Biochemistry - Lipids

Where does Beta-oxidation occur?

The mitochondrial matrix

What is required before a fatty acid can be oxidozed?

Activation via acyl-coA synthase of a fatty acid to fatty acyl-CoA which uses 2 ATP

How are long chain fatty acids transported into the mitochondria from the cytosol?

Carnitine shuttle: via carnitine acyltransferase I - translocase - carnitine  acyltransferase II

What are the 4 recurring steps of beta-oxidation?

1. Oxidation (FADH2 formed)

2. Hydration

3. Oxidation (NADH formed)

4. Thiolysis (CoA in, acetyl-CoA out)

What is the net yield of Palmitate (16:0)"?

106 ATP (8 acetyl-CoA > TCA; + 7FADH2 and 7NADH from beta-oxidation)

What is the final product of Odd chain fatty acid oxidation?

Propionyl-CoA (3C) converted to Succinyl-CoA via vit. B12

Why do we need extra enzymes when it comes to oxidation on unsaturated FA?

Natural double bonds are cis, beta-oxidation requires trans Δ2 - so need isomerase and reductase 

Where does fatty acid synthesis occur?

Cytosol

How does acetyl-CoA get from the mitochondria to the cytosol?

exported as citrate in the citrate shuttle (acetyl-CoA + OAA > citrate > crosses > broken back down to acetyl-CoA

What is the committed step of fatty acid synthesis?

Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA (requires ATP and biotin)

What is the main elongating unit in FA synthesis?

Malonyl-CoA - adds 2C per cycle

What reducing power is required for FA synthesis?

NADPH (from the pentose phosphate pathway or malic enzyme)

What enzyme complex carries out FA synthesis?

Fatty acid Synthase (FAS) - has 7 catalytic domains on one polypeptide

What are the 4 recurring steps of FA synthesis?

1. condensation

2. reduction (NADPH)

3. Dehydration

4. Reduction (NADPH)

What is the main product of FA synthesis?

palmitate (16:0)

What are the cofactors of beta-oxidation compared to FA synthesis?

Beta-oxidation: NADH and FADH2

FA synthesis: NADPH

What are the substrates of beta-oxidation vs. FA synthesis?

Oxidation: fatty acyl-CoA

synthesis: acetyl-coA and malonyl-CoA

What is the energy requirements of beta-oxidation vs. synthesis?

oxidation: yields ATP (catabolic)

synthesis: requires ATP for malonyl-CoA (anabolic)

Acyl carrier protein (ACP)

FAS1 domain that carries reaction intermediates from one active site to the next

Malonyl/acetyl - CoA-ACP transferase (MAT)

FAS1 domain that catalyzes the transfer of acetyl and malonyl groups from coA to FAS1

KR (beta-ketoacyl-ACP reductase)

FAS1 domain that catalyzes reduction of beta-ketobutyryl ACP

ER (enoyl-ACP reductase)

FAS1 domain that catalyzes the reduction of trans-delta2-butenoyl-ACP

TE (Thioesterase)

Domain of FAS1 that catalyzes the release of palmitate from FAS1 by hydrolysis (end step)

DH (beta-hydroxyacyl-ACP dehydratase)

Domain of FAS1 that catalyzes dehydration of delta-beta-hydroxybutyryl ACP

KS (beta-ketoacyl-ACP synthase)

FAS1 domain that Catalyzes condensation of acetyl and malonyl groups

Common properties of naturally occurring fatty acids

• Not soluble in water

• saturated = solid at room temp

• unsaturated = liquid at room temp

• amphipathic molecule: carboxylic acid is hydrophilic, fatty acid tail is hydrophobic

• reduced form of carbon (why it produces more energy)

Structure of TAGs

• 3 fatty acids each ester linked to the glycerol backbone

• The simplest lipids constructed from fatty acids

What are the main functions of TAGs

• energy storage in adipocytes

• insulation

What are the advantages of storing energy as TAGs?

• carbon atoms of FA are more reduced than those of sugars, so oxidation of TAGs yields more than twice as much energy per gram

• They are unhydrated so fat is easier to store because you don’t have to carry the extra weight of water of hydration that is associated with stored polysaccharides

What are the 3 general classes of structural lipids and their subclasses?

1. Phospholipids - glycerophospholipids and sphingolipids

2. Glycolipids - sphingoglycolipids

3. sterols

Glycerophospholipids general structure

• Fatty acids joined through ester linkage to C1 and C2 of the glycerol backbone

• glycerol-3-phosphate (glycerol = C3H8O3 - attached to a phosphate group)

• polar head group joined to phosphatidic acid through a phosphodiester linkage

• head group can be neutral, negatively charged or positively charged

Sphingolipids 

• large class of membrane glycolipids and phospholipids

• derived from the compound sphingosine 

• A fatty acid is attached at C2 by an amide linkage to form the parent compound ceramide

General structure of Sphingolipids

• Fatty acid joined through amide linkage to C2 of glycerol backbone

• Sphingosine (look for o, N, OH and unsaturated fatty acid)

• polar head group joined through glycosidic linkage or phosphodiester linkage

What are the 3 subclasses of sphingolipids?

1. Sphingomyelins: head group is phosphocholine or phosphoethanolamine

2. Glycosphingolipids: head group contains one or more sugars bound to ceramide in glycosidic linkage

3. Gangliosides: head group contains complex oligosaccharides

How are sphingolipids involved in cell recognition?

Glycosphingolipids are often enriched on the extracellular surface of the cell membrane and play a role in cell recognition and cell-to-cell communication 

• Also can determine ABO blood type on the surface of erythrocytes

Sterols

• Organic compounds within the general formula C17H28O

• structure contains the rigid steroid nucleus with 4-fused rings

What is the general structure of a biological membrane?

• phospholipids form a bilayer in which proteins are embedded

• the phospholipid bilayer is semipermeable, flexible and self-repairing

• lipids and proteins form a fluid mosaic

• the bilayer exhibits structural and functional asymmetry

Liquid ordered state (L0)

• favored at low temps and by long chain saturated fatty acids 

• All motion of individual molecules is constrained

Liquid disordered state (Ld)

• Favored at high temps by short chain unsaturated fatty acids

• hydrocarbon chains are in constant motion

Why do we want our membrane to be maintained between Lo and Ld states?

• We need the membrane to be fluid enough to transport and allow lateral movement

• Too much movement will make it difficult to maintain a polarized cell

How are lipids distributed on membranes?

Outer leaflet: phosphatidylcholine, sphingomyelin

inner leaflet: phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol

Hormone-sensitve lipase

Enzyme responsible for mobilizing Stored TAGs in adipose tissue by hydrolyzing the TAG to release free fatty acids and glycerol into the blood

Lipoprotein Lipase (LPL)

Enzyme responsible for hydrolyzing the TAGs in circulating lipoproteins (chylomicrons and VLDL) so tissues can take up the released fatty acids

Citrate shuttle

A way to transport acetyl-CoA from the mitochondria into the cytosol

• citrate is transported from the mitochondria to the cytosol for fatty acid synthesis

• ATP-citrate lyase converts citrate to acetyl-CoA + OAA in the cytosol

• The reaction consumes 1 ATP per citrate 

Membrane Rafts

Lipid microdomains that form within the cell membrane

• rich in long, saturated fatty acids, cholesterol and lipidated GPI-anchored proteins

• facilitates transient segregation of specific membrane receptors and signaling proteins

• sit in a more ordered state

Phosphatidylinositol

• Minor component of cytoplasmic side of cell membrane

• phospholipase C cleaves PIP2 to generate second messengers IP3 and DAG

• generates a signal molecule from a membrane lipid

Phosphatidylinositol signaling pathway

1. Agonist binds specific receptor on cell membrane (agonist can be a hormone)

2. Receptor activates associated G-protein (GDP-GTP exchange)

3. Activated G-protein activates PLC

4. PLC cleaves PIP2 generating IP3 and DAG

5. IP3 activates IP-gated Ca2+ channels - releasing Ca2+ 

6. DAG and Ca2+ activate PKC at surface of plasma membrane 

7. PKC phosphorylates cellular targets mediating diverse effects

Eicosanoids

• Autocrine and paracrine signaling molecules

• Synthesized from arachidonate and other polyunsaturated FA

• diverse biological functions involved in reproduction, inflammation, fever, pain, blood clotting, blood pressure, etc

Steroid Hormones

• oxidized derivatives of sterols

• diffuse into target tissues where they bind highly specific nuclear receptors to regulate changes in gene expression

Lipid nanoparticle (LNP)

A particle comprised primarily of lipids and engineered to encapsulate and protect therapeutic payloads for targeted delivery into cells - DNA or RNA

PEGylated lipids

Prevent aggregation and immune activation

Cationic Ionizable lipids

Charged at acidic PH and neutral at physiological PH. Promote the loading of Nucleic acid payloads and release into cytoplasm 

• complexes with the negatively charged mRNA

• see them in covid vaccine and gene therapy 

Lipid Absorption

1. Bile salts emulsify fats into micelles

2. intestinal lipases hydrolyze TAGs to monoacylglycerol and free FAs

3. Monoacylglycerols and free FAs are transported into enterocytes and converted into TAGs

4. TAGs are resynthesized and packaged into chylomicrons for transport to lymph system

Chylomicrons

Transport dietary TAGs to peripheral tissues

• hydrophobic core rich in TAGs

• Hydrophobic surface containing phospholipids and apolipoproteins

How are TAGs from chylomicrons taken in by target tissues?

At target tissues TAGs are hydrolyzed to free FAs and monoacylglycerol by LPL

• LPL is activated by apoC-II

Perilipins

Coat the surface of lipid droplets and are regulatory proteins that function to restrict mobilization of lipids

How are fatty acids transported in blood?

Transported by serum Albumin after being released from adipocytes

• FA dissociate from serum albumin and enter target cells through plasma membrane transporters

Exogenous Pathway

Describes the transport of dietary lipids within chylomicrons to extrahepatic tissues and uptake of remnants in liver

Endogenous pathway

The transport of lipids between the liver and peripheral tissues by VLDL and LDL, once excess lipids have been metabolized to TAG and cholesterol esters within the liver

• The loss of TAG from VLDL produces LDL

• LDL not endocytosed by peripheral tissues returns to liver

Reverse cholesterol transport

The transport of excess cholesterol in extrahepatic tissues back to the liver as HDL

• HDL is produced by the liver and accumulates cholesterol from chylomicron and VLDL remnants

Enterohepatic circulation

The synthesis, secretion and reabsorption of bile salts by the liver and gallbladder

• LCAT catalyzes the formation of cholesterol esters from cholesterol and lecithin

TAG Cycle

The break down and resynthesis of TAGs in a continuous cycle

1. FA released from TAGs in adipose tissue are released into blood

2. some of these FA are used as fuel by peripheral tissues, but most are taken up by the liver

3. FA are resynthesized to TAG in the liver

4. TAGs are transported back to adipose tissue

What are the 3 stages of oxidizing fatty acids?

1. Beta-oxidation - forms acetyl-CoA

2. Citric acid cycle - acetyl-CoA is oxidized to CO2

3. Oxidative phosphorylation - reduced electron carriers are oxidized within the ETC driving the synthesis of ATP

First step of Beta-oxidation

Oxidation: dehydration of fatty-acyl-CoA produces a C=C bond between the alpha and beta carbons producing a trans-delta2-enoyl-CoA

• catalyzed by acyl-CoA dehydrogenase

Second step of beta-oxidation

Hydration: H2O is added across the double bond between alpha and beta carbons to form L-beta-hydroxy-acyl-CoA

• catalyzed by enoyl-CoA hydratase

• beta carbon is now HC-OH

Step 3 of beta-oxidation

Second oxidation: L-beta-hydroxyacyl-CoA is dehydrogenated to form beta-ketoacyl-CoA

• catalyzed by beta-hydroxy-CoA dehydrogenase

• H on beta carbon leaves - beta carbon is now Rc=o

Fourth step of beta-oxidation

Thiolysis: beta-ketoacyl-CoA reacts with a molecule of free CoA forming acetyl-CoA forming acetyl-CoA and acyl-CoA

• catalyzed by acyl-CoA acetyltransferase 

• alpha carbon leaves in the newly formed acetyl-CoA

General equation for beta oxidation (example palmitic acid)

palmitoyl-CoA + CoA + FAD + NAD+ +H2O > Marisotyl-CoA (14C) + Acetyl-CoA + FADH2 =NADH + H+

Where do the ATP come from in the complete Oxidation of palmitate?

• Beta-oxidation: FADH2- 7×1.5 = 10.5 + NADH - 7×2.5 = 17.5

• Citric Acid Cycle: 1 acetyl-CoA produces 10 ATP - 3 NADH produced, 1 FADH2, 1 GTP = 8×10 = 80 ATP

• Total = 108 ATP - 2ATP from Acyl-CoA Synthetase = 106 ATP

How does oxidation of unsaturated fatty acids differ?

Requires 2 additional reactions:

• when the double bond gets to the beta-carbon, it needs to be modified so that the cis double bond is in the trans configuration

• an isomerase repositions the double bond by generating trans-delta2-dodeanoyl-CoA

• will yield less energy - around 1.5 ATP less

How does the oxidation of odd chain fatty acids differ?

Complete oxidation requires 3 extra steps 

• the cleavage of the final 5 carbon product yields acetyl-CoA and propionyl-CoA (3C)

• Propionyl-CoA is metabolized by a separate pathway within the mitochondrial matrix

Propionyl-CoA Metabolism pathway

1. propionyl-CoA is carboxylated to form methylmalonyl-CoA via the activity of HCO3-, biotin and ATP and enzyme propionyl-CoA carboxylase

2. D-methylmalonyl-CoA is epimerized to L-stereoisomer by methylmalonyl-CoA epimerase

3. L-methylmalonyl-CoA is rearranged to Succinyl-CoA using the enzyme methylmalonyl-CoA mutase and coenzyme B12

What has to happen for long chain fatty acids to be oxidized?

Beta oxidation occurs in peroxisomes

• Pathway is very similar to regular beta-oxidation except that the first reaction produces H2O2

• Once acetyl-CoA is formed it is exported to mitochondria 

Citrate shuttle

In the mitochondrial matrix: citrate synthase generates citrate from acetyl-CoA and OAA

• Citrate crosses the mitochondrial membrane via the citrate transporter

Cytosol: citrate lyase generates acetyl-CoA and OAA requiring the use of 1 ATP- OAA carries on to form malate, acetyl-CoA goes to fatty acid synthesis

How is Malate formed from oxaloacetate?

• Malate dehydrogenase generates malate from OAA using NADH (NADH > NAD+)

• malate can cross the mitochondrial membrane and regenerate OAA in the mitochondrial matrix or generate NADPH in cytosol

How is malate used to form NADPH? (primary pathway of malate)

• Malate is oxidized to pyruvate via malic enzyme - this reaction uses NADP+ and produces NADPH and CO2

• pyruvate enters mitochondria via pyruvate transporter 

• pyruvate carboxylase generates OAA from pyruvate requiring ATP

Synthesis of malonyl-CoA from Acetyl-CoA

Requires the enzyme acetyl-CoA carboxylase (ACC)

1. Biotin carboxylase domain catalyes addition of a carboxyl group from HCO3- to biotin within the biotin carrier protein domain - requires ATP to activate bicarbonate

2. Biotin carrier protein transfers carboxyl group to transcarboxylase domain

3. Transcarboxylase domain catalyzes transfer of carboxyl group to acetyl-CoA forming Malonyl-CoA

What are the 3 domains of Acetyl-CoA Carboxylase?

• biotin carboxylase domain

• biotin carrieer potein domain

• Transcarboxylase domain

Cholesterol Esters

• Formed when the hydroxyl group on cholesterol is esterified with a fatty acid

• entirely hydrophobic

• serves as storage and transport forms of cholesterol

• found in the core of lipoproteins

How are fatty acids longer than 16 carbons made?

• Palmitate is the precursor for long chain FA

• palmitate can be lengthened by fatty acid elongation systems

• Does not use ACP, 2C transferred to acetyl-CoA

How are unsaturated fatty acids synthesized?

• palmitate and stearate are precursors to palmitoleate and oleate (both have double bonds between C9 and C10)

• Double bond is introduced by an oxidative reaction catalyzed by fatty acyl-CoA desaturase

Fatty acyl-CoA desaturase

• Enzyme that catalyzes the double bond in unsaturated fatty acid synthesis through an oxidative reaction

• 4 enzymes active at C4, C5, C6, C9

Why are some fatty acids considered essential?

• human enzymes cannot introduce a double beyond C9

• alpha-linolenate 18:3 (9,12,15)

• linoleate 18:2 (9,12)

• important precursors to a wide range of unsaturated fatty acids that function as signaling molecules

Eicosanoids

• A family of autocrine and paracrine signaling molecules derived from fatty acids

• synthesized in response to stimuli such as chemokines and cytokines

Synthesis of eicosanoids

Synthesized mainly from arachidonic acid 

• phospholipase A2 catalyzes hydrolysis of an ester bond releasing a fatty acid from a glycerophospholipid

• rxn: (glycerophospholipid + H2O > lysophospholipid + arachidonate

• COX metabolizes arachidonate to PGH2

• PGH2 is a precursor to other prostaglandins and thromboxanes 

Prostaglandins

vasodilators, mediate inflammation and pain

Thromboxanes

Vasoconstrictors, mediate blood clotting 

Aspirin

• NSAID

• COX inhibitor

• irreversible inhibition

Ibuprofin

• NSAID

• COX inhibitor

• reversible competitive inhibition

Cholesterol

crucial component of cellular membranes, important precursor to steroid hormones and bile acids.

• Synthesized from acetyl-CoA

Cholesterol synthesis

1. Condensation of 3 acetate units to form a 6-carbon intermediate mevalonate

2. conversion of mevalonate to activated isoprene

3. polymerization of six 5-carbon isoprene units to form squalene

4. cyclization of squalene to form the four rings of the steroid nucleus within cholesterol

Stage 1 of cholesterol synthesis

• 2 molecules of acetyl-CoA condense to form acetoacetyl-CoA

• Acetoacetyl-CoA condenses with a third molecule of acetyl-CoA to yield HMG-CoA

• HMG-CoA is reduced to mevalonate

Stage 2 of cholesterol synthesis

• 3 phosphate groups are transferred to mevalonate

• The intermediate 3-phospho-5-pyrophosphomevalonate releases CO2 and phosphate producing an activated isoprene

• Lots of ATP used in this stage

Stage 3 of cholesterol Synthesis

• Two activated isoprenes condense forming geranyl pyrophosphate (10 carbons)

• Geranyl pyrophosphate condenses with another activated isoprene forming farnesyl pyrophosphate (15C)

• Two molecules of farnesyl pyrophosphate condense forming squalene (30C)

Stage 4 of cholesterol Synthesis

• Squalene monooxygenase catalyzes the addition of one oxygen atom from O2 to the end of squalene forming an epoxide

• Cyclization results in the formation of lanosterol which contains the 4 rings characteristic of the steroid nucleus

• Several additional reactions add and reposition methyl groups to form cholesterol

Metabolic fates of cholesterol

• Bile acids

• Cholesterol esters

• Steroid hormones

Regulation of cholesterol synthesis

• Insulin: promotes activation of HMG-CoA reductase

• Glucagon: promotes inactivation of HMG-CoA reductase

• AMPK: promotes inactivation of HMG-CoA reductase

• Oxysterol: promotes proteolysis of HMG-CoA reductase

What is the important point of regulation in cholesterol synthesis?

• Activation of HMG-reductase

• This is the step that statins inhibit

Mechanism of action of statins

• The most widely used drugs for lowering serum cholesterol levels           

• Competitive inhibitors of HMG-CoA reductase resembling the mevalonate

Ketone bodies

• Produced in the liver as an alternative fate for acetyl-CoA

• Major fuel source for the brain when glucose is unavailable because fatty acids cannot cross the blood brain barrier