoproteins - amphipathic helices

apolipoprotein A-1

1. Transport - LipoproteinsThe Five Classes of Lipoproteins

Chylomicrons: diet-derived lipids to body cellsVery Low Density Lipoprotein (VLDL): synthesized lipids from the liver to body cellsIntermediate and Low Density Lipoproteins (IDL/LDL): cholesterol round the bodyHigh Density Lipoprotein (HDL): cholesterol from the body back to the liver for breakdown and excretion

NOTE: apolipoprotein C-II (a lipase!) in the chylomicron and other lipoproteins

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1. Chylomicrons

Mucosal cellsconvert FA toTAG andpackage themintochylomicrons.

These aredelivered totarget tissuesby blood/lymph system.

1. Chylomicrons

ApoC-II(lipoproteinlipase)hydrolyzesTAG to FA

apoC-II

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1. VLDL - Endogenous Lipids

Endogenouslipids arepackaged intoVLDLanddelivered totarget tissues.

1. LDL & HDL

Delivery ofcholesterol byLDLto targettissues

HDLtransport

cholesterolback to theliver (forconversion tobile acids)

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2. Fatty Acid Breakdown

2. Key Chemistry

The 6 Modification Reactions

how to make/break C-C bonds?

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thioester

Assembly and Breakdown Reactions

in fatty acid synthesis, KS typically uses acyl-acyl carrierprotein (ACP)

in other related chemistry, KS uses acyl-CoA in place of ACP

2. Key Chemistry

Coenzyme A vs. Acyl-Carrier Protein(ACP)

~20

2. Carrier Molecules

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ACP vs. CoA

~20

phosphopantetheineattachment site

(a serine)

MW = 8800 Da MW = 767 Da

ACP: either freeor integral to an

enzyme

2. Carrier Molecules

An Item of Interest

Roy Vagelos: The Role of the Acyl CarrierProtein in Fatty Acid Synthesis

The transacylases, condensing enzyme (KAS), andreductase (ER) were first characterized by the Vageloslab at Washington University. 1966 - Chair of the Dept. of Biological Chemistry 1973 - Division of Biology & Biomedical Sciences 1975 - Merck (1984-1994 CEO) - Invermectin - river

blindness

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2. Fatty Acid BreakdownThree Stages and Three Systems


1. Mobilization of TAG (hormone-sensitive lipase)2. Activation (linkage to CoA)3. Transport into the mitochondria4. "-oxidation (removal of 2C units)5. Into the TCA cycle and on to the electron transport chain

The Basic Steps

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The Basic Steps

1. Mobilization of TAG to FA


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The Basic Steps

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2. ActivationSynthesis of Fatty Acyl-CoA by Acyl-CoA Synthetase

Fatty Acid + CoA + ATP -> acyl-CoA + AMP + PPi


NOTE: synthetase means ATP consumed; synthase uses other means

C-terminaldomain

N-terminaldomain

Multiple isoforms with varied specificity to acceptfatty acids of varied lengths

2. Activation

Formation coupled to cleavage of ATP to AMP and PPi coupled to formation ofa 'high-energy' thioester linkage.


Acyl-CoA Synthetasetwo-step reaction - adenylation and nucleophilic attack

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2. Activation2. Fatty Acid Breakdown

Acyl-CoA Synthetasemoving a fatty acid into a soluble CoA molecule

ATP binding triggers closure of the C-terminal domain& opening of the fatty acid-binding tunnel

The opened binding tunnel conveys the fatty acid into the enzyme



moving a fatty acid into a soluble CoA molecule

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Release of pyrophosphate after the formation of a fatty acyl-AMP

(adenylated intermediate) & CoA binding

2. Fatty Acid Breakdown2. Activation

Synthesis of Fatty Acyl-CoA by Acyl-CoA Synthetasemoving a fatty acid into a soluble CoA molecule

Opening of the N- and C-terminal domains and release of

products



moving a fatty acid into a soluble CoA molecule

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The Basic Steps

3. Across the Membrane ...K ~ 1; energetically O-acyl of carnitine equals thioester of CoA


eq

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1. acyl group to carnitine2. transport to the matrix

3. acyl group to CoA4. carnitine returned to cytosol

2. Fatty Acid Breakdown3. Across the Membrane ...


The core beta-oxidation reactions arein the mitochondria ...

3. Across the Membrane ...

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The Basic Steps

4. "-oxidation

Goals:1) generate Acetyl-CoA

2) repeat the cycle tocontinue generation of

acetyl-CoA!


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multiple isoforms withdistinct specificity:

short (C4-C6)medium (C6-C10)long (C10-C12)

very long (C12-C18+)

4. Acyl-CoA Dehydrogenase

next, from the double-bond ...build up the ketoacyl structure


add a hydroxyl group and aproton to the unsaturated

beta-carbon

4. Enoyl-CoA Hydratase

recall: fumarase (TCA)

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oxidation of the alcohol to aketone via NAD+

4. Hydroxyacyl-CoA Dehydrogenase

4. Thiolase

the value of the ketoacylgroup


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ketoacyl-CoA acetyl-CoA + fatty acyl-CoA (C-2)

two forms of thiolase1) degradative (breakdown of ketoacyl)2) biosynthetic (formation of ketoacyl)

2. Fatty Acid Breakdown4. Thiolase

ketoacyl-CoA acetyl-CoA + fatty acyl-CoA (C-2)

the chemical logic of a degradative thiolase1) use the ketoacyl structure to break off an acetyl group

2) reform a shorter acyl-CoA


4. Thiolase

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4. Thiolase, first half ...2. Fatty Acid Breakdown

active sitecysteine

enolate

thioesterenzymeintermediate


4. Thiolase, second half ...

active sitebase provides

proton

CoA enters &thiol exchange

enolate

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The Basic Steps

5. links to electron transport & TCA

into TCA


link acyl-CoAdehydrogenase to

electron transportregeneration of FAD is

coupled to ATP synthesis

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2. Fatty Acid BreakdownWhat if a fatty acid has an odd-number of carbons?

convert tosuccinyl-CoA

(TCA)

2. Fatty Acid BreakdownWhat if the fatty acid is unsaturated (i.e., contains double

bonds)?

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how much energy comes frompalmitate or a C16 fatty acid?

What#s the worth?


1 round of "-oxidation yields: 1 NADH 1 FADH2 1 acetyl-CoA

1 FADH2 + 3 NADH

For a C16 fatty acid (palmitoyl-CoA) (7 rounds): 7 NADH, 7 FADH2, 8 acetyl-CoA

8 GTP, 24 NADH, 8 FADH2

106 ATP equivalents


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2. Fatty Acid BreakdownWhere does this happen in cells?

In mammals, the beta-oxidation enzymes are in the

mitochondria, but very long chain (C22+) fatty acids requireperoxisomes

peroxisome: an organelle containingspecialized enzymes that oxidizedifferent molecules (fatty acids &

amino acids).

Generation of hydrogen peroxide

during some reactions (catalase toconvert H2O2to H2O and O2)

1. Very Long Chain Acyl-CoA Synthetase - links the fatty acidto a CoA molecule

2. Transport by a peroxisomal carnitine acyltransferase

3. Oxidation cycle requires and additional peroxisomalenzymes:

acyl-CoA oxidaseand peroxisomalthiolasecapable ofhandling very long fatty acid chains

"-oxidation in peroxisomes


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Major differences:

1) oxidation in peroxisomesis not coupled to ATPsynthesis

instead electrons aretransferred to oxygen

(yields hydrogen peroxide)

2) No TCA enzymes, soacetyl-CoA is exported

acyl-CoAoxidase

thiolase

bifunctionalhydratase/

dehydrogenase

"-oxidation across organisms


In mammals:mostly in mitochondria but with specializedenzymes in peroxisome for very long chain FA

In plants: the peroxisomes are the major site of fatty acidbreakdown. Not for energy, but for the synthesis of glucoseas a biosynthetic molecule (sucrose, amino acids, energy,

nucleotides, metabolites)

In bacteria:the beta-oxidation enzymes are soluble/cytosolic

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2. Fatty Acid Breakdownalso very different organization of the four activities:

1. acyl-CoA dehydrogenase2. enoyl-CoA hydratase

3. hydroxyacyl-CoA dehydrogenase

4. thiolase

Gram-positive bacteria Gram-negative bacteria

bifunctional hydratase/dehydrogenase

2. Fatty Acid Breakdownalso very different organization of the four activities:1. acyl-CoA dehydrogenase

2. enoyl-CoA hydratase3. hydroxyacyl-CoA dehydrogenase

4. thiolase

the plant peroxisomal system uses atetrafunctionalenzyme that includes

the bifunctional hydratase/dehydrogenaseand

two auxiliary enzymes (anepimerase and isomerase) needed

to deal with multiple C=C bonds

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3. Ketone Bodies

3. Ketone BodiesIn mitochondria of liver and kidney,

acetyl-CoA derived from fatty acid oxidation can beconverted to "ketone bodies"

transported fromthe liver

used as an energysource in brain and

skeletal muscle(fasting conditions)

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3. Ketogenesis

acetoacetate

acetyl-CoA + acetyl-CoA

acetone

hydroxybutyrate

acetoacetate

acetyl-CoA + acetyl-CoA

liver

target

conversion of acetyl-CoA to "ketones"

During fasting or

starvation (lowcarbohydrateintake)

oxaloacetate levelsfall & lowers fluxthrough citratesynthase

elevated acetyl-CoA

3. Acetoacetate

biosynthetic

only in livermitochondria

(does not compete withcholesterol synthesis)

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3. Hydroxybutyrate

acetone + CO2

ketosis: acetoacetateproduced faster than it is

metabolized.

acetone breath - a symptom

of diabetes

spontaneous

(slow)

excreted/exhaled

3. Back to Acetyl-CoA

degradative

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3. Acetyl-CoAkey intermediate between fat and carbohydrate metabolism

Animals readily convert carbohydrate to fat, but cannot carry outnet conversion of fat to carbohydrate

4. Fatty Acid Biosynthesis

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1. Transport of acetyl-CoA into the cytosol (mammals)

2. Production of malonyl-CoA: acetyl-CoA carboxylase3. Synthesis of fatty acids: fatty acid synthase (7 reactions)4. Esterification into triacylglycerides

The Basic Steps

Keep in mind plants are a little different ... As are many bacteria ... Some pathogens (Mycoplasma genitalium and M.

pneumoniae) are entirely dependent on host for fatty acid


1. Transport of acetyl-CoA into the cytosol (mammals)2. Production of malonyl-CoA: acetyl-CoA carboxylase3. Synthesis of fatty acids: fatty acid synthase (7 reactions)4. Esterification into triacylglycerides

The Basic Steps

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4. Transport Systemsrecall gluconeogenesis

dicarboxylatetransporters

4. Citrate Transport

used for FAsynthesis

used for FAsynthesis

moving acetyl-CoA from mitochondrion to cytosol

TCA cycle

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4. Tricarboxylate Transport

All of the synthesis reactions are inthe cytosol ... (for mammals)

4. Plants - It's the Chloroplast

TAG = triacylglycerolsDAG = diacylglycerolPA = phosphatidic acidLPA = lysophosphatidic acidFA = fatty acid

Chloroplast:fatty acidsynthesis

EndoplasmicReticulum:

fatty acids toTAGs

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1. Transport of acetyl-CoA into the cytosol (mammals)

2. Production of malonyl-CoA: acetyl-CoA carboxylase3. Synthesis of fatty acids: fatty acid synthase (7 reactions)4. Esterification into triacylglycerides

The Basic Steps

4. Acetyl-CoA Carboxylasefirst committed step: making malonyl-CoA

biotin-carboxylaseactive site

carboxytransferaseactive site

similar mechanistic sequence as pyruvate carboxylase

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One large multifunctional ACCase

4. Acetyl-CoA CarboxylaseIn bacteria and plantchloroplasts ...

Mammals, fungi, plant cytosol

Multi-subunit ACCase

Biotin carboxylase (dimer, 2 x 49 kDa)Carboxyltransferase, alpha (dimer, 2 x 35 kDa)Carboxyltransferase, beta (dimer, 2 x 33 kDa)Biotin Carboxyl Carrier Protein

(BCCP) (dimer, 2 x 17 kDa)

4. Acetyl-CoA Carboxylase

citrate allosterically increases activity

long-chain acyl-CoA#s are feedback inhibitors

AMP-dependent kinase phosphorylates to inactivate

glucagon/epinephrine via cAMP-dependent pathway (PKA) topromote phosphorylation and inhibition

(energy demand -> breakdown of stored lipids)

insulin enhances activity by promoting dephosphorylation (glucose -> insulin release -> fuel storage/FA synthesis)

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4. Fatty Acid Synthase

the logic: generate a

ketoacyl group andthen reduce it to-CH2-CH2-

& repeat

(opposite of theoxidation reactions)


the 7 step program ...1. malonyl/acetyl-CoA/ACP transacylase (MAT)2. !-ketoacyl-ACP synthase (KS)3. ketoreductase (KR)4. dehydrase (DH)5. enoyl-reductase (ER)6. repeat steps 2-5, as needed7. thioesterase (TE)

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~20

phosphopantetheineattachment site

(a serine)

MW = 8800 Da MW = 767 Da

ACP: either freeor integral to anenzyme


CoA

MAT: malonyl/acetyl-CoA/ACP transacylase

1. MAT: loading acetyl or malonyl groups from CoAon to the ACP

priming theketosynthase

feeding the elongationreactions


acetyl- and malonyl-CoAs have distinct purposes

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priming theketosynthase with an acetyl group


2. KS: extending the primed acetyl group


enolate

decarboxylationof the

ketoacid

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4. Fatty Acid Synthasedecarboxylation ofmalonyl-ACP(CoA)

elongation of the chain



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3-5. removing the ketone ... KR, DH, ER




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6-7. repeat and release fatty acid - thioesterase



(FAS) OrganizationIn microbes and plants,

each FAS reaction catalyzed by an isolated protein(including ACP)

in bacteria, cytosolic monofunctional FAS proteinsin plants, chloroplast-localized monofunctional proteins

the differences in FAS has pharmaceutical applications

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4. Organization of FAS2500 kDa (6 alpha;

6 beta)2 x 534 kDa

4. Fungal FAScentral wheel with two domes

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4. Fungal FAS

4. Fungal FAS - the ACP

each ACP is tethered,but can access each active site in the complex

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4. Fungal FAS - ACP into KS

2500 kDa 2 x 534 kDa

4. Mammalian FAS

two open reactionchambers:

one side (top) formodifying reactions

&the other (bottom)

for condensationreactions

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4. Mammalian FAS

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4. Oxidation vs. Synthesis

4. Odds & Ends

FAS gets to palmitate (C16): longer chain fatty acids needspecialized "elongases" or ELO proteins

and to introduce unsaturation (C=C): desaturases -membrane bound di-iron proteins

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4. Esterification

a series of acyl transferasesto generate phosphatidic acid

from PAtomembrane lipidsortriacylglycerides

oxidation is controlled by the rate of TAG hydrolysis inadipose tissue by hormone-sensitive lipase

glucagon stimulates the lipase by cAMP-dependentphosphorylation (i.e., more FA & oxidation increases)

insulin - reduces cAMP levels and inactivates the lipase

(i.e., TAG synthesisincreases)

"-oxidation vs. synthesis

5. Regulation

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5. Regulation2. controlled byserum FA levels

1. cAMPactivates

hormone-sensitivelipase

Notice the reciprocalaction of cAMP

6. Membrane Lipids

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6. PhospholipidsTypes of Phospholipids

An "average mammalian cell" contains ...45-55% Phosphatidyl Choline 5-10% sphingolipids

15-25% Phosphatidyl Ethanolamine5-10% Phosphatidyl Serine1-2% Phosphatidic Acid

6. Phospholipids

Phosphatidylcholinephosphocholine

40% to 60% of phospholipid in animals & plants

major phospholipid circulating in plasma

choline not made by animalsrequires dietary sources or recycling

lipid tail

head group

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6. Phospholipids

Activationof the"HeadGroup"to yield

PtdE & PtdC

6. Phospholipids

"Head Group"exchange

PtdEA -> PtdSer

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7. Cholesterol Synthesis

all 27 carbons of cholesterol are derived from acetyl-CoA


1) Acetyl-CoA conversion to HMG-CoA2) HMG-CoA to isopentenyl pyrophosphate via

mevalonate

3) condensation of 6 isoprenes to squalene (C30)

4) squalene cyclization to lanosterol

5) processing to cholesterol (and on to bile acids/steroids)

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7. Cholesterol Synthesis1) Acetyl-CoA conversion to HMG-CoA - cytosol

(recall the mito. ketone bodies pathway)

key precursor

7. Cholesterol Synthesis2) HMG-CoA to isopentenyl pyrophosphate via mevalonate

NOTE: there is a non-mevalonate pathway toIPP in bacteria (not in mammals) - 'antibiotic'

target. only discovered in the 1990's

The rate-limiting step in cholesterol

synthesis is HMG-CoA reductase

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7. Cholesterol Synthesis2) HMG-CoA Reductase Inhibitors: the Statins

KmHMG-CoA: ~4 uM

Kistatins

: ~10 nM

7. Cholesterol Synthesis3) condensation of 6 isoprenes to squalene (C30)a. conversion of IPP to DMAP

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C5 C5

C10

3) condensation of 6 isoprenes to squalene (C30)b. prenyltransferase

Note: both DMAP & IPP are needed

7. Cholesterol Synthesis3) condensation of 6 isoprenes to squalene (C30)b. prenyltransferase (carbocation intermediate)

removal of PPi from DMAP generates carbocation

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7. Cholesterol SynthesisC5 C5

C10

C5

C15

C15

C30

7. Cholesterol Synthesis4) squalene cyclization to lanosterola. epoxide formation

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7. Cholesterol Synthesis4) squalene cyclization to lanosterol

b. carbocation cascade - squalene cyclase

epoxide

formation

carbocationcascade

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7. Cholesterol Synthesis5) processing to cholesterol

bile acidssteroids

(androgens,progestins,estrogens)

7. Cholesterol Regulation

HMG-CoA Reductase

short-term: phosphorylation to yield a less activeenzyme(AMP-dependent kinase)

long-term: control enzyme amount in cell (main control!)

Gene Expression regulated by the SREBP system

SREBP = Sterol RegulatoryElement Binding Protein(regulatory domain and ahelix-loop-helix (bHLH)

domain

SCAP = SREBP cleavage- activating protein (sterol-

sensing domain)

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7. Cholesterol Regulation

SRE = sterol regulatory element

SREBP = SRE binding protein(regulatory domain and ahelix-loop-helix (bHLH)

domain

high cholesterol - SREBP/SCAPin ER as an inactive proform

SCAP = SREBP cleavage- activating protein (sterol- sensing domain)

SRE = sterol regulatory elementSREBP = SRE binding protein

(regulatory domain and ahelix-loop-helix (bHLH)

domainSCAP = SREBP cleavage- activating protein (sterol- sensing domain)

low cholesterol - 1) vesicle transport of

SREBP/SCAP to Golgi 2) proteolysis by S1P

releases N-terminal 3) proteolysis by S2P

releases bHLHtranscriptionfactor from the membrane

4) binding to SREand geneinduction

7. Cholesterol Regulation low cholesterol - activation of transcriptionfactor and HMG-CoA gene expression

ER to Golgi

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a) cholesterol transport and efflux depends on liver LDL receptors and ABCtransporter

b) familial hypercholesterolemia (high blood cholesterol) - absence of LDLreceptors [or high dietary intake can suppresse LDL receptor synthesis]

c) Tangier disease (high cell cholesterol; low HDL) - lack of efflux

7. Cholesterol Disorders

8. Organelle Summary

Documents

9. Lipids Chapter 17-19 (1)