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Ch 17-19: Lipids

1) 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

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

saturated

unsaturated

1. Triacylglycerols (TAGs)

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 palmitate ! or C16 fatty acid compared to glucose?)

1. Lipids As Energy
Energy yields: 1 g fatty acid = 9 kcal 1 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

1. Metabolic Orientation

1. Digestion
TAGs cannot be absorbed by the intestine. Bile Acids:
emulsication of lipids in the small intestine; gall bladder production

Intestinal Lipases:
TAGs to free fatty acids and monoglycerides
exogenous = diet endogenous = synthesized

1. Bile Acids
Problem: TAGs are water insoluble and digestive enzymes are water soluble Bile acids: amphipathic detergent like molecules - emulsify lipids; and essential for downstream processing

Gallbladder removal patients: difculty digesting lipids

Action of bile salts in emulsifying fats in the intestine:


bile acid ionizes to its cognate bile salt

1. Bile Acids

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

1. Intestinal Lipase
hydrolyzes fatty acid from TAG

Keep in mind: what the fatty acid is depends on the composition of the TAG

Most lipids are absorbed as free fatty acids and 2-monoglycerides, but also small fraction as free glycerol and diglycerides.

1. Intestinal Lipase
the hydrolysis reaction
409258ad.2.jpg (JPEG Image, 600x567 pixels)

(think back to serine proteases)

http://www.nature.com/nature/journal/v409/n6817/images/4092...

How do you move a hydrophobic molecule into a hydrophilic one?

1. Intestinal Lipase

Lipase forms a complex with co-lipase, and is only functional at the water-fat interface.

1. Interfacial Catalysis
Co-lipase: prevents inhibition resulting from binding of bile acids to lipase notice the lid movement protein-lipid interaction opens the lipase active site

1. Absorption
Bile acids and fatty acid binding proteins allow transport of FA micelles across the mucosa of the intestinal wall.

1. Chylomicrons

Mucosal cells convert FA to TAG and package them into chylomicrons.

1. Transport - Lipoproteins
Lipoproteins are lipidprotein complexes that allow movement of apolar lipids through aqueous environments.

Generalized structure of a plasma lipoprotein: The spherical particle, part of which is shown, contains neutral lipids in the interior and phospholipids, cholesterol, and protein at the surface.

1. Transport - Lipoproteins
LDL cartoon

type of apolipoprotein varies with lipoprotein

1. Apolipoproteins
Coating the surface of lipoproteins - amphipathic helices

apolipoprotein A-1

1. Transport - Lipoproteins
The Five Classes of Lipoproteins

Chylomicrons: diet-derived lipids to body cells Very Low Density Lipoprotein (VLDL): synthesized lipids from the liver to body cells Intermediate and Low Density Lipoproteins (IDL/LDL): cholesterol round the body High 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

1. Chylomicrons
Mucosal cells convert FA to TAG and package them into chylomicrons. These are delivered to target tissues by blood/ lymph system.

1. Chylomicrons

ApoC-II (lipoprotein lipase) hydrolyzes TAG to FA

apoC-II

1. VLDL - Endogenous Lipids

Endogenous lipids are packaged into VLDL and delivered to target tissues.

1. LDL & HDL


Delivery of cholesterol by LDL to target tissues HDL transport cholesterol back to the liver (for conversion to bile acids)

2. Fatty Acid Breakdown

2. Key Chemistry
The 6 Modication Reactions

how to make/break C-C bonds?

2. Key Chemistry
Assembly and Breakdown Reactions

thioester

in fatty acid synthesis, KS typically uses acyl-acyl carrier protein (ACP) in other related chemistry, KS uses acyl-CoA in place of ACP

2. Carrier Molecules
Coenzyme A vs. Acyl-Carrier Protein (ACP)
~20

2. Carrier Molecules
ACP vs. CoA
ACP: either free or integral to an enzyme ~20

phosphopantetheine attachment site (a serine)

MW = 8800 Da

MW = 767 Da

An Item of Interest
Roy Vagelos: The Role of the Acyl Carrier Protein in Fatty Acid Synthesis
The transacylases, condensing enzyme (KAS), and reductase (ER) were rst characterized by the Vagelos lab 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

2. Fatty Acid Breakdown


Three Stages and Three Systems

1. The "-oxidation process (acetyl-CoA & NADH/FADH2) 2. Oxidation of acetyl groups in the TCA cycle 3. Electrons from 1 & 2 pass to O2 via transport chain for ATP synthesis

2. Fatty Acid Breakdown


The Basic Steps
1. 2. 3. 4. 5. Mobilization of TAG (hormone-sensitive lipase) Activation (linkage to CoA) Transport into the mitochondria "-oxidation (removal of 2C units) Into the TCA cycle and on to the electron transport chain

2. Fatty Acid Breakdown


The Basic Steps
1. 2. 3. 4. 5. Mobilization of TAG (hormone-sensitive lipase) Activation (linkage to CoA) Transport into the mitochondria "-oxidation (removal of 2C units) Into the TCA cycle and on to the electron transport chain

2. Fatty Acid Breakdown


1. Mobilization of TAG to FA

2. Fatty Acid Breakdown

2. Fatty Acid Breakdown


The Basic Steps
1. 2. 3. 4. 5. Mobilization of TAG (hormone-sensitive lipase) Activation (linkage to CoA) Transport into the mitochondria "-oxidation (removal of 2C units) Into the TCA cycle and on to the electron transport chain

2. Fatty Acid Breakdown


2. Activation
Synthesis of Fatty Acyl-CoA by Acyl-CoA Synthetase Fatty Acid + CoA + ATP -> acyl-CoA + AMP + PPi C-terminal domain N-terminal domain

Multiple isoforms with varied specicity to accept fatty acids of varied lengths
NOTE: synthetase means ATP consumed; synthase uses other means

2. Fatty Acid Breakdown


2. Activation
Acyl-CoA Synthetase two-step reaction - adenylation and nucleophilic attack

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

2. Fatty Acid Breakdown


2. Activation
Acyl-CoA Synthetase moving a fatty acid into a soluble CoA molecule

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

2. Fatty Acid Breakdown


2. Activation
Synthesis of Fatty Acyl-CoA by Acyl-CoA Synthetase moving a fatty acid into a soluble CoA molecule

The opened binding tunnel conveys the fatty acid into the enzyme

2. Fatty Acid Breakdown


2. Activation
Synthesis of Fatty Acyl-CoA by Acyl-CoA Synthetase moving a fatty acid into a soluble CoA molecule

Release of pyrophosphate after the formation of a fatty acyl-AMP (adenylated intermediate) & CoA binding

2. Fatty Acid Breakdown


2. Activation
Synthesis of Fatty Acyl-CoA by Acyl-CoA Synthetase moving a fatty acid into a soluble CoA molecule

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

2. Fatty Acid Breakdown


The Basic Steps
1. 2. 3. 4. 5. Mobilization of TAG (hormone-sensitive lipase) Activation (linkage to CoA) Transport into the mitochondria "-oxidation (removal of 2C units) Into the TCA cycle and on to the electron transport chain

2. Fatty Acid Breakdown


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

2. Fatty Acid Breakdown


3. Across the Membrane ...

1. acyl group to carnitine 2. transport to the matrix 3. acyl group to CoA 4. carnitine returned to cytosol

2. Fatty Acid Breakdown


3. Across the Membrane ...

The core beta-oxidation reactions are in the mitochondria ...

2. Fatty Acid Breakdown


The Basic Steps
1. 2. 3. 4. 5. Mobilization of TAG (hormone-sensitive lipase) Activation (linkage to CoA) Transport into the mitochondria "-oxidation (removal of 2C units) Into the TCA cycle and on to the electron transport chain

2. Fatty Acid Breakdown


4. "-oxidation

Goals: 1) generate Acetyl-CoA 2) repeat the cycle to continue generation of acetyl-CoA!

2. Fatty Acid Breakdown


4. Acyl-CoA Dehydrogenase
multiple isoforms with distinct specicity: short (C4-C6) medium (C6-C10) long (C10-C12) very long (C12-C18+)

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

2. Fatty Acid Breakdown


4. Enoyl-CoA Hydratase

add a hydroxyl group and a proton to the unsaturated beta-carbon recall: fumarase (TCA)

2. Fatty Acid Breakdown


4. Hydroxyacyl-CoA Dehydrogenase

oxidation of the alcohol to a ketone via NAD+

2. Fatty Acid Breakdown


4. Thiolase

the value of the ketoacyl group

2. Fatty Acid Breakdown


4. Thiolase
ketoacyl-CoA <-> acetyl-CoA + fatty acyl-CoA (C-2) two forms of thiolase 1) degradative (breakdown of ketoacyl) 2) biosynthetic (formation of ketoacyl)

2. Fatty Acid Breakdown


4. Thiolase
ketoacyl-CoA <-> acetyl-CoA + fatty acyl-CoA (C-2) the chemical logic of a degradative thiolase 1) use the ketoacyl structure to break off an acetyl group 2) reform a shorter acyl-CoA

2. Fatty Acid Breakdown


4. Thiolase, rst half ...
active site cysteine

thioesterenzyme intermediate enolate

2. Fatty Acid Breakdown


4. Thiolase, second half ...
enolate

active site base provides proton CoA enters & thiol exchange

2. Fatty Acid Breakdown


The Basic Steps
1. 2. 3. 4. 5. Mobilization of TAG (hormone-sensitive lipase) Activation (linkage to CoA) Transport into the mitochondria "-oxidation (removal of 2C units) Into the TCA cycle and on to the electron transport chain

2. Fatty Acid Breakdown


5. links to electron transport & TCA

link acyl-CoA dehydrogenase to electron transport regeneration of FAD is coupled to ATP synthesis

into TCA

2. Fatty Acid Breakdown


What if a fatty acid has an odd-number of carbons?

convert to succinyl-CoA (TCA)

2. Fatty Acid Breakdown


What if the fatty acid is unsaturated (i.e., contains double bonds)?

2. Fatty Acid Breakdown

What#s the worth?


how much energy comes from palmitate or a C16 fatty acid?

2. Fatty Acid Breakdown


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

2. Fatty Acid Breakdown


Where does this happen in cells?
In mammals, the beta-oxidation enzymes are in the mitochondria, but very long chain (C22+) fatty acids require peroxisomes

peroxisome: an organelle containing specialized enzymes that oxidize different molecules (fatty acids & amino acids). Generation of hydrogen peroxide during some reactions (catalase to convert H2O2 to H2O and O2)

2. Fatty Acid Breakdown


"-oxidation in peroxisomes
1. Very Long Chain Acyl-CoA Synthetase - links the fatty acid to a CoA molecule 2. Transport by a peroxisomal carnitine acyltransferase 3. Oxidation cycle requires and additional peroxisomal enzymes: acyl-CoA oxidase and peroxisomal thiolase capable of handling very long fatty acid chains

acyl-CoA oxidase

Major differences: 1) oxidation in peroxisomes is not coupled to ATP synthesis instead electrons are transferred to oxygen (yields hydrogen peroxide) 2) No TCA enzymes, so acetyl-CoA is exported

bifunctional hydratase/ dehydrogenase

thiolase

2. Fatty Acid Breakdown


"-oxidation across organisms
In mammals: mostly in mitochondria but with specialized enzymes in peroxisome for very long chain FA In plants: the peroxisomes are the major site of fatty acid breakdown. Not for energy, but for the synthesis of glucose as a biosynthetic molecule (sucrose, amino acids, energy, nucleotides, metabolites) In bacteria: the beta-oxidation enzymes are soluble/cytosolic

2. Fatty Acid Breakdown


also very different organization of the four activities: 1. acyl-CoA dehydrogenase 2. enoyl-CoA hydratase 3. hydroxyacyl-CoA dehydrogenase 4. thiolase
Gram-positive bacteria Gram-negative bacteria

bifunctional hydratase/dehydrogenase

2. Fatty Acid Breakdown


also very different organization of the four activities: 1. acyl-CoA dehydrogenase 2. enoyl-CoA hydratase 3. hydroxyacyl-CoA dehydrogenase 4. thiolase

the plant peroxisomal system uses a tetrafunctional enzyme that includes the bifunctional hydratase/ dehydrogenase and two auxiliary enzymes (an epimerase and isomerase) needed to deal with multiple C=C bonds

3. Ketone Bodies

3. Ketone Bodies
In mitochondria of liver and kidney, acetyl-CoA derived from fatty acid oxidation can be converted to "ketone bodies"

transported from the liver used as an energy source in brain and skeletal muscle (fasting conditions)

3. Ketogenesis
conversion of acetyl-CoA to "ketones"
During fasting or starvation (low carbohydrate intake) oxaloacetate levels fall & lowers flux through citrate synthase elevated acetyl-CoA

acetyl-CoA + acetyl-CoA liver acetoacetate acetone

hydroxybutyrate

acetoacetate target acetyl-CoA + acetyl-CoA

3. Acetoacetate
biosynthetic

only in liver mitochondria (does not compete with cholesterol synthesis)

3. Hydroxybutyrate

spontaneous (slow) excreted/exhaled

acetone + CO2

ketosis: acetoacetate produced faster than it is metabolized. acetone breath - a symptom of diabetes

3. Back to Acetyl-CoA

degradative

3. Acetyl-CoA
key intermediate between fat and carbohydrate metabolism

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

4. Fatty Acid Biosynthesis

4. Fatty Acid Biosynthesis


The Basic Steps
1. 2. 3. 4. Transport of acetyl-CoA into the cytosol (mammals) Production of malonyl-CoA: acetyl-CoA carboxylase Synthesis of fatty acids: fatty acid synthase (7 reactions) Esterication into triacylglycerides

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

4. Fatty Acid Biosynthesis


The Basic Steps
1. 2. 3. 4. Transport of acetyl-CoA into the cytosol (mammals) Production of malonyl-CoA: acetyl-CoA carboxylase Synthesis of fatty acids: fatty acid synthase (7 reactions) Esterication into triacylglycerides

4. Transport Systems
recall gluconeogenesis
dicarboxylate transporters

4. Citrate Transport
moving acetyl-CoA from mitochondrion to cytosol

TCA cycle

used for FA synthesis

used for FA synthesis

4. Tricarboxylate Transport

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

4. Plants - It's the Chloroplast


Chloroplast: fatty acid synthesis Endoplasmic Reticulum: fatty acids to TAGs
TAG = triacylglycerols DAG = diacylglycerol PA = phosphatidic acid LPA = lysophosphatidic acid FA = fatty acid

4. Fatty Acid Biosynthesis


The Basic Steps
1. 2. 3. 4. Transport of acetyl-CoA into the cytosol (mammals) Production of malonyl-CoA: acetyl-CoA carboxylase Synthesis of fatty acids: fatty acid synthase (7 reactions) Esterication into triacylglycerides

4. Acetyl-CoA Carboxylase
rst committed step: making malonyl-CoA

biotin-carboxylase active site

carboxytransferase active site

similar mechanistic sequence as pyruvate carboxylase

4. Acetyl-CoA Carboxylase
In bacteria and plant chloroplasts ... 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)

Mammals, fungi, plant cytosol One large multifunctional ACCase

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) to ! promote phosphorylation and inhibition ! (energy demand -> breakdown of stored lipids) insulin enhances activity by promoting dephosphorylation ! (glucose -> insulin release -> fuel storage/FA synthesis)

4. Fatty Acid Biosynthesis


The Basic Steps
1. 2. 3. 4. Transport of acetyl-CoA into the cytosol (mammals) Production of malonyl-CoA - acetyl-CoA carboxylase Synthesis of fatty acids - fatty acid synthase (7 reactions) Esterication into triacylglycerides

4. Fatty Acid Synthase


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

4. Fatty Acid Synthase


the logic: generate a ketoacyl group and then reduce it to -CH2-CH2& repeat (opposite of the oxidation reactions)

4. Fatty Acid Synthase


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

4. Fatty Acid Synthase


MAT: malonyl/acetyl-CoA/ACP transacylase ACP: either free or integral to an enzyme

~20

CoA

phosphopantetheine attachment site (a serine)

MW = 8800 Da

MW = 767 Da

4. Fatty Acid Synthase


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

priming the ketosynthase

feeding the elongation reactions

acetyl- and malonyl-CoAs have distinct purposes

4. Fatty Acid Synthase


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

4. Fatty Acid Synthase


2. KS: extending the primed acetyl group

priming the ketosynthase elongation

4. Fatty Acid Synthase


priming the ketosynthase with an acetyl group

4. Fatty Acid Synthase


2. KS: extending the primed acetyl group decarboxylation of the ketoacid

enolate

4. Fatty Acid Synthase


decarboxylation of malonyl-ACP(CoA) elongation of the chain

4. Fatty Acid Synthase


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

4. Fatty Acid Synthase


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

4. Fatty Acid Synthase


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

4. Fatty Acid Synthase


6-7. repeat and release fatty acid - thioesterase

4. Fatty Acid Synthase (FAS) Organization


In microbes and plants, each FAS reaction catalyzed by an isolated protein (including ACP)

in bacteria, cytosolic monofunctional FAS proteins in plants, chloroplast-localized monofunctional proteins the differences in FAS has pharmaceutical applications

4. Organization of FAS

4. Organization of FAS
macromolecular complexes in fungi (yeast) and mammals

Nenad Ban (ETH-Zurich)

4. Organization of FAS
2500 kDa (6 alpha; 6 beta) 2 x 534 kDa

4. Fungal FAS
central wheel with two domes

4. Fungal FAS each chamber with


3 double-tethered ACP

4. Fungal FAS - the ACP

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

4. Fungal FAS - ACP into KS

4. Mammalian FAS
2500 kDa 2 x 534 kDa

two open reaction chambers: one side (top) for modifying reactions & the other (bottom) for condensation reactions

4. Mammalian FAS

4. Oxidation vs. Synthesis

4. Odds & Ends


FAS gets to palmitate (C16): longer chain fatty acids need specialized "elongases" or ELO proteins and to introduce unsaturation (C=C): desaturases membrane bound di-iron proteins

4. Esterication

a series of acyl transferases to generate phosphatidic acid

from PA to membrane lipids or triacylglycerides

5. Regulation
"-oxidation vs. synthesis
oxidation is controlled by the rate of TAG hydrolysis in ! adipose tissue by hormone-sensitive lipase glucagon stimulates the lipase by cAMP-dependent ! phosphorylation (i.e., more FA & oxidation increases) insulin - reduces cAMP levels and inactivates the lipase ! (i.e., TAG synthesis increases)

5. Regulation
2. controlled by serum FA levels

1. cAMP activates hormonesensitive lipase

Notice the reciprocal action of cAMP

6. Membrane Lipids

6. Phospholipids
Types of Phospholipids

An "average mammalian cell" contains ... 45-55% Phosphatidyl Choline ! ! ! 5-10% sphingolipids 15-25% Phosphatidyl Ethanolamine 5-10% Phosphatidyl Serine 1-2% Phosphatidic Acid

6. Phospholipids
Phosphatidylcholine
phosphocholine

head group lipid tail 40% to 60% of phospholipid in animals & plants major phospholipid circulating in plasma choline not made by animals requires dietary sources or recycling

6. Phospholipids

Activation of the "Head Group" to yield PtdE & PtdC

6. Phospholipids

"Head Group" exchange PtdEA -> PtdSer

7. Cholesterol Synthesis

all 27 carbons of cholesterol are derived from acetyl-CoA

7. Cholesterol Synthesis
1) Acetyl-CoA conversion to HMG-CoA 2) 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)

7. Cholesterol Synthesis
1) Acetyl-CoA conversion to HMG-CoA - cytosol (recall the mito. ketone bodies pathway)

key precursor

7. Cholesterol Synthesis
2) HMG-CoA to isopentenyl pyrophosphate via mevalonate The rate-limiting step in cholesterol synthesis is HMG-CoA reductase

NOTE: there is a non-mevalonate pathway to IPP in bacteria (not in mammals) - 'antibiotic' target. only discovered in the 1990's

7. Cholesterol Synthesis
2) HMG-CoA Reductase Inhibitors: the Statins KmHMG-CoA: ~4 uM Kistatins: ~10 nM

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

7. Cholesterol Synthesis
3) condensation of 6 isoprenes to squalene (C30) b. prenyltransferase C5 C5

C10

Note: both DMAP & IPP are needed

7. Cholesterol Synthesis
3) condensation of 6 isoprenes to squalene (C30) b. prenyltransferase (carbocation intermediate)
removal of PPi from DMAP generates carbocation

7. Cholesterol Synthesis
C5 C5

C10 C5 C15 C15

C30

7. Cholesterol Synthesis
4) squalene cyclization to lanosterol a. epoxide formation

7. Cholesterol Synthesis
4) squalene cyclization to lanosterol b. carbocation cascade - squalene cyclase

epoxide formation carbocation cascade

7. Cholesterol Synthesis
5) processing to cholesterol

bile acids steroids (androgens, progestins, estrogens)

7. Cholesterol Regulation
HMG-CoA Reductase
short-term: phosphorylation to yield a less active enzyme(AMP-dependent kinase) long-term: control enzyme amount in cell (main control!) Gene Expression regulated by the SREBP system
SREBP = Sterol Regulatory Element Binding Protein (regulatory domain and a helix-loop-helix (bHLH) domain SCAP = SREBP cleavageactivating protein (sterolsensing domain)

7. Cholesterol Regulation
high cholesterol - SREBP/SCAP ! in ER as an inactive proform
SRE = sterol regulatory element SREBP = SRE binding protein (regulatory domain and a helix-loop-helix (bHLH) domain SCAP = SREBP cleavageactivating protein (sterolsensing domain)

7. Cholesterol Regulation
low cholesterol - activation of transcription factor and HMG-CoA gene expression
SRE = sterol regulatory element SREBP = SRE binding protein (regulatory domain and a helix-loop-helix (bHLH) domain SCAP = SREBP cleavageactivating protein (sterolsensing domain) low cholesterol ! 1) vesicle transport of ! SREBP/SCAP to Golgi ! 2) proteolysis by S1P ! releases N-terminal ! 3) proteolysis by S2P ! releases bHLH transcription ! factor from the membrane ! 4) binding to SRE and gene ! induction

ER to Golgi

7. Cholesterol Disorders
a) cholesterol transport and efux depends on liver LDL receptors and ABC ! transporter b) familial hypercholesterolemia (high blood cholesterol) - absence of LDL ! receptors [or high dietary intake can suppresse LDL receptor synthesis] c) Tangier disease (high cell cholesterol; low HDL) - lack of efux

8. Organelle Summary