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17| Fa'y Acid Catabolism
© 2013 W. H. Freeman and Company
21| Lipid Biosynthesis
Oxida=on of fa'y acids is a major energy source in many organisms
• About one-‐third of our energy needs comes from dietary triacylglycerols
• About 80% of energy needs of mammalian heart and liver are met by oxida<on of fa=y acids!!
• Many hiberna<ng animals, such as grizzly bears, rely almost exclusively on fats as their source of energy (and water during their long-‐term sleep)
Fats provide efficient fuel storage
• The advantage of fats over polysaccharides: – Fa=y acids carry more energy per carbon because they are more reduced
– Fa=y acids carry less water along because they are nonpolar (aggregate in lipid droplets and are unsolvated)
• Glucose and glycogen are for short-‐term energy needs, quick delivery
• Fats are for long-‐term (months) energy needs, good storage, slow delivery
Fat Storage in White Adipose Tissue
Nuclei
“Squeezed”
Dietary fa'y acids are absorbed in the vertebrate small intes=ne
Emulsification by biological detergents (bile)
Breakdown of TAG to DAG, MAG, FFA and glycerol
Uptake by intestinal cells
Chylomicrons (lipoproteins)
Bloodstream to target tissues
2nd breakdown of TAG
Used for energy (muscles) or reesterified for energy (adipose)
Remaining chylomicrons go to liver and enter by RME à used for ketone bodies synthesis. When diet contains more f.a. than needed, liver converts them to TAG and packages them into VLDL to be transported to adipocytes
Lipids are transported in the blood as chylomicrons
Apoliporpotein + lipids particles = lipoprotein Lipoproteins range in density: VLDL to VHDL
Hormones trigger mobiliza=on of stored triacylglycerols
• Hydrolysis of TAGs is catalyzed by lipases -‐ can produce MAGs, DAGs, FFA and glycerol
• Some lipases are regulated by hormones glucagon and epinephrine Recall: • Epinephrine means: “We need energy now” • Glucagon means: “We are out of glucose”
Hormones trigger mobiliza=on of stored triacylglycerols
• Perilipins – proteins that coat lipid droplets and restrict access to lipids to prevent premature mobiliza<on
• ê[glc]blood è glucagon èè PKA è phosphoryla<on of hormone-‐sensi<ve lipase & perilipin è dissocia<on of CGI and ac<va<on of adipose triacylglycerol lipase monoacylglycerol lipase hydrolyzes MAGs Serum albumin binds up to 10 f.a. noncovalently
Glycerol from fats enters glycolysis
• Only 5% of biologically-‐ac<ve energy of TAG is in glycerol
• Glycerol kinase ac<vates glycerol at the expense of ATP
• Subsequent reac<ons recover more than enough ATP to cover this cost
• Allows limited anaerobic catabolism of fats
Fa'y Acid Transport into Mitochondria
• Fats are degraded into fa=y acids and glycerol in the cytoplasm of adipocytes
• Fa=y acids are transported to other <ssues for fuel • β-‐oxida<on of fa=y acids occurs in mitochondria
• Small (< 12 carbons) fa=y acids diffuse freely across mitochondrial membranes
• Larger fa=y acids (most free fa=y acids) are transported via acyl-‐carni<ne/carni<ne transporter (carni=ne shu'le)
• Three steps:
Conversion of a fa'y acid to a fa'y acyl–CoA (1)
Nucleophilic attack by f.a. anion
Phosphoester linkage between f.a. carboxyl and α phosphate of ATP
Thioester linkage between f.a. carboxyl and thiol group of CoA-SH Hydrolysis of PPi to 2Pi is highly exergonic and pulls the first reaction forward
Acyl-‐Carni=ne/Carni=ne Transport
(2)
(3)
Transesterification to carnitine
Transesterification to CoA
2 separate pools of CoA: Matrix CoA à used mostly in oxidative degradation (pyr, f.a., a.a.) Cytosolic CoA à used in biosynthesis of f.a.
Carnitine-mediated entry is the rate limiting step for oxidation of f.a. in mito
Stages of Fa'y Acid Oxida=on
• Stage 1 consists of oxida<ve conversion of two-‐carbon units into acetyl-‐CoA via β-‐oxida<on with concomitant genera<on of NADH and FADH2
– involves oxida<on of β carbon to thioester of fa=y acyl-‐CoA
• Stage 2 involves oxida<on of acetyl-‐CoA into CO2 via citric acid cycle with concomitant genera<on NADH and FADH2
• Stage 3 generates ATP from NADH and FADH2 via the respiratory chain
Stages of Fa'y Acid Oxida=on
The β-‐Oxida=on Pathway Each pass removes one acetyl moiety in the form of acetyl-‐CoA.
Palmitate (C16) undergoes seven passes through the oxidative sequence
Formation of each acetyl-CoA requires removal of 4 H atoms {2 e– pairs and 4 H+})
Step 1: Dehydrogena=on of Alkane to Alkene
• Catalyzed by isoforms of acyl-‐ CoA dehydrogenase (AD) on the mitochondrial inner membrane – Very-‐long-‐chain AD (VLCAD, 12–18 carbons)
– Medium-‐chain AD (MCAD, 4–14 carbons)
– Short-‐chain AD (SCAD, 4–8 carbons) • Results in trans double bond, different from naturally occurring
unsaturated fa=y acids, between α and β C
• Analogous to succinate dehydrogenase reac<on in the CAC – Electrons from bound FAD transferred directly to the electron-‐ transport
chain via electron-‐transferring flavoprotein (ETF)
Step 2: Hydra=on of Alkene
• Catalyzed by two isoforms of enoyl-‐CoA hydratase: – Soluble short-‐chain hydratase (crotonase) – Membrane-‐bound long-‐chain hydratase, part of trifunc<onal complex
• Water adds across the double bond yielding alcohol
• Analogous to fumarase reac<on in the CAC – Same stereospecificity
Step 3: Dehydrogena=on of Alcohol
• Catalyzed by β-‐hydroxyacyl-‐CoA dehydrogenase • The enzyme uses NAD cofactor as the hydride acceptor
• Only L-‐isomers of hydroxyacyl CoA act as substrates
• Analogous to malate dehydrogenase reac<on in the CAC
• The first three steps create a much less stable C-‐C bond, where the α C is bound to 2 carbonyl groups
Step 4: Transfer of Fa'y Acid Chain
• Catalyzed by acyl-‐CoA acetyltransferase (thiolase) via covalent mechanism – The carbonyl carbon in β-‐ketoacyl-‐CoA is electrophilic – Ac<ve site thiolate acts as nucleophile and releases acetyl-‐CoA
– Terminal sulfur in CoA-‐SH acts as nucleophile and picks up the fa=y acid chain from the enzyme
• The net reac<on is thiolysis of a carbon-‐carbon bond
Trifunc=onal Protein (TFP)
• Hetero-‐octamer – Four α subunits
• enoyl-‐CoA hydratase ac<vity • β-‐hydroxyacyl-‐CoA dehydrogenase ac<vity • Responsible for binding to membrane
– Four β subunits • long-‐chain thiolase ac<vity
• May allow substrate channeling • Associated with mitochondrial inner membrane • Processes fa=y acid chains with 12 or more carbons • Shorter chains are processed by soluble individual enzymes in the matrix
Similar mechanisms introduce carbonyls in other metabolic pathways
Fa'y Acid Catabolism for Energy • For palmi<c acid (C16)
– Repea<ng the above four-‐step process six more <mes (7 total) results in eight molecules of acetyl-‐CoA • FADH2 is formed in each cycle (7 total) • NADH is formed in each cycle (7 total)
• Acetyl-‐CoA enters citric acid cycle and further oxidizes into CO2 – This makes more GTP, NADH, and FADH2
• Electrons from all FADH2 and NADH enter ETC
• Transfer of e–s from FADH2 and NADH to O2 yields 1 H2O per pair (camels and hiberna<ng animals!)
Palmitoyl-‐CoA + 7CoA + 7O2 + 28Pi+ 28ADP à 8 acetyl-‐CoA + 28ATP + 7H2O (β oxida<on) Palmitoyl-‐CoA + 23O2 + 108Pi+ 108ADP à CoA + 108ATP + 16CO2 + 23H2O (full oxida<on)
NADH and FADH2 serve as sources of ATP
Oxida=on of Unsaturated Fa'y Acids • Naturally occurring Unsaturated Fa=y acids contain cis double bonds – Are NOT a substrate for enoyl-‐CoA hydratase
• Two addi<onal enzymes are required – Isomerase: converts cis double bonds star<ng at carbon 3 to trans double bonds
– Reductase: reduces cis double bonds not at carbon 3
• Monounsaturated fa=y acids require the isomerase • Polyunsaturated fa=y acids require both enzymes
Oxida=on of Monounsaturated Fa'y Acids
Oleate (18:1 Δ9)
converted to oleoyl-CoA and imported into mito via carnitine shuttle
Oxida=on of Polyunsaturated
Fa'y Acids
Linoleate (Δ9,Δ12)
First double bond requires isomeriza=on
Second requires reduc=on/isomeriza=on
Oxida=on of odd-‐numbered fa'y acids
• Most dietary fa=y acids are even-‐numbered • Many plants and some marine organisms also synthesize odd-‐numbered fa=y acids
• Propionyl-‐CoA forms from β-‐oxida<on of odd-‐numbered fa=y acids
• Bacterial metabolism in the rumen of ruminants also produces propionyl-‐CoA
• Oxida<on is iden<cal to even-‐numbered long-‐chain fa=y acids, but the last pass through β-‐oxida<on is a fa=y acyl-‐CoA with a 5-‐C fa=y acid that is cleaved to give acetyl-‐CoA and propionyl-‐CoA
Carboxyla=on of Propionyl-‐CoA
Isomeriza=on to Succinyl-‐CoA à CAC
Isomeriza=on in propionate oxida=on requires coenzyme B12
Complex Cobalt-‐Containing Compound: Coenzyme B12
• Very unstable bond • Breaks to yield –CH2
. and Co3+
• Used to transfer the hydrogen atom to a different C in the molecule (isomerization)
• No mixing of the transferred H atom with the hydrogen of the solvent (H2O)
• The formation of this complex cofactor occurs in one of two known reactions that cleaves a triphosphate from ATP
Regula=on of Fa'y Acid Synthesis and Breakdown
Cytosol
• Occurs only when need for energy requires it • 2 pathways for f.a.CoA in liver: TAG synthesis in cytosol or f.a. oxida<on in mito • Transfer into mito is rate limi<ng, once f.a. are in mito they WILL undergo oxida<on
Concn increases when CHO is well-‐supplied Inhibi<on of shu=le ensures oxida<on of f.a. is inhibited when ñenergy
ñ[NADH]/[NAD+] ý ñAcetyl-CoA ý
Gene=c defects in fa'y acyl-‐CoA dehydrogenases
• Inability to oxidize fats for energy has serious effects on health
• More than 20 human gene<c defects in f.a. transport and metabolism occur
• MCAD (medium chain acyl-‐CoA dehydrogenase) deficiency is the most common syndrome in European popula<ons
-‐ Unable to oxidize f.a. of 6 – 12 Cs -‐ If diagnosed aoer birth, the infant can be treated with low fat, high carbohydrate diet
β-‐Oxida=on in Mitochondria vs. Peroxisomes
• Differ in the first step: -‐ passes e–s directly to O2 forming H2O2 which is quickly removed by the ac<on of catalase -‐ energy is lost as heat instead of producing ATP • Differ in f.a. specificity: -‐ more ac<ve on very long f.a. and branched f.a. (α oxida=on) -‐ process long chain f.a. into shorter ones which are exported to mito to complete oxida<on • Zellweger syndrome – inability to m make peroxisomes
ω oxida=on • In the ER of liver and kidney • For f.a. with 10 – 12 Cs • Addi<on of OH by a mixed func=on oxidase (cytochrome P450)
• Alcohol dehydrogenase oxidizes OH to aldehyde
• Aldehyde dehydrogenase oxidizes aldehyde to acid
• CoA can a=ach to either end and β oxida<on resumes
Forma=on of Ketone Bodies
• Entry of acetyl-‐CoA into citric acid cycle requires oxaloacetate
• When oxaloacetate is depleted, acetyl-‐CoA is converted into ketone bodies (acetone, acetoacetate and D-‐β-‐hydroxybutyrate) – Frees Coenzyme A for con<nued β-‐oxida<on
– Acetone is exhalled – Acetoacetate and β-‐HB are transported in the blood
• Under starva<on condi<ons, the brain can use ketone bodies for energy
• The first step is reverse of the last step in the β-‐oxida<on: thiolase reac<on joins two acetate units
Release of Free Coenzyme A
Another condensation with acetyl-CoA yields HMG-CoA
Forma=on of Ketone Bodies
Cleaved into acetoacetate and acetyl-CoA
Specific for the D-isomer; don’t confuse it with L-β-hydroxyacyl-CoA DH of β oxidation
Untreated diabetes à [acetoacetate] is high à more acetone produced à exhaled (odor)
Ketone Bodies as fuel In extrahepatic tissues: Ketone bodies can be used as fuels in all tissues except the liver The liver is a producer, not a consumer, of ketone bodies
ß CAC Found in all tissues except the liver
Liver is the source of ketone bodies • Produc<on of ketone
bodies increases during starva<on (and diabetes)
• Ketone bodies are released by liver to bloodstream
• Organs other than liver can use ketone bodies as fuels
• High levels of acetoacetate and β-‐hydroxybutyrate lower blood pH dangerously (acidosis)
• Acidosis due to ketone bodies -‐ ketoacidosis
Lipids fulfill a variety of biological func=ons
• Energy storage • Cons<tuents of membranes • Anchors for membrane proteins • Cofactors for enzymes • Signaling molecules • Pigments • Detergents • Transporters • An<oxidants
Catabolism and anabolism of fa'y acids proceed via different pathways
• Catabolism of fa=y acids (excergonic and oxida=ve) – produces acetyl-‐CoA – produces reducing power (NADH and FADH2)
– ac<va<on of fa=y acids by CoA – takes place in the mitochondria
• Anabolism of fa=y acids (endergonic and reduc=ve) – requires acetyl-‐CoA and malonyl-‐CoA
– requires reducing power from NADPH
– ac<va<on of fa=y acids by 2 different –SH groups on protein – takes place in cytosol in animals, chloroplast in plants
Subcellular localiza=on of lipid metabolism
Overview of Fa'y Acid Synthesis
• Fa=y acids are built in several passes, processing one acetate unit at a <me.
• The acetate is coming from ac<vated malonate in the form of malonyl-‐CoA.
• Each pass involves reduc<on of a carbonyl carbon to a methylene carbon.
Malonyl-‐CoA is formed from acetyl-‐CoA and bicarbonate
• The reac<on carboxylates acetyl CoA • Catalyzed by acetyl-‐CoA carboxylase (ACC)
– Enz has three subunits: • One unit has Bio<n covalently linked to Lys • Bio<n carries CO2 • In animals, all three subunits are on one polypep<de chain
– HCO3− (bicarbonate) is the source of CO2
The Acetyl-‐CoA Carboxylase (ACC) Reac=on • Two-‐step rxn similar to carboxyla<ons catalyzed by pyruvate carboxylase (gluconeogenesis) and propionyl-‐CoA carboxylase (odd f.a. metabolism)
• CO2 binds to bio<n - CO2 is ac<vated by a=achment to N in ring of bio<n
Synthesis of fa'y acids is catalyzed by fa'y acid synthase (FAS)
• FAS system: – Catalyzes a repea<ng four-‐step sequence that elongates the fa=y acyl chain by two carbons at each step
– Uses NADPH as as the electron donor – Uses two enzyme-‐bound -‐SH groups as ac<va<ng groups
• FAS I in vertebrates and fungi • FAS II in plants and bacteria
FAS I vs. FAS II
FAS I • Single polypep<de chain in
vertebrates • Leads to single product:
palmitate 16:0 • C-‐15 and C-‐16 are from the
acetyl CoA used to prime the rxn
FAS II • Made of separate, diffusible
enzymes • Makes many products
(saturated, unsaturated, branched, many lengths, etc.)
• Mostly in plants and bacteria
Fa'y Acid Synthesis • Overall goal: a=ach two-‐C acetate unit from malonyl-‐CoA to a
growing chain and then reduce it • Reac<on involves cycles of four enzyme-‐catalyzed steps
– Condensa<on of the growing chain with ac<vated acetate – Reduc<on of carbonyl to hydroxyl – Dehydra<on of alcohol to trans-‐alkene – Reduc<on of alkene to alkane
• The growing chain is ini<ally a=ached to the enzyme via a thioester linkage
• During condensa<on, the growing chain is transferred to the acyl carrier protein (ACP)
• Aoer the second reduc<on step, the elongated chain is transferred back to fa=y acid synthase
The General Four-‐Step Fa'y Acid Synthase I Reac=on in Mammals (1)
Prep: Malonyl CoA and acetyl CoA (or longer fa=y acyl chain) are bound to FAS I
-‐ bind via thioester terminus of a Cys of the FAS -‐ ac<vates the acyl group
Step 1: Condensa<on rxn a=aches two C from malonyl CoA to the a=ached acetyl-‐CoA (or longer fa=y acyl chain)
-‐ also releases CO2 from malonyl-‐CoA -‐ the decarboxyla<on facilitates the rxn -‐ creates β-‐keto intermediate
Step 1 of FAS I: Elonga=on
Step 2: 1st Reduc<on: NADPH reduces the β-‐keto intermediate to an alcohol
Step 3: Dehydra<on: OH group from C-‐2 and H from neighboring CH2 are eliminated, crea<ng double bond (trans-‐alkene)
Step 4: 2nd Reduc<on: NADPH reduces double bond to yield saturated alkane Step 5: Transloca<on: The growing chain is moved from ACP to –SH on FAS
The General Four-‐Step Fa'y Acid Synthase I Reac=on in Mammals
Steps 2-‐4 of the FAS I rxn
Overall Palmitate Synthesis
Acyl Carrier Protein (ACP) serves as a shu'le in fa'y acid synthesis
• Contains a covalently a=ached prosthe<c group 4’-‐phosphopantetheine – Flexible arm to tether acyl chain while carrying intermediates from one enzyme subunit to the next
• Delivers malonate to the fa=y acid synthase
• Shu=les the growing chain from one ac<ve site to another during the four-‐step reac<on
Charging ACP and FAS I with acyl groups ac=vates them
• Two thiols must be charged with the correct acyl groups before condensa<on rxn can begin – Thiol from 4’-‐phosphopantethine in ACP
– Thiol from Cys in fa=y acid synthase
1) Acetyl group of acetyl-‐CoA is transferred to ACP – Catalyzed by malonyl/acetyl-‐CoA transferase (MAT) – ACP passes this acetate to the Cys of the β-‐ketoacyl-‐ACP synthase (KS)
domain of FAS I
2) ACP –SH group is re-‐charged with malonyl from malonyl-‐CoA
Charging, Ac=va=on with ACP, and the Four-‐Step Sequence of Mammalian Fa'y
Acid Synthesis
• Ac<vated acetyl and malonyl groups form acetoacetyl-‐ACP and CO2 – Claisen condensa<on rxn
• Catalyzed by β-‐ketoacyl-‐ACP synthase (KS)
• Coupling condensa<on to decarboxyla<on of malonyl-‐CoA makes the rxn energe<cally favorable
• Carbonyl at C-‐3 is reduced to form D-‐β-‐hydroxybutyryl-‐ACP – NADPH is e− donor
• Catalyzed by β-‐ketoacyl-‐ACP reductase (KR)
• OH and H removed from C-‐2 and C-‐3 of β-‐hydroxybutyryl-‐ACP to form trans-‐Δ2-‐butenoyl-‐ACP
• Catalyzed by β-‐hydroxyacyl-‐ACP dehydratase (DH)
• NADPH is the electron donor to reduce double bond of trans-‐Δ2-‐butenoyl-‐ACP to form butyryl-‐ACP
• Catalyzed by enoyl-‐ACP reductase (ER)
Enzymes in Fa'y Acid Synthase
• Condensa<on with acetate – β-‐ketoacyl-‐ACP synthase (KS)
• Reduc<on of carbonyl to hydroxyl – β-‐ketoacyl-‐ACP reductase (KR)
• Dehydra<on of alcohol to alkene – β-‐hydroxyacyl-‐ACP dehydratase (DH)
• Reduc<on of alkene to alkane – enoyl-‐ACP reductase (ER)
• Chain transfer/charging – Malonyl/acetyl-‐CoA ACP transferase
The Transferase and FAS rxns are repeated in new rounds
• Product of first round is butyryl-‐ACP – (bound to phosphopantetheine-‐SH group of ACP)
• Butyrul gp is transferred to the Cys of β-‐ketoacyl-‐ACP synthase – In the first round, acetyl-‐CoA was bound here
• New malonyl-‐CoA binds to ACP • Aoer new round of four steps, six-‐C product is made (bound to ACP)
Beginning of the Second Round of Fa'y Acid Synthesis
Stoichiometry of Synthesis of Palmitate (16:0)
1) 7 acetyl-‐CoAs are carboxylated to make 7 malonyl-‐CoAs… using ATP
7 AcCoA + 7 CO2 + 7 ATP à 7 malCoA + 7 ADP + 7 Pi
2) Seven cycles of condensa<on, reduc<on, dehydra<on and reduc<on…using NADPH to reduce the β-‐keto group and trans-‐double bond
AcCoA + 7 malCoA + 14 NADPH + 14 H+ àPalmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O
Note: Eukaryotes have one addi<onal energy cost. (Next slide)
Acetyl-‐CoA is transported into the cytosol for fa'y acid synthesis
• In nonphotosynthe<c eukaryotes… • Acetyl-‐CoA is made in the mitochondria • But fa=y acids are made in the cytosol • So Acetyl-‐CoA is transported into the cytosol with a cost of 2 ATPs
• Therefore, cost of FA synthesis is 3 ATPs per 2-‐C unit
Fa'y acid synthesis occurs in cell compartments where NADPH levels are high
• Cytosol for animals, yeast • Chloroplast for plants
• Sources of NADPH: – In adipocytes: pentose phosphate pathway and malic enzyme
– NADPH is made as malate converts to pyruvate + CO2
– In hepatocytes and mammary gland: pentose phosphate pathway • NADPH is made as glucose-‐6-‐phosphate converts to ribulose 6-‐phosphate
– In plants: photosynthesis
Pathways for NADPH Produc=on
Acetyl-‐CoA, generated in the mitochondria, is shu'led to the cytosol as citrate
• In most eukaryotes, the acetyl-‐CoA for lipid synthesis is made in the mitochondria – But lipid synthesis occurs in the cytosol
• And there is no way for acetyl-‐CoA to cross mitochondrial inner membrane to the cytosol
• So acetyl-‐CoA is converted to citrate – Acetyl-‐CoA + oxaloacetate à citrate
• Same rxn as occurs in CAC • Catalyzed by citrate synthase • Citrate passes through citrate transporter
Citrate is cleaved to regenerate acetyl-‐CoA
• Citrate (now in cytosol) is cleaved by citrate lyase – Regenerates acetyl-‐CoA and oxaloacetate – Rxn requires ATP – Acetyl-‐CoA can now be used for lipid synthesis
• What happens to the oxaloacetate because there is no oxaloacetate transporter either?
Oxaloacetatecyt is converted to malate
• Malate dehydrogenase in cytosol reduces oxaloacetate to malate
• Two poten<al fates for malate: – Can be converted to NADPHcyt and pyruvatecyt via the malic enzyme • NADPH used for lipid synthesis • Pyruvatecyt sent back to mito via pyruvate transporter • Converted back to oxaloacetatemito by pyruvate carboxylase, requires ATP
– Can be transported back to mito via malate -‐α-‐ketoglutarate transporter • Malatemito is reoxidized to oxaloacetatemito
Shu'le for Transfer of Acetyl Groups from Mitochondria to Cytosol
Fa'y acid synthesis is =ghtly regulated via ACC
• Acetyl CoA carboxylase (ACC) catalyzes the rate-‐limi<ng step – ACC is feedback-‐inhibited by palmitoyl-‐CoA – ACC is acEvated by citrate
• Remember citrate is made from acetyl-‐CoAmito
• Citrate signals excess energy to be converted to fat – When [acetyl-‐CoA]mito ↑, converted to citrate…citrate exported to cytosol
Importance of Citrate to Regula=on of Fa'y Acid Synthesis
• In animals, citrate s<mulates fa=y acid synthesis! – Precursor for acetyl-‐CoA
• Sent to cytosol and cleaved to become AcCoA when AcCoA and ATP ↑ (energy excess)
– Allosteric ac<vator of ACC – Inhibitor of PFK-‐1
• Reduces glycolysis
ACC is also regulated by covalent modifica=on
• Inhibited when energy is needed • Glucagon and epinephrine:
– reduce sensi<vity of citrate ac<va<on – lead to phosphoryla<on and inac<va<on of ACC via PKA • ACC is ac<ve as dephosphorylated monomers • When phosphorylated, ACC polymerizes into long inac<ve filaments
• Dephosphoryla<on reverses the polymeriza<on
Regula=on of Fa'y Acid Synthesis in Vertebrates
Addi=onal Modes of Regula=on in Fa'y Acid Synthesis
• Changes in gene expression – Example: Fa=y acids (and eicosanoids) bind to transcrip<on factors called Peroxisome Proliferator-‐Ac=vated Receptors (PPARs) à inducing gene expression of some genes
• Reciprocal regula<on – Malonyl-‐CoA inhibits fa=y acid import into mito
• One of many ways to ensure that fat synthesis and oxida<on don’t occur simultaneously
Palmitate can be lengthened to longer-‐chain fa'y acids
• Elonga<on systems in the endoplasmic re<culum and mitochondria create longer fa=y acids
• As in palmitate synthesis, each step adds units of 2 C
• Stearate (18:0) is the most common product
Palmitate and stearate can be desaturated
• Palmitate(16:0)àpalmitoleate(16:1; Δ9) • Stearate (18:0)àoleate (18:1; Δ9)
– Catalyzed by fa=y acyl-‐CoA desaturase in animals • Also known as the fa=y acid desaturases • Requires NADPH; enzyme uses cytochrome b5 and cytochrome b5 reductase
Note that this is a Δ9-‐desaturase! It reduces the bond between C-‐9 and C-‐10.
Vertebrate fa'y acyl desaturase is a non-‐heme, iron-‐containing, mixed func=on
oxidase
• O2 accepts four electrons from two substrates • Two electrons come from saturated fa=y acid • Two electrons come from ferrous state of cytochrome b5
Desatura=on of a Fa'y Acid by Fa'y Acyl-‐CoA Desaturase
Plants can desaturate posi=ons beyond C-‐9
• Humans have Δ4, Δ5, Δ6, and Δ9 desaturases but cannot desaturate beyond Δ9
• Plants can produce: – linoleate 18:2(Δ9,12) – α-‐linolenate 18:3 (Δ9,12,15)
• These fa=y acids are “essen=al” to humans – Polyunsaturated fa=y acids (PUFAs) help control membrane fluidity
– PUFAs are precursors to eicosanoids • Implica<ons of stearoyl-‐ACP desaturase (SCD) on obesity
– SCD1-‐mutant mice are resistant to diet-‐induced obesity!
Oxidases, Monooxygenases, and Dioxygenases
Many enzymes use oxygen as an e− acceptor, but not all of them incorporate oxygen into the product.
• Oxidases do not incorporate oxygen into the product – Oxygen atoms usually end up in H2O2
• Oxygenases do incorporate oxygen into the product – Monooxygenases incorporate one of the oxygen atoms into the product
– Dioxygenases incorporate both oxygen atoms into the product
Monooxygenases incorporate one oxygen into the product
AH + BH2 + O-‐O àA-‐OH + B + H2O • Product is ooen hydroxylated, so also called hydroxylases or mixed-‐func=on oxygenases
– Example: Phenylanine hydroxylase hydroxylates phenylalanine to form tyrosine
– Deficiency causes phenylketonuria (PKU)
Cytochrome P450s are monooxygenases
• Important in drug metabolism • Hydroxylate nonpolar molecules
– usually inac<va<ng them and making them more H2O-‐soluble for excre<on
• If two drugs (or alcohol and a drug) use the same P450, they will compete, and levels of the drug or alcohol will not be cleared as quickly – Can be deadly
Dioxygenases incorporate two oxygens in the product
• Usually metalloproteins – Ac<ve sites have Fe or Mn ions
• Rxns ooen involve opening an aroma<c ring
• Example: Tryptophan 2,3-‐dioxygenase
Eicosanoids are potent short-‐range hormones made from arachidonate
• Eicosanoids are paracrine signaling molecules • They include prostaglandins, leukotrienes, thromboxanes
• Created from arachidonic acid, 20:4 (Δ5,8,11,14) • Arachidonate is incorporated into the phospholipids of membranes
• In response to s<muli (hormone, etc.), phospholipase A2 is ac<vated and a=acks the C-‐2 fa=y acid, releasing arachidonate
Prostaglandins are made by prostaglandin H2 synthase (cyclooxygenase, COX)
• COX (aka PGH2 synthase) is a bifunc<onal ER enzyme:
• Step 1: cyclooxygenase ac<vity of PGH2 synthase adds 2 O2 to form PGG2
• Step 2: peroxidase ac<vity converts peroxide to alcohol, creates PGH2
• PGH2 is precursor to other eicosanoids
Conversion of Arachidonate to Prostaglandins and Other Eicosanoids
• Thromboxane synthase present in thrombocytes converts PGH2 to thromboxane A2
• Induce the constric<on of blood vessels and blood clovng
• Low doses of aspirin reduce the risk of heart a=acks and strokes by reducing thromboxane produc<on
PGH2 synthase has two isoforms
• COX-‐1 catalyzes synthesis of prostaglandins that regulate gastric mucin secreEon
• COX-‐2 catalyzes synthesis of prostaglandins that mediate pain, inflammaEon, and fever
NSAIDs inhibit cyclooxygenase ac=vity
• Aspirin (Acetylsalicylate) is an irreversible inhibitor – Acetylates a Ser in the ac<ve site – Blocks ac<ve site in both COX isozymes
• Ibuprofen and naproxen are compe<<ve inhibitors – Resemble substrate, also block the ac<ve site in both isozymes
– Undesired side effects such as stomach irritaEon, why?
A Few NSAIDs that Inhibit PGH2
Arachidonate (substrate)
Advil, motrin Aleve
COX-‐2-‐specific inhibitors have a checkered history
• Developed to inhibit prostaglandin forma<on without harming stomach
• Includes Vioxx, Bextra, and Celebrex • Vioxx and Bextra removed from market due to increased rates of stroke and heart a=ack – May disrupt balance between blood-‐thinning prostacyclin and blood-‐clovng thromboxanes
Leukotriene synthesis also begins with arachidonate
• O2 is added to arachidonate via lipoxygenases • Creates species that differ in the posi<on of the OOH group • Not inhibited by NSAIDs
Biosynthesis of Triacylglycerols • Synthesized or ingested fa=y acids are either stored for energy or used in membranes depending on the needs of the organism
• Animals and plants store fat for fuel
– Plants: in seeds, nuts – Typical 70-‐kg human has ~15 kg fat
• Enough to last 12 wks • Compare with 12 hrs’ worth glycogen in liver and muscle
• Animals and plants and bacteria make phospholipids for cell membranes
The precursor for the backbone of fat and phospholipids is glycerol 3-‐phosphate
• Both pathways start by the forma<on of fa=y acyl esters of glycerol
• The substrates are fa=y acyl-‐CoAs and L-‐glycerol 3-‐phosphate
• Most glycerol 3-‐phosphate comes from dihydroxyacetone phosphate (DHAP) from glycolysis – via glycerol 3-‐phosphate dehydrogenase
• Some glycerol 3-‐phosphate made from glycerol – via glycerol kinase – Minor pathway in liver and kidney only
Acyl transferases a'ach two fa'y acids to glycerol 3-‐phosphate
• Phospha<dic acid is the precursor to TAGs and phospholipids – Made of glycerol 3-‐phosphate + 2 fa=y acids
– Fa=y acids are a=ached by acyl transferases
– Release of CoA
To make TAG, phospha=dic acid is dephosphorylated and acylated
• Phospha<dic acid phosphatase (lipin) removes the 3-‐phosphate from the phospha<dic acid – Yields 1,2-‐diacylglycerol
• Third carbon is then acylated with a third fa=y acid – Yields triacylglycerol
Conversion of Phospha=dic Acid into Triacylglycerol
Regula=on of Triacylglycerol Synthesis by Insulin
• Insulin results in s<mula<on of triacylglycerol synthesis
• Lack of insulin results in: – Increased lipolysis – Increased fa=y acid oxida<on
• Some<mes to ketones, if citric acid cycle intermediates (oxaloacetate) that react with acetyl CoA are depleted
– Failure to synthesize fa=y acids
Regula=on of Fat Metabolism by Glucagon and Epinephrine
• Glucagon and epinephrine result in s<mula<on of triacylglycerol breakdown (mobiliza<on of fa=y acids) – Also decrease glycolysis – Also increase gluconeogenesis
Triacylglycerol breakdown and re-‐synthesis create a fu=le cycle
• Seventy-‐five percent of free fa=y acids (FFA) released by lipolysis are reesterified to form TAGs rather than be used for fuel – Some recycling occurs in adipose <ssue – Some FFA from adipose cells are transported to liver, remade into TAG, and re-‐deposited in adipose cells
• Although the distribu<on between these two paths may vary (the flux of FFA into and out of the adipose), overall, the percentage of FFA being esterified remains at ~75%.
The Triacylglycerol Cycle * In mammals, TAG molecules are broken down and resynthesized in a TAG cycle even during starva<on.
Benefits of this fu=le cycle?
• Recycling con<nues even in starvaEon • Specula<on:
– energy reserve for “fight or flight” crises that might occur during fas<ng
• The total # of FFA in flux may change but the % recycled remains – unless a pharamacological interven<on happens (i.e., thiazolidinedione drugs, type 2 DM)
What is the source of the glycerol 3-‐phosphate needed for fa'y acid
reesterifica=on?
• During lipolysis (s<mulated by glucagon or epinephrine), glycolysis is inhibited – So DHAP is not readily available to make glycerol 3-‐phosphate
• And adipose cells don’t have glycerol kinase to make glycerol 3-‐phosphate on-‐site
• So cells make DHAP via glyceroneogenesis
Glyceroneogenesis makes DHAP for glycerol 3-‐phosphate genera=on
• Glyceroneogenesis contains some of the same steps of gluconeogenesis – Converts pyruvate à DHAP – Basically, a shortened version of gluconeogenesis in the liver and adipose <ssue
• Explains why adipose cells express pyruvate carboxylase and PEPCK even though fat cells don’t make glucose
Glyceroneogenesis
Regula=on of PEPCK expression is =ssue-‐dependent
• Cor<sol and glucagon both increase PEPCK expression in liver. – Results in more TAG synthesis, so more released to the blood
• Cor<sol and other glucocor<coids decrease PEPCK expression in adipose <ssue – ↓ glyceroneogenesis in adipose means less recycling; more FFA are released into the blood
– Most glycerol freed from TAG in adipose is sent to liver and converted to glucose
Regula=on of Glyceroneogenesis via Glucocor=coid Hormones
Cor=sol and glucagon can elevate blood sugar
1) ↑ PEPCK expression in liver à↑ gluconeogenesis (so ↑ [glucose])
2) ↓ PEPCK expression in adipose <ssue à glycerol freed, sent to liver, converted to glucose
3) Plus, the FFA associated with increased flux through TAG cycle à interfere with glucose uptake in muscle, keep [glucose]blood high à may lead to insulin resistance (type 2 DM)
Thiazolidinedione drugs target insulin resistance by increasing glyceroneogenesis
• Elevated FFA levels seem to promote insulin resistance
• Thiazolidinediones upregulate PEPCK in adipose <ssue via PPARγ, lead to ↑ glyceroneogenesis,↑ resynthesis of TAG in adipose <ssue and ↓ release of FFA
• Thus the drugs promote sensi<vity to insulin
Thiazolidinediones/Glitazones
Have this group in common
Avandia (Rosiglitazone) – removed from market due to associa<on with heart a=ack
Pioglitazone (Actos)
Regula=on of Glyceroneogenesis via Thiazolidinediones
Biosynthesis of Membrane Phospholipds
• Begin with phospha<dic acid or diacylglycerol
• A=ach head group to C-‐3 OH group – C-‐3 has OH, head group has OH
– New phospho-‐head group created when phosphoric acid condenses with these two alcohols
– Eliminates two H2O
Further Details on A'aching the Head Group
• Either one of the alcohols is ac<vated by a=aching to CDP (cy<dine diphosphate)
• The free (not bound to CDP) alcohol then does nucleophilic a=ack on the CDP-‐ac<vated phosphate
• Releases CMP and a glycerophospholipid E. coli: CDP-DAG
Eukaryotes: both
Synthesis of Phospha=dylethanolamine and Phospha=dylcholine in Yeast
• Phospha<dylserine is decarboxylated to phospha7dylethanolamine – phospha<dylserine decarboxylase
• Phospha<dylethanolamine acted on by S-‐adenosylmethionine (methyl group donor), adds three methyl groups to amino group à phopsha7dylcholine (lecithin) – Catalyzed by methyltransferase
Phospholipid Synthesis in Mammals • Phospha7dylserine isn’t synthesized from CDP-‐diacylglycerol as it is in yeast and bacteria
• Made “backwards” from PE or PC via head group exchange rxns – Catalyzed by specific synthases – Pathway “salvages” the choline
Sphingolipids are made in four steps
1) Synthesis of sphinganine from palmitoyl-‐CoA and serine
2) A=achment of fa'y acid via amide linkage 3) Desatura=on of N-‐acylsphinganine
(dihydroceramide) • Yields N-‐acylsphingosine (ceramide)
4) A=achment of head group • Can yield a cerebroside or ganglioside
ER
Golgi
Phospholipids must be transported from the ER to membranes
• Phospholipids are: – synthesized in the smooth ER – transported to Golgi complex for addi<onal synthesis
• Must be inserted into specific membranes in specific propor7ons but can’t diffuse because they are nonpolar
• So transported in membrane vesicles that fuse with target membrane
• Details of the process are not well-‐understood
Four Steps of Cholesterol Synthesis
1) Three acetates condense to form 5-‐C mevalonate
2) Mevalonate converts to phosphorylated 5-‐C isoprene
3) Six isoprenes polymerize to form the 30-‐C linear squalene
4) Squalene cyclizes to form the four rings that are modified to produce cholesterol
Step 1: Forma=on of Mevalonate from Acetyl-‐CoA
• 2 Acetyl-‐CoAs àAcetoacetyl-‐CoA – Catalyzed by acetyl-‐CoA acyl transferase
(thiolase)
• Acetyl-‐CoA + Acetoacetyl-‐CoA à β-‐hydroxyl-‐β-‐methylglutaryl-‐CoA (HMG-‐CoA) – Catalyzed by HMG-‐CoA synthase
• NOT the mitochondrial HMG-‐CoA synthase used in ketone body forma<on
• HMG-‐CoA + 2 NADPH àmevalonate – Catalyzed by HMG-‐CoA reductase – Rate-‐limi7ng step and point of
regula7on! – HMG-‐CoA reductase is a target for some
cardiovascular drugs
Sta=n drugs inhibit HMG-‐CoA reductase to lower cholesterol
• Sta<ns resemble HMG-‐CoA and mevalonate à compe<<ve inhibitors of HMG-‐CoA reductase
• First sta<n, lovasta<n, was found in fungi • Lowers serum cholesterol by ~20 – 40% • Also reported to improve circula<on, stabilize plaques by removing chol from them, reduce vascular inflamma<on
• Most circulaEng chol comes from internal manufacture rather than the diet
Step 2: Conversion of Mevalonate to Two Ac=vated Isoprenes
• 3 PO43− transferred stepwise from
ATP to mevalonate • Decarboxyla<on and hydrolysis
creates a diphosphorylated 5-‐C product (isoprene) with a double bond
• Isomeriza<on to a second isoprene
• The two “ac<vated” isoprene units are Δ3-‐isopentyl pyrophosphate and dimethylallyl pyrophosphate
Step 3: Six Ac=vated Isoprene Units Condense to Form Squalene
• The two isoprenes join head -‐to-‐tail, displacing one set of diphosphates à forms10-‐C geranyl pyrophopshate
• Geranyl pyrophosphate joins to another isopentenyl pyrophosphate à forms 15-‐C farnesyl pyrophosphate
• Two farnesyl pyrophosphates join head-‐to-‐head to form phosphate-‐free squalene
Step 4: Conversion of Squalene to Four-‐Ring Steroid Nucleus
• Squalene monooxygenase adds one oxygen to the end of the squalene chain à forms squalene 2,3-‐epoxide
• Here pathways diverse in animal cells vs. plant cells
• The cycliza<on product in animals is lanosterol, which converts to cholesterol
• In plants, the epoxide cyclizes to other sterols such as s<gmasterol
Conversion of Squalene to Cholesterol
Fates of Cholesterol Aner Synthesis
• In vertebrates, most cholesterol synthesized in the liver, then exported: - As bile acids, biliary cholesterol or cholesteryl esters
• Other <ssues convert cholesterol into steroid hormones, etc.
Bile Acids Assist in Emulsifica=on of Fats
• Bile is stored in the gall bladder, secreted into small intes<ne aoer fa=y meal
• Bile acids such as taurocholic acid emulsify fats – Surround droplets of fat, increase surface area for a=ack by lipases
Cholsteryl esters are more nonpolar than cholesterol
• Contain a fa=y acid esterified to the oxygen – Comes from a fa=y acyl-‐CoA – Makes the cholesterol more hydrophobic, unable to enter membranes
• Transported in lipoproteins to other <ssues or stored in liver
Cholesterol and other lipids are carried on lipoprotein par=cles
• Lipids are carried through plasma on spherical par<cles – Surface is made of apolipoprotein and phospholipid monolayer
– Interior contains cholesterol, TAGs, cholesteryl esters
Four Major Classes of Lipoprotein Par=cles
• Named based on posi<on of sedimenta<on (density) in centrifuge
• Large enough to see in electron microscope • Includes:
– Chylomicrons (largest and least dense) – Very low-‐density lipoproteins (VLDL) – Low-‐density lipoproteins (LDL) – High-‐density lipoproteins (HDL) – smallest, most dense
Electron Microscope Pictures of Lipoproteins
Apolipoproteins in Lipoproteins
• “Apo” for “without”… – So “apolipoprotein” refers to the protein part of a lipoprotein par<cle
• Provide sites for the par<cle to bind to cell surface receptors, ac<vate enzymes, etc.
• At least ten have been characterized in humans
Chylomicrons carry fa'y acids to =ssues
• Have more TAG and less protein à hence, least dense.
• Have ApoB-‐48, ApoE, and ApoC-‐II
• ApoC-‐II ac<vates lipoprotein lipase to allow FFA release for fuel in adipose <ssue, heart, and skeletal muscle
Chylomicron remnants deposit their cholesterol in the liver
• When chylomicrons are depleted of their TAG, “remnants” go to liver
• ApoE receptors in liver bind the remnants, take them up by endocytosis
• Remnants release their cholesterol in the liver
VLDLs transport endogenous lipids
• Cholesteryl esters and TAGs from excess FA and cholesterol are packed into very low-‐density lipoproteins (VLDL)
• Excess carbohydrate in the diet can also be made into TAG in the liver and packed into VLDL
• Contain apoB-‐100, apoC-‐I, apoC-‐II, apoC-‐III, and apoE
VLDLs take TAGs to adipose =ssue and muscle
• Again, ApoC-‐II ac<vates lipoprotein lipase to release free fa=y acids
• Adipocytes take up the FFA, reconvert them to TAGs, and store them in lipid droplets
• Muscle uses the TAG for energy
VLDL remnants become LDL
• Removal of TAG from VLDL produces LDL • Because TAG removed, LDL is enriched in cholesterol/chloesteryl esters
• ApoB-‐100 is the major apolipoprotein
LDLs carry cholesterol from liver to muscle and adipose =ssue
• Muscle and adipose <ssue have LDL receptors, recognize apoB-‐100 à Enable myocytes and adipocytes to take up cholesterol via receptor-‐mediated endocytosis
Cholesterol Uptake by Receptor-‐Mediated Endocytosis
Familial hypercholesterolemia is associated with LDL receptor muta=ons
• Muta<ons in LDL receptor prevent normal uptake of LDL by liver and other <ssues
• LDL accumulates in blood • Heterozygous individuals have risk of heart a=ack greater than normal
• Homozygous individuals have much increased risk of heart a=ack
HDL carries out reverse cholesterol transport
• HDLs contain a lot of protein – Including ApoA-‐I and lecithin-‐cholesterol acyl transferase (LCAT) • Catalyzes the forma<on of cholesteryl esters from lecithin and cholesterol
• Enzyme converts chol of chylomicron and VLDL remnants to cholesteryl esters
• HDL picks up cholesterol from cells and returns them to the liver
Five Modes of Regula=on of Cholesterol Synthesis and Transport
1) Covalent modifica<on of HMG-‐CoA reductase
2) Transcrip<onal regula<on of HMG-‐CoA gene
3) Proteoly<c degrada<on of HMG-‐CoA reductase
4) Ac<va<on of ACAT, which increases esterifica<on for storage
5) Transcrip<onal regula<on of the LDL receptor
Regula=on of Cholesterol Metabolism
HMG-‐CoA reductase is most ac=ve when dephosphorylated
1) AMP-‐dependent protein kinase -‐ when AMP rises, kinase phosphorylates the enzyme à ac<vity ↓, cholesterol synthesis ↓
2) Glucagon, epinephrine -‐ cascades lead to phosphoryla<on, ↓ ac<vity
3) Insulin -‐ cascades lead to dephosphoryla<on,↑ ac<vity
Covalent modifica=on provides short-‐term regula=on.
LOW Energy Level
Longer-‐term Regula=on of HMG-‐CoA Reductase through Transcrip=onal Control
• Sterol regulatory element-‐binding proteins (SREBPs) – When sterol levels are high, SREBP is in ER membrane with other proteins
– When sterol levels decline, complex is cleaved, moves to the nucleus
– SREBP ac<vates transcrip<on of HMG-‐CoA reductase and LDL receptor as well as other genes à more cholesterol produced and imported
Regula=on of Cholesterol Synthesis by SREBP
Regula=on of HMG-‐CoA Reductase by Proteoly=c Degrada=on
• Insig (insulin-‐induced gene protein) senses cholesterol levels. – Binds to HMG-‐Co-‐A reductase, – Triggers ubiquina<on of HMG-‐CoA reductase – Targets the enzyme for degrada<on by proteasomes
– Also prevents the synthesis of HMG-‐CoA reductase (complexing and inhibi<ng SREBP)
Cardiovascular disease (CVD) is mul=-‐factorial
• Very high LDL-‐cholesterol levels tend to correlate with atherosclerosis – Although many heart a=ack vic<ms have normal cholesterol, and many people with high cholesterol do not have heart a=acks
• Low HDL-‐cholesterol levels are nega<vely associated with heart disease
How Plaques Form
• LDL with partly oxidized fa=y acyl groups s<cks to the lining of arteries
• A=racts macrophage cells of the immune system
• These cells don’t regulate their uptake of sterols, so they accumulate cholesterol and cholesteryl esters
• The macrophages become foam cells (named for appearance)
How Plaques Form (cont.)
• Foam cells undergo apoptosis • Remnants accumulate, along with scar <ssue, etc.
• Can occlude a blood vessel or break off and travel to another artery
• Occlusion of blood vessels in the heart cause heart a=ack; occlusion in the brain causes stroke
Familial Hypercholesterolemia
• Due to gene<c muta<on in LDL receptor • Impairs receptor-‐mediated uptake of cholesterol from LDL
• Cholesterol accumulates in the blood and in foam cells
• Regula<on mechanisms based on cholesterol sensing inside the cell don’t work
• Homozygous individuals can experience severe CVD as youths
Reverse cholesterol transport by HDL explains why HDL is cardioprotec=ve
• HDL picks up cholesterol from non-‐liver <ssues, including foam cells at growing plaques
• ABC (ATP-‐Binding Casse=e) transporters bring cholesterol from inside the cell to the plasma membrane
• HDL carries cholesterol back to liver
Reverse Cholesterol Transport
Ques=on 7 (Take home exam) Due: NEXT WEEK ([email protected])
• Please solve ques=ons: 1. 6 (uncouplers) 2. 17 (ATP turnover) 3. 22 (alanine) 4. 24 (diabetes) For wri[en answers, I prefer to have them typed in Word. I can accept the assignment in one file sent to my email. For answers that require solving mathemaEcally, you can either type them or write them down and scan them.