Lipid MetabolismLipid Metabolism

http://www.expasy.org/cgi_bin/search_biochem_index/

http://www.tcd.ie/Biochemistry/IUBMB_Nicholson/

http://www.genome.ad.jp/kegg/metabolism.html/

Metabolic Functions of Eukaryotic OrganellesMetabolic Functions of Eukaryotic Organelles

Lipid Digestion, Absorption, and TransportLipid Digestion, Absorption, and Transport

triacylglycerols (fats) triacylglycerols (fats) constitute 90% of dietary constitute 90% of dietary lipidlipid

major form of metabolic major form of metabolic energy storage in energy storage in animalsanimals

~6x energy yield vs CHO ~6x energy yield vs CHO and protein since it is and protein since it is nonpolar and stored in nonpolar and stored in anhydrous stateanhydrous state

Lipid DigestionLipid Digestion

occurs at lipid-water interfacesoccurs at lipid-water interfaces enhanced by the emulsifying enhanced by the emulsifying

action of bile salts (bile acids)action of bile salts (bile acids) pancreatic lipase requires pancreatic lipase requires

activation activation triacylglycerol lipasetriacylglycerol lipase hydrolysis of TAGs at 1 and 3 hydrolysis of TAGs at 1 and 3

positionspositions complex with colipase (1:1)complex with colipase (1:1) mixed micelles of mixed micelles of

phosphatidylcholinephosphatidylcholine bile saltsbile salts

1LPA.pdb

Major Bile Acids and their Major Bile Acids and their ConjugatesConjugates

amphipathic detergent-like amphipathic detergent-like molecules that solubilize fat molecules that solubilize fat globulesglobules

cholesterol derivativescholesterol derivatives synthesized in the liver and synthesized in the liver and

secreted as glycine or secreted as glycine or taurine conjugatestaurine conjugates

exported into gallbladder for exported into gallbladder for storagestorage

secreted into small secreted into small intestine, where lipid intestine, where lipid digestion and absorption digestion and absorption occuroccur

Substrate Binding to Substrate Binding to Phospholipase APhospholipase A22

Bile Acids and Fatty Acid-binding Bile Acids and Fatty Acid-binding Protein Facilitate Absorption of LipidsProtein Facilitate Absorption of Lipids

Rat Intestinal Fatty Acid-Binding Rat Intestinal Fatty Acid-Binding Protein (2IFB.pdb)Protein (2IFB.pdb)

a cytoplasmic protein that a cytoplasmic protein that increases the “solubility” of lipids increases the “solubility” of lipids and protects the cell from their and protects the cell from their detergent-like effectsdetergent-like effects

131-residue protein with 10 131-residue protein with 10 antiparallel antiparallel ββ sheets sheets

palmitate (yellow) occupies a palmitate (yellow) occupies a gap between two gap between two ββ strands strands

palmitate’s carboxyl group palmitate’s carboxyl group interacts with Arg 106, Gln 115, interacts with Arg 106, Gln 115, and two bound Hand two bound H22O molecules O molecules while its tail is encased by while its tail is encased by aromatic side chainsaromatic side chains 2IFB.pdb

LipoproteinsLipoproteins lipoproteins are globular lipoproteins are globular

micelle-like particlesmicelle-like particles nonpolar core of TAGs and nonpolar core of TAGs and

cholesteryl esterscholesteryl esters surrounded by amphiphilic surrounded by amphiphilic

coating of protein, coating of protein, phospholipid, and cholesterolphospholipid, and cholesterol

intestinal mucosal cells intestinal mucosal cells convert FA to TAGs and convert FA to TAGs and package them with package them with cholesterols, into lipoproteins cholesterols, into lipoproteins called called chylomicronschylomicrons

LDL (Low Density Lipoprotein)

• has 1500 cholesteryl ester

• surrounded by ~800 phospholipid, ~500 cholesterol molecules and 1 apolipoprotein B-100

Characteristics of the Major Classes Characteristics of the Major Classes of Lipoproteins in Human Plasmaof Lipoproteins in Human Plasma

VLDL, IDL, and LDL are synthesized by the liver to transport VLDL, IDL, and LDL are synthesized by the liver to transport endogenous TAGs and cholesterol from liver to other tissuesendogenous TAGs and cholesterol from liver to other tissues

HDL transport cholesterol and other lipids from tissues back to HDL transport cholesterol and other lipids from tissues back to the liverthe liver

ApolipoproteinsApolipoproteins

protein components of lipoprotein (apoproteins)protein components of lipoprotein (apoproteins) apoproteins coat lipoprotein surfacesapoproteins coat lipoprotein surfaces Apolipoprotein A-I (apoA-I), in chylomicrons and HDLApolipoprotein A-I (apoA-I), in chylomicrons and HDL apoA-I is a 29-kD polypeptide with a twisted elliptical shape apoA-I is a 29-kD polypeptide with a twisted elliptical shape

(1AV1.pdb)(1AV1.pdb) pseudocontinuous pseudocontinuous -helix that is punctuated by a kink at Pro -helix that is punctuated by a kink at Pro

residuesresidues

1AV1.pdb

Transport of Plasma TAGs and CholesterolTransport of Plasma TAGs and Cholesterol

Receptor-Mediated Endocytosis of LDLReceptor-Mediated Endocytosis of LDL

Fatty Acid OxidationFatty Acid Oxidation

Fatty Acid ActivationFatty Acid Activation

FA must be “primed” before it can be oxidizedFA must be “primed” before it can be oxidized ATP-dependent acylation reaction to form fatty acyl-CoAATP-dependent acylation reaction to form fatty acyl-CoA activation is catalyzed by acyl-CoA synthetases activation is catalyzed by acyl-CoA synthetases

(thiokinanases)(thiokinanases) Fatty acid + CoA + ATP Fatty acid + CoA + ATP acyl-CoA + AMP + PP acyl-CoA + AMP + PPii

Transport across Mitochondrial MembraneTransport across Mitochondrial Membrane

FAs are activated for oxidation in the cytosol but they are FAs are activated for oxidation in the cytosol but they are oxidized in the mitochondrionoxidized in the mitochondrion

Long-chain fatty acyl-CoA cannot directly cross the inner Long-chain fatty acyl-CoA cannot directly cross the inner membrane of mitochondrionmembrane of mitochondrion

Acyl group is first transferred to carnitineAcyl group is first transferred to carnitine

Translocation process is mediated by a specific carrier protein Translocation process is mediated by a specific carrier protein (1) the acyl group of a cytosolic acyl-CoA is transferred to carnitine, thereby (1) the acyl group of a cytosolic acyl-CoA is transferred to carnitine, thereby

releasing the CoA to its cytosolic poolreleasing the CoA to its cytosolic pool(2) the resulting acyl-carnitine is transported into the mitochondrial matrix (2) the resulting acyl-carnitine is transported into the mitochondrial matrix

by the carrier proteinby the carrier protein(3) the acyl group is transferred to a CoA molecule from the mitochondrial (3) the acyl group is transferred to a CoA molecule from the mitochondrial

poolpool(4) the product carnitine is returned to the cytosol(4) the product carnitine is returned to the cytosol

Transport across Mitochondrial MembraneTransport across Mitochondrial Membrane

ββ Oxidation Oxidation

Acyl-CoA DehydrogenaseAcyl-CoA Dehydrogenase

mitochondria contain 4 acyl-CoA mitochondria contain 4 acyl-CoA dehydrogenasesdehydrogenases

FADHFADH22 resulting from the resulting from the oxidation of the fatty acyl-CoA oxidation of the fatty acyl-CoA substrate is reoxidized by the substrate is reoxidized by the mitochondrial ETCmitochondrial ETC

ribbon diagram of the active site ribbon diagram of the active site of the enzyme (MCAD) with of the enzyme (MCAD) with flavin ring (green), octanoyl-CoA flavin ring (green), octanoyl-CoA substate (blue-white)substate (blue-white)

the octanoyl-CoA binds such the octanoyl-CoA binds such that its Cthat its C-C-Cββ bond is bond is sandwhiched between the sandwhiched between the carboxylate group of Glu 376 carboxylate group of Glu 376 (red)(red)

3MDE.pdb

Enoyl-CoA HydrataseEnoyl-CoA HydrataseAdds water across the double bondAdds water across the double bond

at least three forms of the enzyme are at least three forms of the enzyme are known known

aka crotonases aka crotonases Normal reaction converts Normal reaction converts transtrans-enoyl-CoA -enoyl-CoA

to to LL---hydroxyacyl-CoA -hydroxyacyl-CoA

Hydroxyacyl-CoA Hydroxyacyl-CoA DehydrogenaseDehydrogenase

Oxidizes the Oxidizes the -Hydroxyl Group-Hydroxyl Group This enzyme is completely specific for This enzyme is completely specific for

L-hydroxyacyl-CoA L-hydroxyacyl-CoA D-hydroxylacyl-isomers are handled D-hydroxylacyl-isomers are handled

differently differently

Mechanism of Mechanism of ββ-Ketoacyl-CoA Thiolase-Ketoacyl-CoA Thiolase

Thiolase reaction occurs via Claisen Thiolase reaction occurs via Claisen ester cleavageester cleavage

(1) an active site thiol group adds to the (1) an active site thiol group adds to the ββ--keto group of the substrate acyl-CoAketo group of the substrate acyl-CoA

(2) C-C bond cleavage forms an acetyl-(2) C-C bond cleavage forms an acetyl-CoA carbanion intermediate that is CoA carbanion intermediate that is stabilized by stabilized by ee-- withdrawal into this withdrawal into this thioester’s carbonyl group (Claisen ester thioester’s carbonyl group (Claisen ester cleavage)cleavage)

(3) an enzyme acidic group protonoates (3) an enzyme acidic group protonoates the acetyl-CoA carbanion, yielding the acetyl-CoA carbanion, yielding acetyl CoAacetyl CoA

(4) & (5) CoA displaces the enzyme thiol (4) & (5) CoA displaces the enzyme thiol group from the enzyme-thioester group from the enzyme-thioester intermediate, yielding an acyl-CoA that intermediate, yielding an acyl-CoA that is shortened by two C atomsis shortened by two C atoms

FA Oxidation is Highly ExergonicFA Oxidation is Highly Exergonic

each round of each round of ββ oxidation produces: oxidation produces: 1 NADH (3 ATP)1 NADH (3 ATP) 1 FADH1 FADH22 (2 ATP) (2 ATP) 1 acetyl-CoA (12 ATP)1 acetyl-CoA (12 ATP)

oxidation of acetyl-CoA via CAC generates additional : oxidation of acetyl-CoA via CAC generates additional : 1GTP (1ATP) + 3 NADH (9 ATP) + 1 FADH1GTP (1ATP) + 3 NADH (9 ATP) + 1 FADH2 2 (2 ATP)(2 ATP)

e.ge.g. complete oxidation of palmitoyl-CoA (C16 fatty acyl group) . complete oxidation of palmitoyl-CoA (C16 fatty acyl group) involves 7 roundsinvolves 7 rounds 7 NADH + 7 FADH7 NADH + 7 FADH22 + 8 acetyl-CoA (8 GTP + 24 NADH + 8 + 8 acetyl-CoA (8 GTP + 24 NADH + 8

FADHFADH22))

31 NADH (93 ATP) + 15 FADH31 NADH (93 ATP) + 15 FADH22 (30 ATP) + 8 ATP (30 ATP) + 8 ATP

Net yield: 131 ATP – 2 ATP (fatty acyl-CoA formation) = 129 ATPNet yield: 131 ATP – 2 ATP (fatty acyl-CoA formation) = 129 ATP

Oxidation of Unsaturated FAsOxidation of Unsaturated FAs

almost all unsat FAs of almost all unsat FAs of biological origin contain biological origin contain only cis double bond only cis double bond between C9-C10 (between C9-C10 (99))

additional double bonds additional double bonds occur at 3-C intervalsoccur at 3-C intervals

Oxidation of Unsaturated FAsOxidation of Unsaturated FAs

Problem 1: A Problem 1: A Double Double bondbond

Solution: enoyl-CoA Solution: enoyl-CoA isomeraseisomerase

Problem 2: A Problem 2: A 4 Double 4 Double Bond Inhibits Hydratase Bond Inhibits Hydratase ActionAction

Solution: 2,4-dienoyl-CoA Solution: 2,4-dienoyl-CoA reductasereductase

Problem 3: Unanticipated Problem 3: Unanticipated Isomerization of 2,5-enoyl-Isomerization of 2,5-enoyl-CoA by 3,2-enoyl-CoA CoA by 3,2-enoyl-CoA IsomeraseIsomerase

3,5-2,4-dienoyl-CoA 3,5-2,4-dienoyl-CoA isomeraseisomerase

Oxidation of Odd-Chain FAsOxidation of Odd-Chain FAs

some plants and marine some plants and marine organisms synthesize fatty organisms synthesize fatty acids with an odd number of acids with an odd number of carbon atomscarbon atoms

final round of final round of oxidation oxidation forms propionyl-CoAforms propionyl-CoA

propionyl-CoA is converted propionyl-CoA is converted to succinyl-CoA for entry into to succinyl-CoA for entry into the CACthe CAC

Succinyl-CoA cannot be directly Succinyl-CoA cannot be directly consumed by the CACconsumed by the CAC

Succinyl-CoA is converted to malate via CACSuccinyl-CoA is converted to malate via CAC at high [malate], it transported to the cytosol (Recall: Malate-at high [malate], it transported to the cytosol (Recall: Malate-

Aspartate Shuttle)Aspartate Shuttle) where it is oxidatively decarboxylated to pyruvate and COwhere it is oxidatively decarboxylated to pyruvate and CO22 by by

malate dehydrogenase malate dehydrogenase Pyruvate is then completely oxidized via pyruvate Pyruvate is then completely oxidized via pyruvate

dehydrogenase and the CACdehydrogenase and the CAC

Peroxisomal Peroxisomal Oxidation Oxidation

In animals, In animals, oxidation of FAs oxidation of FAs occurs both in peroxisome occurs both in peroxisome and mitochondrionand mitochondrion

Peroxisomal Peroxisomal oxidation oxidation shortens very long chain FAs shortens very long chain FAs (> 22 C atoms) in order to (> 22 C atoms) in order to facilitate mitochondrial facilitate mitochondrial oxidationoxidation

In yeast and plants, FA In yeast and plants, FA oxidation occurs exclusively in oxidation occurs exclusively in the peroxisomes and the peroxisomes and glyoxysomesglyoxysomes

Ketone BodiesKetone Bodies

Ketone BodiesKetone Bodies Acetyl-CoA produced by Acetyl-CoA produced by

oxidation of FAs in mitochondria oxidation of FAs in mitochondria can be converted to can be converted to acetoacetone or D-acetoacetone or D---HydroxybutyrateHydroxybutyrate

important metabolic fuels for important metabolic fuels for heart and skeletal muscleheart and skeletal muscle

brain uses only glucose as its brain uses only glucose as its energy source but during energy source but during starvation, ketone bodies starvation, ketone bodies become the major metabolic become the major metabolic fuelfuel

ketone bodies are water-soluble ketone bodies are water-soluble equivalents of fatty acids equivalents of fatty acids

KetogenesisKetogenesis

(1) 2 Acetyl-CoAs condense to (1) 2 Acetyl-CoAs condense to form acetoacetyl-CoA in a form acetoacetyl-CoA in a thiolase-catalyzed reactionthiolase-catalyzed reaction

(2) a Claisen ester (2) a Claisen ester condensation of the condensation of the acetoacetyl-CoA with a third acetoacetyl-CoA with a third acetyl-CoA to form HMG-acetyl-CoA to form HMG-CoA as catalyzed by HMG-CoA as catalyzed by HMG-CoA synthaseCoA synthase

(3) degradation of HMG-CoA (3) degradation of HMG-CoA to acetoacetate and acetyl-to acetoacetate and acetyl-CoA in a mixed aldol-CoA in a mixed aldol-Claisen ester cleavage Claisen ester cleavage catalyzed by HMG-CoA catalyzed by HMG-CoA lyaselyase

Metabolic Conversion of Ketone Bodies Metabolic Conversion of Ketone Bodies to Acetyl-CoAto Acetyl-CoA

liver releases acetoacetate and liver releases acetoacetate and --hydroxybutyrate, which are carried by hydroxybutyrate, which are carried by the bloodstream to peripheral tissues the bloodstream to peripheral tissues for use as alternative fuelfor use as alternative fuel

reduction of acetoacetate to D-reduction of acetoacetate to D---hydroxybutyrate by hydroxybutyrate by -hydroxybutyrate -hydroxybutyrate dehydrogenasedehydrogenase

a stereoisomer of L-a stereoisomer of L--hydroxyacyl--hydroxyacyl-CoA that occurs in the CoA that occurs in the -oxidation -oxidation pathwaypathway

acetoacetate undergoes acetoacetate undergoes nonenzymatic convertion to acetone + nonenzymatic convertion to acetone + COCO22

ketosis or ketoacidosis, individuals ketosis or ketoacidosis, individuals with sweet smell (acetone) produces with sweet smell (acetone) produces acetoacetate faster than it can acetoacetate faster than it can metabolizemetabolize

Ketone Bodies and DiabetesKetone Bodies and Diabetes"Starvation of cells in the midst of plenty""Starvation of cells in the midst of plenty"

Glucose is abundant in blood, but uptake by Glucose is abundant in blood, but uptake by cells in muscle, liver, and adipose cells is low cells in muscle, liver, and adipose cells is low

Cells, metabolically starved, turn to Cells, metabolically starved, turn to gluconeogenesis and fat/protein catabolism gluconeogenesis and fat/protein catabolism

In type I diabetics, OAA is low, due to excess In type I diabetics, OAA is low, due to excess gluconeogenesis, so Ac-CoA from fat/protein gluconeogenesis, so Ac-CoA from fat/protein catabolism does not go to TCA, but rather to catabolism does not go to TCA, but rather to ketone body production ketone body production

Acetone can be detected on breath of type I Acetone can be detected on breath of type I diabeticsdiabetics

Fatty Acid BiosynthesisFatty Acid Biosynthesis

A Comparison of Fatty Acid A Comparison of Fatty Acid Oxidation and Fatty Acid BiosynthesisOxidation and Fatty Acid Biosynthesis

The DifferencesThe DifferencesBetween fatty acid biosynthesis and breakdownBetween fatty acid biosynthesis and breakdown Intermediates in synthesis are linked to -SH Intermediates in synthesis are linked to -SH

groups of groups of acyl carrier proteinsacyl carrier proteins (as compared to (as compared to -SH groups of CoA-SH groups of CoA

Synthesis in Synthesis in cytosolcytosol; breakdown in ; breakdown in mitochondriamitochondria

Enzymes of synthesis are Enzymes of synthesis are one polypeptideone polypeptide Biosynthesis uses Biosynthesis uses NADPH/NADPNADPH/NADP++; breakdown ; breakdown

uses NADH/NADuses NADH/NAD++

Transfer of Acetyl-CoA from Mitochondrion to Transfer of Acetyl-CoA from Mitochondrion to Cytosol via Tricarboxylate Transport SystemCytosol via Tricarboxylate Transport System

Acetyl-CoA is generated in the Acetyl-CoA is generated in the mitochondrion mitochondrion

when demand for ATP is low, when demand for ATP is low, minimal oxidation of Acetyl-CoA minimal oxidation of Acetyl-CoA via CAC and oxidative via CAC and oxidative phosphorylation, Acetyl-CoA is phosphorylation, Acetyl-CoA is stored as fatstored as fat

but fatty acid synthesis occurs in but fatty acid synthesis occurs in cytosol and inner membrane is cytosol and inner membrane is impermeable to acetyl-CoA impermeable to acetyl-CoA

acetyl-CoA enters the cytosol in acetyl-CoA enters the cytosol in the form of citrate via the the form of citrate via the tricarboxylate systemtricarboxylate system

Activation by Malonyl-CoAActivation by Malonyl-CoA

Acetate Units are Activated for Transfer in Fatty Acetate Units are Activated for Transfer in Fatty Acid Synthesis by Malonyl-CoAAcid Synthesis by Malonyl-CoA

Fatty acids are built from 2-C units - acetyl-CoAFatty acids are built from 2-C units - acetyl-CoA Acetate units are activated for transfer by Acetate units are activated for transfer by

conversion to conversion to malonyl-CoAmalonyl-CoA DecarboxylationDecarboxylation of malonyl-CoA and of malonyl-CoA and reducing reducing

powerpower of NADPH drive chain growth of NADPH drive chain growth Chain grows to 16-carbonsChain grows to 16-carbons Other enzymes add double bonds and more CsOther enzymes add double bonds and more Cs

Acetyl-CoA CarboxylaseAcetyl-CoA Carboxylase

Catalyzes the first committed step of FA Catalyzes the first committed step of FA biosynthesis (also a rate-controlling step)biosynthesis (also a rate-controlling step)

Reaction mechanism is similar propionyl-CoA Reaction mechanism is similar propionyl-CoA carboxylase and pyruvate carboxylasecarboxylase and pyruvate carboxylase

2 Steps:2 Steps: a COa CO22 activation activation a carboxylationa carboxylation

Phosphopantetheine Group in Phosphopantetheine Group in Acyl-Carrier Protein and in CoAAcyl-Carrier Protein and in CoA

ACP, like CoA, forms thioesters with acyl groupsACP, like CoA, forms thioesters with acyl groups Ser (OH) of ACP is esterified to the Ser (OH) of ACP is esterified to the

phosphopantetheine group, whereas in CoA it is phosphopantetheine group, whereas in CoA it is esterified to AMPesterified to AMP

Acetyl-CoA CarboxylaseAcetyl-CoA CarboxylaseThe "ACC enzyme" commits acetate to fatty The "ACC enzyme" commits acetate to fatty

acid synthesisacid synthesis Carboxylation of acetyl-CoA to form malonyl-Carboxylation of acetyl-CoA to form malonyl-

CoA is the irreversible, CoA is the irreversible, committed stepcommitted step in fatty in fatty acid biosynthesisacid biosynthesis

ACC uses bicarbonate and ATP (AND ACC uses bicarbonate and ATP (AND biotinbiotin!)!) E.coliE.coli enzyme has three subunits enzyme has three subunits Animal enzyme is one polypeptide with all Animal enzyme is one polypeptide with all

three functions - biotin carboxyl carrier, biotin three functions - biotin carboxyl carrier, biotin carboxylase and transcarboxylasecarboxylase and transcarboxylase

Reaction Cycle for Reaction Cycle for the Biosynthesis the Biosynthesis

of Fatty Acidsof Fatty Acids

Fatty acid synthesis (mainly Fatty acid synthesis (mainly palmitic acid) from acetyl-CoA palmitic acid) from acetyl-CoA and malonyl-CoA involves 7 and malonyl-CoA involves 7 enzymatic reactionsenzymatic reactions

In In E. coliE. coli, 7 different enzymes, 7 different enzymes In yeast and animal, fatty acid In yeast and animal, fatty acid

synthase (FAS), a synthase (FAS), a multifunctional enzyme multifunctional enzyme catalyzes FAs synthesiscatalyzes FAs synthesis

Fatty Acid Biosynthesis IFatty Acid Biosynthesis I

Fatty Acid Biosynthesis IIFatty Acid Biosynthesis II

Fatty Acid Biosynthesis IIIFatty Acid Biosynthesis III

Fatty Acid Synthesis in Fatty Acid Synthesis in Bacteria and PlantsBacteria and Plants

Separate enzymes in a complexSeparate enzymes in a complex See Figure 25.7See Figure 25.7 Pathway initiated by formation of acetyl-ACP Pathway initiated by formation of acetyl-ACP

and malonyl-ACP by and malonyl-ACP by transacylasestransacylases Decarboxylation drives the condensation of Decarboxylation drives the condensation of

acetyl-CoA and malonyl-CoAacetyl-CoA and malonyl-CoA Other Other three stepsthree steps are VERY familiar! are VERY familiar! Only differences: D configuration and NADPHOnly differences: D configuration and NADPH Check equations on page 811!Check equations on page 811!

Fatty Acid Synthesis in Fatty Acid Synthesis in AnimalsAnimals

Fatty Acid Synthase - a multienzyme complexFatty Acid Synthase - a multienzyme complex Dimer of 250 kD multifunctional polypeptidesDimer of 250 kD multifunctional polypeptides Note the roles of Note the roles of active site serinesactive site serines on AT & on AT &

MTMT Study the mechanism in Figure 25.11 - note Study the mechanism in Figure 25.11 - note

the roles of ACP and KSasethe roles of ACP and KSase Steps 3-6Steps 3-6 repeat to elongate the chain repeat to elongate the chain

Fatty Acid SynthaseFatty Acid Synthase MAT (malonyl/acetyl-CoA-ACP MAT (malonyl/acetyl-CoA-ACP

transacetylase)transacetylase) KS (KS (-ketoacyl-ACP synthase)-ketoacyl-ACP synthase) DH (DH (-hydroxyacyl-ACP -hydroxyacyl-ACP

dehydrase)dehydrase) ER (enoyl-ACP reductase)ER (enoyl-ACP reductase) KR (KR (-ketoacyl-ACP reductase)-ketoacyl-ACP reductase) TE (Palmitoyl thioesterase)TE (Palmitoyl thioesterase)

Further Processing of FAsFurther Processing of FAs Additional elongation - in mitochondria and ERAdditional elongation - in mitochondria and ER Introduction of cis double bonds - do you need Introduction of cis double bonds - do you need

OO22 or not? or not?

E.coliE.coli add double bonds while the site of attack add double bonds while the site of attack is still near something functional (the thioester)is still near something functional (the thioester)

Eukaryotes add double bond to middle of the Eukaryotes add double bond to middle of the chain - and need power of Ochain - and need power of O22 to do it to do it

Polyunsaturated FAs - plants vs animals...Polyunsaturated FAs - plants vs animals...

Desaturation Desaturation

Unsaturated fatty acids are produced by terminal Unsaturated fatty acids are produced by terminal desaturasesdesaturases

99-, -, 66-, -, 55-, -, 44-fatty acyl-CoA desaturases-fatty acyl-CoA desaturases

Regulation of Fatty Acid Regulation of Fatty Acid MetabolismMetabolism

Sites of Regulation of Fatty Acid MetabolismsSites of Regulation of Fatty Acid Metabolisms

Hormones regulate fatty acid Hormones regulate fatty acid metabolismmetabolism

[glucose] [glucose] : : cells cells glucagon glucagon (fatty acid oxidation)(fatty acid oxidation)

[glucose] [glucose] : : cells cells insulin (fatty insulin (fatty acid biosynthesis)acid biosynthesis)

Short-term regulationShort-term regulationsubstrate availability, allosteric substrate availability, allosteric

interactions and covalent modificationsinteractions and covalent modificationsresponse time < 1 minresponse time < 1 min

Long-term regulationLong-term regulation [enzyme] depends on rates of protein [enzyme] depends on rates of protein

synthesis and/or breakdownsynthesis and/or breakdownprocess requires hours or daysprocess requires hours or days

Epinephrine & norepinephrine Epinephrine & norepinephrine activate “hormone-sensitive” lipase, activate “hormone-sensitive” lipase, releases FAs, which are exported to releases FAs, which are exported to the liver for degradationthe liver for degradation

Regulation of FA SynthesisRegulation of FA SynthesisAllosteric modifiers, phosphorylation and Allosteric modifiers, phosphorylation and

hormoneshormones Malonyl-CoA blocks the carnitine Malonyl-CoA blocks the carnitine

acyltransferase and thus acyltransferase and thus inhibits beta-oxidationinhibits beta-oxidation Citrate Citrate activates acetyl-CoA carboxylaseactivates acetyl-CoA carboxylase Fatty acyl-CoAsFatty acyl-CoAs inhibit acetyl-CoA carboxylase inhibit acetyl-CoA carboxylase Hormones regulate ACCHormones regulate ACC Glucagon activatesGlucagon activates lipases/inhibits ACC lipases/inhibits ACC Insulin inhibitsInsulin inhibits lipases/activates ACC lipases/activates ACC

Biosynthesis of Complex Biosynthesis of Complex LipidsLipids

Synthetic pathways depend on organismSynthetic pathways depend on organism Sphingolipids and triacylglycerols only Sphingolipids and triacylglycerols only

made in eukaryotesmade in eukaryotes PE accounts for 75% of PLs in PE accounts for 75% of PLs in E.coliE.coli No PC, PI, sphingolipids, cholesterolNo PC, PI, sphingolipids, cholesterol in in

E.coliE.coli But some bacteria do produce PCBut some bacteria do produce PC

Glycerolipid BiosynthesisGlycerolipid BiosynthesisCTP drives formation of CDP complexesCTP drives formation of CDP complexes

Phosphatidic acid (PA) is the precursor for all Phosphatidic acid (PA) is the precursor for all other glycerolipids in eukaryotesother glycerolipids in eukaryotes

See Figure 25.18See Figure 25.18 PA is made either into DAG or CDP-DAGPA is made either into DAG or CDP-DAG Note the roles of Note the roles of CDP-cholineCDP-choline and and CDP-CDP-

ethanolamineethanolamine in synthesis of PC and PE in in synthesis of PC and PE in Figure 25.19Figure 25.19

Note exchange of ethanolamine for serine Note exchange of ethanolamine for serine (25.21)(25.21)

Other PLs from CDP-DAGOther PLs from CDP-DAGFigure 25.22Figure 25.22

CDP-diacylglycerolCDP-diacylglycerol is used in eukaryotes is used in eukaryotes to produce:to produce: PI in one stepPI in one step PG in two stepsPG in two steps Cardiolipin in three stepsCardiolipin in three steps

Plasmalogen BiosynthesisPlasmalogen BiosynthesisDihydroxyacetone phosphate is the Dihydroxyacetone phosphate is the

precursorprecursor Acylation activates and an exchange Acylation activates and an exchange

reaction produces the ether linkagereaction produces the ether linkage Ketone reduction is followed by acylationKetone reduction is followed by acylation CDP-ethanolamineCDP-ethanolamine delivers the delivers the

headgroupheadgroup A desaturase produces the double bond A desaturase produces the double bond

in the alkyl chainin the alkyl chain

Sphingolipid BiosynthesisSphingolipid BiosynthesisHigh levels made in neural tissueHigh levels made in neural tissue

Initial reaction is a condensation of serine and Initial reaction is a condensation of serine and palmitoyl-CoApalmitoyl-CoA

3-ketosphinganine synthase is 3-ketosphinganine synthase is PLP-dependentPLP-dependent Ketone is reduced with help of Ketone is reduced with help of NADPHNADPH Acylation is followed by double bond formationAcylation is followed by double bond formation See Figure 25.25See Figure 25.25 Resulting ceramide is precursor for other Resulting ceramide is precursor for other

sphingolipidssphingolipids

Eicosanoid BiosynthesisEicosanoid Biosynthesis

PLAPLA22 releases arachidonic acid - a releases arachidonic acid - a

precursor of eicosanoidsprecursor of eicosanoids Eicosanoids are Eicosanoids are local hormoneslocal hormones The endoperoxide synthase oxidizes The endoperoxide synthase oxidizes

and cyclizesand cyclizes Tissue injury and inflammation triggers Tissue injury and inflammation triggers

arachidonate release and eicosanoid arachidonate release and eicosanoid synthesissynthesis

Eicosanoid BiosynthesisEicosanoid Biosynthesis Aspirin and other nonsteroid anti-Aspirin and other nonsteroid anti-

inflammatory agents inhibit the inflammatory agents inhibit the cyclooxygenasecyclooxygenase Aspirin covalentlyAspirin covalently Others noncovalentlyOthers noncovalently

Cholesterol BiosynthesisCholesterol BiosynthesisOccurs primarily in the liverOccurs primarily in the liver

Biosynthesis begins in the cytosol with the Biosynthesis begins in the cytosol with the synthesis of mevalonate from acetyl-CoAsynthesis of mevalonate from acetyl-CoA

First step is a thiolase reactionFirst step is a thiolase reaction Second step makes HMG-CoASecond step makes HMG-CoA Third step - Third step - HMG-CoA reductaseHMG-CoA reductase - is the - is the

rate-limiting step in cholesterol rate-limiting step in cholesterol biosynthesisbiosynthesis

HMG-CoA reductase is site of action of HMG-CoA reductase is site of action of cholesterol-lowering drugscholesterol-lowering drugs

Regulation of HMG-CoA Regulation of HMG-CoA ReductaseReductase

As rate-limiting step, it is the principal site of As rate-limiting step, it is the principal site of regulation in cholesterol synthesisregulation in cholesterol synthesis

1) 1) PhosphorylationPhosphorylation by cAMP-dependent by cAMP-dependent kinases inactivates the reductasekinases inactivates the reductase

2) 2) Degradation Degradation of HMG-CoA reductase - of HMG-CoA reductase - half-life is 3 hrs and depends on cholesterol half-life is 3 hrs and depends on cholesterol levellevel

3) 3) Gene expressionGene expression (mRNA production) is (mRNA production) is controlled by cholesterol levelscontrolled by cholesterol levels

The thiolase brainteaser...The thiolase brainteaser...An important puzzleAn important puzzle

If acetate units can be condensed by If acetate units can be condensed by thiolase to give acetoacetate in the 1st thiolase to give acetoacetate in the 1st step of cholesterol biosynthesis, why not step of cholesterol biosynthesis, why not also use thiolase for FA synthesis, also use thiolase for FA synthesis, avoiding complexity of FA synthase?avoiding complexity of FA synthase?

Solution: Subsequent reactions drive Solution: Subsequent reactions drive cholesterol synthesis, but cholesterol synthesis, but eight successive eight successive thiolasethiolase reactionsreactions would be very would be very unfavorable energetically for FA synthesisunfavorable energetically for FA synthesis

Squalene from MevalonateSqualene from MevalonateDriven by ATP hydrolysis, decarboxylation Driven by ATP hydrolysis, decarboxylation

and PPand PPii hydrolysis hydrolysis

Six-carbon mevalonate makes five carbon Six-carbon mevalonate makes five carbon isopentenyl PPisopentenyl PPii and dimethylallyl PP and dimethylallyl PP ii

Condensation of 3 of these yields farnesyl Condensation of 3 of these yields farnesyl PPPPii

Two farnesyl PPTwo farnesyl PPii s link to form squalene s link to form squalene

Bloch and LangdonBloch and Langdon were first to show that were first to show that squalene is derived from acetate units and squalene is derived from acetate units and that cholesterol is derived from squalenethat cholesterol is derived from squalene

Cholesterol from SqualeneCholesterol from Squalene

At the endoplasmic reticulum membraneAt the endoplasmic reticulum membrane Squalene monooxygenase converts Squalene monooxygenase converts

squalene to squalene to squalene-2,3-epoxidesqualene-2,3-epoxide A cyclase converts the epoxide to lanosterolA cyclase converts the epoxide to lanosterol Though lanosterol looks like cholesterol, Though lanosterol looks like cholesterol, 20 20

more stepsmore steps are required to form cholesterol! are required to form cholesterol! All at/in the endoplasmic reticulum All at/in the endoplasmic reticulum

membranemembrane

Inhibiting Cholesterol SynthesisInhibiting Cholesterol SynthesisMerck and the Lovastatin story...Merck and the Lovastatin story...

HMG-CoA reductase is the key - the rate-HMG-CoA reductase is the key - the rate-limiting step in cholesterol biosynthesislimiting step in cholesterol biosynthesis

Lovastatin (mevinolin)Lovastatin (mevinolin) blocks HMG-CoA blocks HMG-CoA reductase and prevents synthesis of reductase and prevents synthesis of cholesterolcholesterol

Lovastatin is an (inactive) lactoneLovastatin is an (inactive) lactone In the body, the lactone is hydrolyzed to In the body, the lactone is hydrolyzed to

mevinolinic acid, a competitive mevinolinic acid, a competitive (TSA!)(TSA!) inhibitor inhibitor of the reductase, of the reductase, KKii = 0.6 nM! = 0.6 nM!

Biosynthesis of Bile AcidsBiosynthesis of Bile AcidsCarboxylic acid derivatives of cholesterolCarboxylic acid derivatives of cholesterol

Essential for the digestion of food, especially Essential for the digestion of food, especially for solubilization of ingested fats for solubilization of ingested fats

Synthesized from cholesterolSynthesized from cholesterol Cholic acid conjugates with taurine and glycine Cholic acid conjugates with taurine and glycine

to form to form taurocholic and glycocholic acidstaurocholic and glycocholic acids First step is oxidation of cholesterol by a First step is oxidation of cholesterol by a

mixed-function oxidasemixed-function oxidase

Steroid Hormone SynthesisSteroid Hormone SynthesisDesmolase (in mitochondria) forms Desmolase (in mitochondria) forms pregnenolone, precursor to all otherspregnenolone, precursor to all others

Pregnenolone migrates from mitochondria to Pregnenolone migrates from mitochondria to ER where progesterone is formedER where progesterone is formed

Progesterone is a branch pointProgesterone is a branch point - it produces - it produces sex steroids (testosterone and estradiol), and sex steroids (testosterone and estradiol), and corticosteroids (cortisol and aldosterone) corticosteroids (cortisol and aldosterone)

Anabolic steroids are illegal and dangerousAnabolic steroids are illegal and dangerous Recall the Ben Johnson story....Recall the Ben Johnson story....