lipid metabolism student edition 7/3/14 version lipid metabolism student edition 7/3/14 version...

Lipid Metabolism Student Edition

7/3/14 version

Pharm. 304 Biochemistry

Fall 2014

Dr. Brad Chazotte 213 Maddox Hall

[email protected]

Web Site: http://campbell.edu/faculty/chazotte

Original material only ©2014 B. Chazotte

http://campbell.edu/faculty/chazotte

LIPID METABOLISM GOALS

• Learn how lipids in vertebrates are transported and enter metabolic pathways from the diet

• Learn how fatty acids undergo β-oxidation • Learn how fatty acids are biosynthesized.• Understand the control of fatty acid metabolism and apply

it to metabolic regulation• Ketone body anabolism and catabolism• Have a general knowledge of the synthesis of other lipids• Learn the metabolism of cholesterol

A Very Brief Recap from Previous Lipid Lectures

Voet, Voet, & Pratt 2013 Table 9-1Horton et al 2012 Fig 9.2

Lipids Are A Diverse Class of Compounds

Biologically Relevant Fatty Acids

Summary of Lipid Metabolism

Horton et al., 2012 Figure 16.XX

Lipid Digestion, Absorption & Transport

Voet, Voet, and Pratt., 2013 Page 678

Triacylgylcerols

Comprise ~90% dietary lipids

Major form of energy storage in humans – highly reduced structures

Hydrophobic molecules

Consist of glycerol triesters of fatty acids

Lipid Sources• fats obtained from the diet• fats obtained from lipid droplet storage in cells, e.g. adipocytes• fats synthesized in one organ for transport to another

Lehninger, 2005 Figure 17.1, 17.2

Overview of Dietary & Storage Lipid Processing in Vertebrates

Voet, Voet, and Pratt, 2013 Figure 20.1

Major Bile Salts

Chylomicron

Lehninger, 2005 Figure 17.3

Mobilization of Traiacylgycerols Stored in Adipose Tissue

Need 2012 figure! Missing other lipases)

Berg, Tymoczko & Stryer 2012 Figure 22.7

Destinations of Storage Triacylglycerol Catabolism

Glycerol Oxidation



Glycerol released from the catabolism of triacylglycerol is phosphorylated by glycerol kinase

The resulting glycerol-3-P converted by glycerol-3-P dehydrogenase to dihydroxyacetone phosphate.

Triose phosphate isomerase does the final conversion to D-glyceraldehyde-3-P which proceeds through glycolysis or, if needed, gluconeogenesis

Where does glycerol from triacylglyerols go?

Voet, Voet, and Pratt, 2013 Table 20-1

Human Plasma Lipoprotein’s Major Classes Characteristics

Voet, Voet, and Pratt, 2013 Figure 20-5Lehninger, 2005 Figure 17.2

Voet, et al., 2008 Figure 20-7

Overview of Plasma Triacylgycerol & Cholesterol Transport in Humans


Cholesterol is transported in the blood via lipoprotein particles. HDL are involved in cholesterol transport from the tissues to the liver.VLDLs, IDLS, and LDLs are involved in transport of cholesterol from the liver to the tissues via the circulatory system and they are synthesized in the liver


Low Density Lipoprotein (LDL)Receptor-mediated Endocytosis

• Receptors synthesized on ER• Processed by Golgi• Inserted into plasma membrane and

located at clathrin-coated pits• Upon binding LDL receptors bud in to

form clathrin-coated vesicles• Vesicles fuse with endosome after

depolymerization of clathrin coat• Endosome pH (~5.0) causes

dissociation of LDL and receptor• Receptors concentrate in membrane

and LDLs concentrate in vesiclular portion

• Endosome fuses with lysosome• Cholesterol either to ER or converted

to cholestryl ester


Subcellular Location of Lipid Metabolism

Voet, Voet, and Pratt, 2013 Figure 20-10

Fatty Acids Must Be “Activated”


Fatty Acid Activation Mechanism Acyl-CoA Synthetase Rx

• Acyl-CoA Synthetases (thiokinases) specific for short, intermediate, & long carbon chain FA’s catalyze the formation of a thioester linkage between fatty acid COOH group and CoA thiol group. Two step reaction.

• Fatty acyl CoA hydrolysis ∆G’˚ = -31 kJ mol-1 • Synthesis ∆G’˚ = -34 kJ mol-1 with formation driven

by hydrolysis of PPi.

• Fatty acyl- CoA esters formed at cytosolic side of mitochondrial outer membrane.

• Transported into mitochondria for energy production (oxidation) or used in cytosol for membrane lipid syntheses.

Intermediate

Voet, Voet , and Pratt, 2013 Figure 20-11

Fatty Acids Need to be Transported into the Mitochondrion


Voet, Voet, and Pratt, 2013 Page 666

Steps: Esterification to CoA (cyto.) Transesterification to carnitine

+ transport across IM Transesterification back to

CoA (mito)

Rate-limiting step for fatty acid oxidation in mitochondria and REGULATORY point.

Needed for transmembrane transit of long chain fatty acids, e.g. palmitic acid

Carnitine & Carnitine Palmitoyl Transferase (CPT) Deficiencies

Devlin , ed. 1997 p. 384

Several diseases result from problems in transport of Long Chain –Fatty Acids across mitochondrial inner membrane. Can be due to:• DEFICIENCIES IN CARNITINE

LEVELS Primary: defect in high-affinity PM carnitine

transporter in muscle, kidney, heart & fibroblasts (but not liver). Treat with dietary carnitine

Secondary: associated with β-oxidation defects giving rise to accumulation of high levels acylcarnitines which are excreted

• DYSFUNCTION IN CPT SYSTEM CPT I: few reports hepatic – likely lethal CPT II: mutations with partial loss of activity.

mutations (>90% loss) affect early infancy- periods of fasting precipitate problems

Clinical Symptoms: mild recurrent muscle cramping to severe weakness & death

Clinical Symptoms: muscle weakness during prolonged exercise

Clinical Symptoms: hypoketotic hypoglycemia, hyperammonemia, cardiac malfunction, sometimes death

Fatty Acid Oxidation

Lehninger, 2013 Table 17.1

β-oxidation: process of successive removal of 2-carbon fragments from fatty acids . In mitochondria, also in peroxisomes.ω-oxidation: in liver ER of some vertebrate species - starts at “opposite” end of FA. Minor pathway in mammals unless β-oxidation is defective.


Stages of Fatty Acid Oxidation

Ultimately energy is obtained from the complete oxidation of a fatty acid which proceeds via electron transport and oxidative phosphorylation to yield ATP

Voet, Voet & Pratt 2013 Figure 20-12

β-Oxidation Pathway for Fatty acyl-CoAs (Overview)

Comprised of 4 Reactions:1. Acyl Co A Dehydrogenase (AD):

causes the formation of a trans-α-β double bond

2. Enoyl CoA hydratase (EH) hydrates the double bond to form 3-L-hydroxyacyl CoA

3. 3-L-hydroxyacyl CoA dehydrogenase (HAD) drives an NAD+-dependent dehydrogenation to form a β-ketotacyl-CoA

4. β-ketotacyl-CoA thiolase (TK) or thiolase, causes a cleavage of a Cα-Cβ in a thiolysis reaction to produce acetyl-CoA and a new 2-carbon fewer acyl CoAThe C2-C3 (α-β) bond is relatively

stable – need to make it less stable to break it. Part of β-oxidation rationale.

Acyl-CoA DehydrogenasesFour acyl-CoA dehydrogenases (isozymes) in mitochondria:• SCAD short chain acyl CoA dehydrogenase specificity

highest for C4 to C6

• MCAD medium chain acyl CoA dehydrogenase specificity higher for C6 to C10

• LCAD long chain acyl CoA dehydrogenase specificity higher for C12 to C18

• VLCAD very long chain acyl CoA dehydrogenase

1st Reaction step in β-oxidation.

Acyl-CoA dehydrogenases send reducing equivalents to the ETF dehydrogenase on the mitochondrial inner membrane and from there to ubiquinone in electron transport. This route in β-oxidation constitutes a major energy flux.

Acyl-CoA Dehydrogenase Deficiencies

MCAD Deficiency has also been found in 10% of sudden infant death syndrome (SIDS) . Declines in glucose catabolism a while after eating leads to an increase in fatty aid oxidation. Imbalance between glucose and fatty oxidation in such infants may give rises to SIDS Voet, Voet & Pratt, 2013 p.668

Either SCAD, MCAD, and LCAD can be deficient as a result of an autosomal recessive genetic defect and impair β-oxidation.

MCAD deficiency is one of the best characterized. Typically manifests itself within 2 years of birth, usually after 12+ hours of fasting. Symptoms can include: vomiting, lethargy and frequently coma accompanied by hypoketoic hypoglycemia and dicarboxylic aciduria. .Devlin 1997 p.385

Voet Voet & Pratt 2013 p.. 668; Devlin 1997, p. 385

β-Oxidation (1): acyl-CoA dehydrogenase Rx


• Dehydrogenation to form a trans double bond.

• Bond is α-β to the carbonyl, i.e. between C2 and C3 (∆2).

• Reducing equivalents are transferred via FAD to the ETF dehydrogenase in the mitochondrial inner membrane – hence to electron transport

• Product: called trans-∆2-Enoyl-CoA

~stable bond

Mitochondrial Trifunctional Protein (TFP)(For long chain fatty acids)

1. β-oxidation steps 2-4 also involve chain-length dependent (short, medium & long) enzymes

2. For fatty acids of 12 or more carbons• TFP heterooctamer of α4β4 subunits;

each α subunit contains an enoyl-CoA hydratase & β-hydroxyacyl-CoA dehydrogenase each β subunit has the thiolase activity• Permits efficient substrate channeling 3. Fatty acids less than 12 carbons are then handled by soluble

enzymes in the mitochondrial matrix

β-Oxidation (2): Enoyl-CoA Hydratase Rx


Three systems of isozymes enoyl-CoA-hydratase that are chain length dependent (short, medium, or long chain).Reaction involves the addition of water to the double bond.Forms the L stereoisomer.

β-Oxidation (3): 3-L-hydroxyacyl-CoA Dehydrogenase Rx


Dehydrogenation Rx

Enzyme absolutely stereospecific for L-stereoisomer of hydroxyacyl CoA

NAD is the electron acceptor

β-Oxidation (4): β-ketoacyl-CoA thiolase Rx


Rx yields an acetyl CoA and an acyl CoA with two carbons less than the previous cycle.

Now β-ketoacyl-CoA C2-C3 bond much less stable due to two carbonyls.

A Claisen ester cleavage Has acetyl CoA carbanion

intermediate.


Various Fatty Acid Types Must be Catabolized Somewhat Differently

Unsaturated Fatty Acids

Odd Chain Fatty Acids

Saturated Fatty Acids

Horton et al., 2013 Chapter 9

“normal” β-oxidation via 2-carbon acetyl CoA fragments

Problem: double bonds

Problem: removing 2-carbon fragments eventually left with 3-carbon fragment

Solution: Two additional reactionsVoet: 4 additional enzymes

Solution: Three additional reactions to convert to citric acid cycle intermediate


The β-Oxidation Pathway


Oxidation of Unsaturated Fatty Acids

Enoyl CoA isomerase converts the cis ∆3 to a trans ∆2.

NADPH-dependent 2,4-dienoyl CoA reductase reduces the ∆4 double bond. Mammalian enzyme yields trans-3-enoyl CoA

3,2-enoyl-CoA isomerase (mammals) reversibly converts ∆2 & ∆3 bonds

3,4-2,4 dienoyl-CoA isomerase isomerizes the 3-5 diene to a 2,4 diene.

2,4 dienoyl-CoA reductase reduces the double bond

3,2-enoyl-CoA isomerase isomerizes the double bond

Goal: convert a molecular structure to one catabolizble by normal β-oxidation enzymes


β-oxidation of odd-numbered fatty acid (Proprionyl-CoA oxidation )

Problem: Successive removal of acetyl-CoA 2-carbon fragments for odd chain fatty acids eventually leaves a 3-carbon fragment, propionyl-CoA.Solution: Convert propionyl-CoA to succinyl-CoA, a citric acid cycle intermediate via 3 enzymes

NOTE: In order for the succinyl-CoA to undergo a NET OXIDATION in the citric acid cycle it must first be converted to pyruvate and then acetyl-CoA


Comparison of Mitochondrial β-oxidation to that in

Peroxisomes

Peroxisomal β-Oxidation Involved in shortening very long (>22 carbons) fatty acids that are subsequently degraded via the mitochondrion. Only slightly different than mitochondrial β-Oxidation



ω-Oxidation of FA in the ER Found in Vertebrates – located in ER

of liver and kidney. Preferred substrates: 10 or 12 carbon. Works from opposite end of fatty acid

compared to β-oxidation Normally a minor pathway in

mammals Can be important if β-oxidation is

defective

Ketone Bodies & Ketogenesis



• Acetyl-CoA can be converted to acetoacetate or D-β-

hydroxybutyrate called “ketone bodies”. “Alternate fuels” • Occurs in liver mitochondria (matrix) – supplied to

peripheral tissues, e.g. heart & skeletal muscle, also brain

under special conditions.

1. Acetyl-CoA acetyl transferase condenses 2 acetyl CoA’s to acetoacetyl-CoA.

2. HMG-CoA synthase condenses a 3rd acetyl CoA to form β-hydroxy-β-methylglutaryl-CoA

3. HMG-CoA lyase degrades HMG-CoA into acetoaetate and acetyl CoA

Acetoacetate underoges nonenzymatic degradation to acetone & CO2.


Ketone Boyd Formation and Export from the Liver


Metabolic Conversion of Ketone Bodies to acetyl CoA

Metabolism of ketone bodies in PERIPHERAL tissues.D-β-hydroxybutyrate is converted to acetoacetate and produces NADH.The acetoacetate product (also a ketone body) is converted to acetoacetyl-CoA and also yields “free” succinate. Two acetyl-CoA’s are produced by the action of thiolase.

The acetyl-CoA’s are available for use in the peripheral tissues.

Fatty Acid Biosynthesis

• Occurs mainly in liver and adipocytes (mammals)

• FA synthesis and degradation occur by two completely separate pathways

• When glucose is plentiful, large amounts of acetyl CoA are produced by glycolysis and can be used for fatty acid synthesis

• Glucose oxidation in the pentose phosphate pathway provides NADPH for FA synthesis


Comparing Fatty Acid β-oxidation and Fatty Acid Biosynthesis

Different: locations acyl carrier group electron donor/acceptor hydration/dehydration Rx

stereochemistry form of C2 units

produced/donated

By being “different” each can be thermodynamically favorable. Each can be separately regulated.



Tricarboxylate Transport System One of reasons needed:

mitochondrial inner membrane impermeable to acetyl-CoA.

When low ATP demand: acetyl-CoA oxidation and oxidative phosphorylation are low. Save mitochondrial acetyl-CoA as fat.

Must transport acetyl-CoA out of the mitochondrion to store as fat.

Transport acetyl-CoA out as citrate


Biosythesis of Fatty Acids: Reaction Sequence Overview

• 7 reactions located in cytosol• Palmitic acid is a main product• Reactions are endergonic & reductive• ATP used for energy, • Reduced electron carries, e.g., NADPH, for

reductive power

Voet, Voet, and Pratt, 2013 Figure 20-27a

Mammalian Fatty Acid Synthase:

Enzyme Structure


Acetyl CoA Carboxylase

1st committed step in fatty acid biosynthesis – irreversible processRate controlling step – allosteric & hormonal control

OXYGENASES

Oxygenases: catalyze oxidative reactions in which oxygen atoms are directly incorporated into the substrate molecule forming a new hydroxyl or carboxyl group.Dioxygenases: both oxygen atoms are incorporated into the organic moleculeMonooxygenases: (more abundant & complex actions) one oxygen incorporated into the organic molecule and the other reduced to water. They require two substrates to serve as reductants. Also called hydroxylases since in most reactions the main substrate becomes hydroxylated. Sometime called mixed-function oxygenases – oxidize two different subsrates

simultaneously. Mixed-Function Oxidases (monooxygenases), e.g. cytochrome P-450 (most numerous & complex monooxygenases). Different classes of monooxygenases depending on nature of cosubstrate: FMNH2 or FADH2 used by some

NADH or NADPH used by some α-ketoglutarate used by some

Lehninger 2005 Chap 21 p 798

Voet, Voet, and Pratt, 2013 Figure 20-26 part 1

Biosythesis of Fatty Acids: Reaction Sequence I

Acyl Carrier Protein (ACP) esterified to acyl group instead of CoA during synthesis of fatty acid. Priming Reaction:Rx1: carried out by malonyl/acetyl CoA-ACP transacylase [MAT]. Initiated with malonyl-CoA. Transfers malonyl CoA to ACP Likewise acetyl-CoA transferred to ACP for cyclic additions. First “Cycle” Reaction:Rx2: β-ketoacyl-ACP synthase [KS] “condensing enzyme”

Where carbanion will form

Where carbanion will attack

To start initial synthesis of afatty acid

Biosythesis of Fatty Acids:

Reaction Sequence II


Rx3: β-ketoacyl-ACP reductase [KR] use NADPH’s reductive power to convert a carbonyl to a hydroxyl group

Rx4: β-hydroxyacyl-ACP dehydrase [DH] removes water to form a double bond beta to the carbonyl group.

Rx5: enoyl-ACP [ER] reductase uses the NADPH’s reductive power to reduce the double bond.

Reload ACP with malonyl group each cycle to be able to add 2-carbon fragment

Biosythesis of Fatty Acids: Reaction Sequence III


Rx6: palmitoyl thioesterase [TE] After completion of the cyclic “growth” the mature/complete fatty acid is removed from ACP by hydrolyzing the thioester bond.

Overall process: 8 acetyl-CoA + 7 ATP + 14 NADPH +14 H+

→ palmitate + 8 CoA + 7 ADP + 7Pi + 14 NADP+ + 6H2O


Fatty Acid Elongation• FA elongase system present in mito & ER – (but

are different)• Mito elongation a reverse of β-oxidation (but

last Rx uses NADPH) Palmitate precursor of longer chain fatty acids Stearate (C18) a major product

Mitochondrial


Terminal Desaturases to produce Unsaturated Fatty Acids


Electron Transfer in Desaturation

Fatty acyl-CoA desaturase

Note: Plants have greater ability to insert double bonds than animals. Thus those plant polyunsaturated fatty acids are Essential Fatty Acids and must be in an animal’s diet

Palmitate and stearate serve as precursors for the two most common animal monounsaturated fatty acids: palmitoleate (C 16:1 ∆9) and oleate (C 18:1 ∆9)

Mixed function oxidases needed forfatty acid desaturation.

Mammalian Terminal Desaturases

Four terminal desaturases of broad chain-length specificity∆9-, ∆6-, ∆5-, and ∆4- fatty acyl-CoA desaturases Generic reaction below right: For the desaturases to function “x” must be ≥ 5 and (CH2)x can contain one or more double bonds but (CH2)y portion is always saturated.Double bonds inserted between existing double bonds in (CH2)x portion and CoA so that the new double bond is 3-carbons closer to the CoA, i.e. not conjugated.Note: In animals never beyond position C9.



Routes of Synthesis of Other Fatty AcidsMammals cannot convert oleate to linoleate or α-linolenate - hence they are ESSENTIAL FATTY ACIDS in the mammalian diet

NOTE: Linoleic acid (∆9,12 –octadecadienoic acid) is a required precursor for prostaglandin and other eicosanoids and is an essential fatty acid.


Lipid Metabolism: Summary

Fatty Acid Metabolism Regulation

Fatty acid oxidation and synthesis regulated by hormones and cellular factors


Fatty Acid Metabolism: Regulation SitesFatty acid oxidation is largely regulated by fatty acids concentration in the blood.- controlled by rate of triglyceride hydrolysis in adipose tissue via hormone-sensitive triacylglycerol lipase (lower figure) .Glucagon/Insulin ratio determines the rate and direction of fatty acid metabolism.During FA biosynthesis malonyl-CoA inhibits CPT1 (mito)– reducing FA oxidation. AMPK activated by AMP and inhibited by ATP. When active it phosphorylates ACC inactivating ACC thus decreasing malonyl-CoA concentration.


Citrate role:Central to diverting cellular metabolism from metabolic fuel oxidation to storage as fatty acids. When mitochondrial acetyl-CoA & ATP increase citrate is transported out of the mitochondrion. Becomes a precursor for acetyl-CoA & allosteric activator for acetyl-CoA carboxylase; inhibits phosphofructokinase-1 reducing glycolysis.

Hormonal Regulation SummarizedFed state: Insulin (levels increase)

• Inhibits hydrolysis of stored Triglycerides

• Stimulates formation of malonyl CoA, which inhibits CPT I

• Fatty acids remain in cytosol (Fatty acid oxidation enzymes are in the mitochondria)

Fasted state: Epinephrine and glucagon increase (insulin decreases)

• Epinephrine activates lipase enzyme to produce more fatty acids

• Glucagon inactivates malonyl CoA synthesis enzyme (leads to increased transport of fatty acids into mitochondria and the b-oxidation pathway) Horton et al 2013 Chapter 16

Gene Expression in Long-Term Regulation of Fatty Acid Metabolism

Stimulation by insulin• Adipose tissue lipoprotein lipase

amount increased by insulin• fatty acid synthesis• fatty acid storage in adipocytes

Inhibition by starvation • acetyl-CoA carboxylase

synthesis and fatty acid synthase amounts decreased

• lipoprotein lipase amount decreased

• fatty acid synthesis• fatty acid storage in adipocytes


Gene expression

Gene expression

Triacylglycerol Biosynthesis


Mito & ER

ER & Peroxisomes

• Synthesized from glycerol & fatty acyl-CoA esters

• In mitochondrion and ER Glycerol-3-P comes from DHAP using a single step

• In peroxisomes from DHAP in a 3-step sequence

• Glyceroneogensis (part of gluconeogenesis) necessary during starvation to provide glycerol.

• Triacylglycerol 1st stage is the acylation of the two free hydroxyl groups of glycerol-3-P by fatty acyl-CoAs to yield diacylglycerol-3-P.

• Diacylglycerols converted to triacylglycerol by transesterification with 3rd fatty acyl-CoA

Rate of triacylglycerol biosynthesis is affected by several hormones. Insulin promotes the conversion of carbohydrate to triacylglycerol

Regulation of Triacylglycerol and Glyceroneogenesis

Lehninger, 2013 Figure 21-19. Lehninger, 2013 Figure 21-22.

Insulin stimulates conversion of dietary carbohydrates and proteins to fat. Diabetics (low insulin secretion or action) have diminished FA synthesis and acetyl CoA from catabolism shunted to ketone body production.

Glucocorticoid hormones stimulate glyceroneogenesis & gluconeogenesis in liver while suppressing glyceroneogenesis in adipose tissue.

Thiazolidinediones used to treat type-2 diabetes- activate a nuclear receptor PPARγ which induces activity of PEP carboxykinase; the latter largely controls the flux through triacylglycerol cycle in liver –limits rate of glyceroneogensis & gluconeogenesis

Phospholipid & Sphingolipid Syntheses

• Lipids synthesized mostly on cytosolic side of ER• 1,2 diacylglycerol and phosphatidic acid are the

precursors of most glycerophospholipids • Palmitoyl-CoA and serine are the precursors of

sphingolipids.


General Structures


Plasmogen & Alkylacylglycerophospholipid Structures

(Eukaryotic membrane lipids)

Sphingolipids & C20 Lipid Syntheses

Sphingolipids Most are glycolipids (carbohydrate polar headgroups) Includes cerebrosides and gangliosides (Lipid Lectures) Built from palmitoyl-CoA and serine

C20-based lipids Prostaglandins, leukotrienes, thromboxane, lipoxin (Lipid lectures) Derivatives of C20 compounds such as arachidonic acid



Ceramide Biosynthesis4 Steps Synthesized by the attachment of carbohydrates units to the C1-hydroxyl of ceramide

1. 3-Ketosphinganine synthase – condenses palmitoyl-CoA with serine

2. 2-Ketosphinganine reductase – uses NADPH to reduce keto group on 3-ketosphinganine

3. Acyl-CoA transferase - transfers an acyl group from acyl-CoA to form an amide bond wit sphinganine’s 2-amino group

4. Dihydroceramide dehydrogenase – uses an FAD-dependent oxidation to convert dihydroceramide to ceramide


Sphingomyelin StructureAn important structural lipid in nerve cell membranesFormed from the reaction of

phosphatidylcholine with the C1-OH group of N-acylsphingnosine.

Eicosanoids

Lehninger 2005 Figure 10.18b


Prostaglandin H2 Synthase Rx

Eicosanoid Synthesis

Eicosanoids: * Potent short range signaling molecules * Hormonal or other stimuli trigger phospholipase A2

* phospholipase A2 releases arachidonic acid from middle carbon of membrane phospholipid glycerol

Formation of prostaglandin H2 (PGH2) first step (precursor of many prostaglandins and thromboxanes)

1. 1st step catalyzed by cyclooxygenase (COX) prostaglandin H2 synthase inserts molecular O2 to form PGG2

2. 2nd step catalyzed by COX’s peroxidase activity to form PGH2 COX enzymes affected by NSAIDS!

Voet, Voet, and Pratt, 2013 Page 699a

Aspirin & Two NSAID’s Inhibition of Cyclooxygenase Rx in Prostaglandin Synthesis

Aspirin – Acetylates a specific Ser residue of prostaglandin H2 synthase which blocks arachidonate from the active site. (Effects on heart attacks and strokes too.)

NSAIDS – bind noncovalently to the enzyme and also block the enzyme active site: e.g. acetaminophen & ibuprofen

Acetaminophen, one of the most commonly used analgesics/antipyretic, in acutality does not bind well to COX-1 and COX-2 (also is not an anti-inflammatory agent). Actually effects “COX-3” which is expressed at high levels in the CNS – target of drugs deceasing fever & pain

Lehninger, 2005 Box 21.2 Figure 2a

COX-1 COX-2

Voet, Voet, and Pratt, 2013 Page 699b

Two NSAID COX-2 Inhibitors of Prostaglandin H2 Synthesis(Vioxx and Celebrex)

Withdrawn 2004 Use more restricted now

Chemists designed inhibitors to COX-2 called coxibs based on 3-D structures of COX-1 & COX-2 . Analgesics

Have not well understood cardiac side effects.

Were considered important for treating inflammatory diseases viz. arthritis

COX-1: constitutively expressed in most mammalian tissues to maintain prostaglandin synthesis needed to maintain homeostasis in organs and tissues.COX-2: expressed only in certain tissues in response to certain stimuli – responsible for elevated PG levels that cause inflammation. COX-3: high expression levels in CNS, re: acetaminophen

COX Isoforms and NSAIDS

Bextra withdrawn ~2004

THROMBOXANESThromboxane synthase (in platelets) coverts PGH2 to thromboxane A2.From thromboxane A2 others are produced.

Thromboxanes contain a ring of 5 or 6 atomsThromboxanes induce blood vesicle constriction and platelet aggregation.Regular low doses of aspirin reduce thromboxane production – reduces heart attack and stroke risk


Arachidonate to Leukotrienes(Brief Overview)

• Leukotrienes are linear compounds. Their synthesis begins with the action of several lipooxygenases that catalyze molecular oxygen incorporation.

• Enzymes are found in leukocytes, heart, brain, ling and spleen are mixed function oxidases of the cytochrome P450 family.

• (Not inhibited by aspirin or NSAIDS.)

Voet, Voet, and Pratt, 2013 Box 20-4 figure 1

Spingolipid Degradations and Lipid Storage Diseases: Diagram

Cholesterol Metabolism

“In a healthy organism, an intricate balance is maintained between biosynthesis, utilization, and transport of cholesterol, keeping harmful deposition to a minimum.”

Voet, Voet and Pratt, 2013 p. 701


Summary of Cholesterol Biosynthesis

There are four major stages:1. Condensation of 3 acetate units to

form mevalonate.2. Conversion of mevalonate to

activated isoprene units.3. Polymerization of six 5-carbon

isoprene units to form 30-carbon linear squalene molecule.

4. Cyclization of squalene to form the four rings of the steroid nucleus with additional modifications


Made from acetyl-CoA


1. Synthesis Mevalonate from acetyl-CoA

Two molecules of acetyl-CoA are condensed to form acetoacetyl-CoA

A third molecule of acetyl-CoA is condensed with acetoacetyl-CoA to form β-hydroxy-β-methylglutaryl-CoA (HMG-CoA)

The reduction of MHG-CoA to mevalonate is a committed step that utilizes the electrons from 2 NADPH molecules..Major regulation point in cholesterol synthesis!.

HMG-CoA: a key cholesterol precursor.

Voet, Voet and Pratt, 2013 Figure 20-37

2. Conversion of mevalonate to activated isoprene units

Isopentenylpyrophosphate formation from HMG-CoA


In stage 2 three Pi groups are transferred from ATPs.

The 1st phosphorylation of the newly added OH group is performed by mevalonate-5-phosphotransferase.

Phosphomevalonate kinase converts the added Pi to PPi.

An ATP-dependent decarboxylation occurs via pyrophosphomevalonate decarboxylase yielding ∆3-isopentyl pyrophosphate (an activated isoprene).

Isoprene carbon skeleton


Pyrophomevalonate decarboxylase reaction in Cholesterol

intermeidate biosynthesis


Intermediate Reactions in Squalene Biosynthesis


3. Formation of Squalene from Isopentenyl pyrophosphate & dimethylallyl phosphate

Head has PPi

Activated isoprenes

2nd head-to tail condensation of geranyl pyrophosphate with isopentenyl phosphate to form C15 farnesyl pyrophosphate

1st head-to tail condensation to produce C10 geranyl pyrophosphate

Head to Head condensation of two farnesyl pyrophosphates by squalene synthetase to form squalene.

4. Conversion of Squalene to the Four Ring Steroid Nucleus


Squalene Epoxidase Reaction



Conversion of Lanosterol to Cholesterol

19 steps!


Lanosterol Conversion to Cholesterol in 19 (simple?) Steps

Transported from the liver in lipoprotein complexes

acyl-CoA:cholesterol acyltransferase

Overview of Isoprenoid Biosynthesis


Regulation of Cholesterol Synthesis

HMG-CoA reductase – rate limiting step for cholesterol biosynthesis – main regulatory site of pathway!!-short-term regulation:

competitive inhibition, allosteric effects, reversible covalent modification by phosphorylation hormones effecting short-term phosphorylation: Glucagon stimulates phosphorylation (inactivation) Insulin promotes dephosphoylation (activation)-long-term regulation:

feedback control of the amount of enzyme present in the cell “transcriptional regulation”*PRIMARY regulatory mechanism*

STATINS INHIBIT!


Cholesterol-mediated Proteolytic Activation of SREBP

• Cholesterol’s cellular concentration effects HMG-CoA reductase transcription.

• HMG-CoA reductase gene + 20 other genes encoding enzymes mediate the uptake and synthesis of cholesterol & unsaturated fatty acids.

Sterol Regulatory Element-Binding Proteins

[Cholesterol] high: SREBP-SCAP remains in ER

[Cholesterol] low: SCAP “escorts” SREBP to the Golgi via COPII-coated vesicles. In Golgi SREBP cleaved by proteases S1P and S2P – SREBP N-terminal domain goes to nucleus and interact with target genes’ SREs

[Cholesterol] = low

[Cholesterol] =high

Some Steroid Hormones Derived from Cholesterol



Competitive Inhibitors of HMG-CoA Reductase for Hypercholesterolemia Treatment

STATINSStatins all contain HMG-like group – competitive inhibitor have much lower Km than regular substrate Bulky hydrophobic groups interfere with HMG-CoA reductase

Regular substrate

(lactones- active as hydroxy acids)


AtherosclerosisAtherosclerotic Plaque in a Coronary Artery

• Slow progressive disease• Deposition of lipid in large blood vessel walls.• Initial event may be association of lipoproteins with

vessel wall proteoglycans• Trapped lipids trigger inflammatory response with

endothelial cells to express adhesion molecule to monocytes

• Monocytes burrow into vessel wall & become macrophages and engorge (oxidized) lipids – “foam cells”

• Foam cells attract more WBC• Vessel forms a plague – cholesterol, cholesterol

esters and dead macrophages surround by smooth muscle cells. (the latter can undergo calcification)

• Restricts or can block blood flow

THE END “So long, farewell, auf wiedersehen,

good-bye” Oscar Hammerstein II

Have a nice life!

lipid metabolism student edition 7/3/14 version lipid metabolism student edition 7/3/14 version...

Documents