15 april 2008 lipid metabolism andy howard introductory biochemistry 15 april 2008

15 April 2008

Lipid Metabolism

Andy HowardIntroductory Biochemistry

15 April 2008

15 April 2008 Lipid Metabolism p. 2 of 85

Making and Breaking Lipids Lipid biosynthesis is a significant route to

the creation of energy-storage molecules, membrane components, and hormones;

Lipid catabolism is a critical energy-producing pathway, and we also need to understand degradation of functional lipids

… but first, a few final slides about plants!


What we’ll discuss End of plant stuff Lipid anabolism

Fatty acid synthesisMaking fats and

phospholipidsEicosanoidsEther lipidsSphingolipids Isoprenoids & steroids

Fatty acid oxidation Sequence of reactions

for saturated FAs Unsaturations Energetics

Phospholipid degradation

Steroid and other degradative systems


Crassulacean acid metabolism

Leaf cells open to CO2 uptake lose a lot of water during the day(high evaporation rate)

Solution: assimilate carbon at night Reactions are as in C4 pathway;

cellular specialization and enzyme regulation are different


Stomata and vacuoles Stomata (spaces between cells that

can open to allow access for respiration) near mesophylls open only at night, enabling PEP carboxylation to oxalacetate and then reduction to malate

Malate stored in central vacuole, then released during the day when the stomata are closed


CAM: day and night

University of Newcastle, Plant Physiology program


iClicker quiz question 1 Oxidation of a 2n-

carbon fatty acid yields (n-1) QH2,(n-1) NADH, and n acetyl CoA. Initiating the process costs 2 ATPs. Assume we can get 10 ATP per acetyl CoA. How much ATP can we get from oxidizing palmitate?

(a) 104 ATP (b) 106 ATP (c ) 108 ATP (d) 112 ATP (e) Undeterminable

given the data supplied


Answer to 1st question Palmitate is a C16 carboxylic acid.

Therefore in the conditions of the problem, 2n = 16, n = 8, n-1 = 7.

Thus we get 7 QH2, 7 NADH,8 acetyl CoA produced by its oxidation

Thus we get 7*2.5 + 7 * 1.5 + 8 * 10 = 17.5 + 10.5 + 80 = 108 ATP produced

Starting the process costs 2 ATP, so the net result is 106 ATP gained


iClicker quiz question 2Why would you not expect to find crassulacean

acid metabolism in tropical plants? (a) Tropical plants do not photosynthesize. (b) Tropical plants cannot develop the stomata

that close off the chloroplast-containing cavities (c) Water conservation is less critical in areas of

high rainfall (d) The waxy coating required to close off the

leaves’ access to O2 would dissolve in the high humidity and high temperature of the tropics

(e) None of the above


Answer: (c)

The primary significance of CAM is conservation of water in regions of low humidity, where evaporation rates are high and water is scarce. Neither of these conditions pertains in the tropics.


Control of CAM PEP carboxylase inhibited by

malate and low pH That prevents activity during

daylight, which would lead to futile cycling and competition for CO2 between PEP carboxylase and RuBisCO


Compartmentation in bacteria In photosynthetic bacteria,

RuBisCO is concentrated in protein microcompartment called a carboxysome

Active carbonic anhydrase there: catalyzes HCO3

- OH- + CO2

That tends to keep the CO2 / O2 ratio high


Lipids:What we won’t cover

Special Cases Locations for synthesis Regulation by hormones Absorption and mobilization Ketone bodies


Lipid Anabolism Generally the starting point for building

up lipids are acetyl CoA and malonyl CoA, and their variants acetyl ACP and malonyl ACP Fatty acids Steroids

These are energy-requiring reactions: the compounds we’re making are reduced

Malonyl CoA


Overview (cf. fig. 16.1)

Bacteria:acetyl CoA + malonyl ACP acetoacetyl ACP + CO2 + CoASH

Eukaryotes:acetyl CoA + ACP

acetyl ACP + CoASHAcetyl ACP + malonyl ACP acetoacetyl

ACP + CO2 + ACP

Acetoacetyl ACP


Making malonyl CoA

Acetyl CoA incorporates an extra methylene via acetyl CoA carboxylase

Biotin- and ATP-dependent enzyme; similar to pyruvate carboxylase

PDB 1w96 (biotin carboxylase domain)183 kDa trimeryeast

1uyr (carboxyl-transferase domain)162 kDa dimer; yeast


Making malonyl ACP

Malonyl CoA:ACPtransacylasetransfers the malonate group from coenzyme A to the acyl carrier protein

Ferredoxin-like protein Similar enzyme converts acetyl

CoA to acetyl ACP

PDB 1NM235 kDa monomerStreptomyces coelicolor


Acyl carrier protein itself

Acts as a template on which acyl chain elongation can occur

Simple protein: 83 amino acids, mostly helical

This is actually an NMR structure

PDB 1OR59.1 kDa monomerStreptomyces


Initiation reaction

We want to start with a four-carbon unit attached to acyl carrier protein

We get that by condensing acetyl CoA or acetyl ACP with malonyl ACP with ketoacyl ACP synthase (KAS) to form acetoacetyl ACP

Intermediate has KAS covalently attached to both substrates

Decarboxylation of enzyme-bound intermediates leads to 4-carbon unit attached to ACP3 + 2 1 + 4


Is this typical? Yes! We’ve carboxylated acetyl CoA to

make malonyl ACP and then decarboxylated the product of malonyl ACP with acetyl CoA / ACP

This provides a favorable free-energy change (at the expense of ATP) for the overall reaction

Similar approach happens in gluconeogenesis(pyruvate oxaloacetate PEP)

Ketoacyl ACP synthasePDB 1HNJ70 kDa dimer;monomer shownE.coli


Elongations in FA synthesis: overview Acetoacetyl ACP: starting point for elongations Pattern in each elongation is

reduction dehydration reduction,resulting in a saturated product

Reenter pathway by condensing with malonyl ACP Elongated product plays the same role that acetyl

CoA or acetyl ACP plays in the initial -ketoacyl ACP synthase reaction: C2n + C3 -> CO2 + C2n+2


1st step: reduce ketone sec-alcohol

Enzyme:3-ketoacylACP reductase

Ketone reacts with NADPH+ H+ to produce sec-alcohol + NADP

D-isomer of sec-alcohol always forms;by contrast, during degradation,L-isomer forms

Enzyme is typical NAD(P)-dependent oxidoreductase

PDB 2C07125 kDa tetramer; Monomer shownPlasmodium falciparum


2nd step: alcohol to enoyl ACP

3-hydroxyacyl ACP dehydratase Eliminates water at beta, alpha

postions to producetrans-2-enoyl ACP:R–CHOH–CH2-CO-S-ACP R–CH=CH–CO-S-ACP + H2O

Note that this is a derivative of atrans-fatty acid; but it’s complexed to ACP!

This form is primarily helical;there is an alternative found in Aeromonas that is an alpha-beta roll structure

PDB 1DCI182 kDa hexamertrimer shownRat mitochondria


3rd step:enoyl CoA to saturated ACP

Enzyme: enoyl-ACP reductase Leaves behind fully saturated FA

complexed to acyl carrier protein:R–CH=CH–CO-S-ACP R–CH2CH2CO-S-ACP

This can then condense with malonyl ACP with decarboxylation to form longer beta-ketoacyl ACP:Rn-ACP + malonyl-CoA -keto-Rn+2-ACP + CO2 + CoASH

Enzyme is FMN-dependent

PDB 2Z6I73 kDa dimerStreptococcus pneumoniae


How does this end? Generally starts at C4 and

goes to C16 or C18. Condensing enzyme won’t fit

longer FAs Completed fatty acid is

cleaved from ACP by action of a thioesterasewith a 3-layer Rossmann fold

Palmitoyl thioesterase IPDB 1EI931 kDa monomerbovine


The overall reaction Acetyl CoA + 7 Malonyl CoA + 14NADPH +

14 H+ 14 NADP + Palmitate + 7CO2 + 8HS-CoA + 6H2O

In bacteria we have separate enzymes:a type II fatty acid synthesis system

In animals we have a type I FA synthesis system: a large, multi-functional enzyme including the phosphopantatheine group by which the ACP attaches


iClicker questionWhat advantage, if any, might be associated with

type I fatty acid synthesis systems? (a) None (b) Reactants remain associated with the

enzymatic complex, reducing diffusive inefficiencies

(c) Lowered probability of undesirable reductions of metabolites

(d) Lowered probability of undesirable oxidations of metabolites

(e) improved solubility of products


Answer: (b)

If the enzyme doesn’t have to find the substrate at the beginning of each reaction, things will proceed more readily.


Activating fatty acids

Activate stearate or palmitatevia acyl CoA synthetase:

R–COO- + CoASH + ATP R–CO–SCoA + AMP + PPi

As usual, PPi hydrolysis drivesthe reaction to the right

PLP-dependent reaction Bacteria have one acyl CoA synthetase Mammals: four isozymes for different FA

lengths (small, medium, long, very long)

PDB 1BS042 kDa monomerE.coli


Extending and unsaturating fatty acids

There are applications for FAs with more than 18 carbons and FAs with >=1 cis double bonds

Elongases and desaturases exist to handle these needs (fig. 16.7)

Desaturase adds a cis-double bond; if the FA already has unsaturations, the new one is added three carbons closer to the carboxyl

Elongases condense FA with malonyl CoA; decarboxylation means we add two carbons


Bacterial Desaturases

Acyl ACP desaturases in bacteria simply add a cis double bond in place of the normal trans double bond at the second phase of elongation; the cis double bond thus created remains during subsequent rounds

Ferritin-like structure

PDB 1ZA0;30 kDa monomerMycobacteriumtuberculosis


Eukaryotic Desaturases

Desaturases like stearoyl ACP desaturase in eukaryotes act on the completed saturated fatty acyl CoA species

Enzyme is ferritin-like or RNR-like

Mammals can’t synthesize linoleate and they need it, so it has to be part of the diet

PDB 1OQ980 kDa dimermonomer showncastor bean


Making arachidonate We can convert dietary linoleate to

archidonyl CoA via desaturation and elongations (fig. 16.7)

The fact that the new double bonds start 3 carbons away from the previous one means they’re not conjugated


Phosphatidates

Phosphatidates are intermediates in making triacylglycerol & glycerophospholipids

Fatty acyl groups esterifying 1 and 2 positions of glycerol, phosphate esterifying 3 position


Making phosphatidates

Glycerol-3-phosphate acyltransferase transfers acyl CoA to 1 position of glycerol-3-phosphate; prefers saturated chains

1-acylglycerol-3-phosphate acyl transferase transfers acyl CoA to 2 position of resulting molecule; prefers unsaturated chains

PDB 1IUQ40 kDa monomer

Cucurbita


Making triacylglycerols Phosphatidate phosphatase gets rid

of the phosphate at the 3 position by hydrolysis to make 1,2-diacylglycerol A bit counterintuitive in making

phospholipids: why get rid of the phosphate when you’re going to put a phosphorylated compound back at 3 position?

But the groups you add already have phosphate on them


Further steps in making triacylglycerols Diacylglycerol acyltransferase catalyzes

reaction between 1,2-diacylglycerol and acyl CoA to form triacylglycerol

See fig. 16.9, left-hand side


Making phospholipids from 1,2-diacylglycerol

1,2-diacylglycerol reacts with CDP-choline to form phosphatidylcholine with liberation of cytidine monophosphate

1,2-diacylglycerol reacts with CDP-ethanolamine to form phosphatidylethanolamine this can be methylated 3 times to make

phosphatidylcholine S-adenosylmethionine is the methyl donor in

that case


How do we get CDP-alcohols?

Easy:CTP + alcohol phosphate CDP-alcohol + PPi

As usual, reaction is driven to the right by hydrolysis of PPi

Enzymes are CTP:phosphoethanolamine cytidylyltransferase and CTP:phosphocholine cytidylyltransferase

CDP-ethanolamine


Making acidic phospholipids

Phosphatidate activated to CDP-diacylglycerol as catalyzed by CTP:phosphatidate cytidylyltransferase with release of PPi (see previous reactions)

This can react with serine or inositol to form the relevant phospholipids; see fig. 16.10.

This route to phosphatidylserine is found only in bacteria


Phosphatidylserine


Phosphatidylinositol Phosphatidylinositol is made by this CDP-

diacylglycerol pathway in bacteria and eukaryotes


Making phosphatidylserine

Alternative approach to phosphatidylserine found in eukaryotes:make phosphatidylethanolamine, then phosphatidylethanolamine:serine transferase swaps serine for ethanolamine

When we do it that way, we can recover phosphatidylethanolamine back by a decarboxylation (or another exchange)

Ethanolamine is just serine without COO- !


Where does this happen?

Mostly in the endoplasmic reticulum in eukaryotes

Biosynthesis enzymes are membrane bound but have their active sites facing the cytosol so they can pick up the water-soluble metabolites from which they can build up phospholipids and other lipids


Making eicosanoids

Classes of eicosanoids: Prostaglandins and thromboxanes Leukotrienes

Remember that we make arachidonate from linoleoyl CoA; eiconsanoids made from arachidonate

Reactions involve formation of oxygen-containing rings; thus the enzymes are cyclooxygenases


What eiconsanoids do They’re like hormones, but they act very

locally: within µm of the cell in which they’re produced

Involved in platelet aggregation, blood clots, constriction of smooth muscles

Mediate pain sensitivity, inflammation, swelling

Therefore enzymes that interconvert them are significant drug targets!


Synthesizing prostaglandins

Prostaglandin H synthase (PGHS) binds on inner surface of ER

Cyclooxygenase activity makes a hydroperoxide; this is converted to PGH2

PGH2 gets converted to other prostaglandins, prostacyclin, thromboxane A2 (fig. 16.12)

Prostaglandin H2

PDB 2OYU132 kDa dimerMonomer shownsheep


How aspirin works

Aspirin blocks irreversibly inhibits the COX activity of PGHS by transferring an acetyl group to an active-site Ser

That blocks eiconsanoid production, which reduces swelling and pain

But there are side effects because some PGHS isozymes are necessary


Cyclooxygenase inhibition Cox-1 is constitutive and regulates secretion of

mucin in the stomach Cox-2 is inducible and promotes inflammation,

pain, fever Aspirin inhibits both: the mucin-secretion

inhibition means that causes bleeding or ulcers in the stomach lining

Other nonsteroidal anti-inflammatories (NSAIDs) besides aspirin compete with arachidonate rather than binding covalently to COX-1 and COX-2


Could we find a COX-2 inhibitor?

This would eliminate the stomach irritation that aspirin causes

Some structure-based inhibitors have been developed

They work as expected; but They also increase risk of cardiovascular

disease Prof. Prancan (Rush U) discussed these

issues in his February 2007 colloquium


Leukotrienes Lipoxygenases convert

arachidonate to these compounds, which contain 3 conjugated double bonds

These compounds interact with GPCRs

Involved in inflammatory and allergic reactions

Also involved in the pathophysiology of asthma

Leukotriene B4

PDB 2P0M146 kDa dimerrabbit


Synthesis of ether lipids

Remember: these arelipids with ether linkages instead of acyl linkages

Begins with dihydroxyacetone phosphate Acyltransferase acylates DHAP C-1 1-alkyl-DHAP synthase swaps an alcohol for the

acyl group at C-1 Keto group at C2 of DHAP is reduced to an

alcohol (NADPH-dependent reaction)


Ether lipids, continued

1-alkylglycerophosphate acyltransferase adds another acyl group at C-2

Dephosphorylated at C-3 (as with phospholipids … take the P off, put it back on …)

Phosphocholine or other phosphate-based ligand added at C-3

Plasmalogens earn a double bond between the two carbons adjacent to the ether oxygen on C-1


Sphingolipid synthesis

These are based formally on sphingosine, a C18unsaturated amino alcohol (fig.16.14)

Condense serine with palmitoyl CoA to make 3-ketosphinganine and CO2

NADPH-reduce this to sphinganine Acetylate the amine group to make N-

acylsphinganine Beta-unsaturate the palmitoyl group to make

ceramide, the basis for all other sphingolipids

ceramide








ceramide


Other sphingolipids

React ceramide with phosphatidylcholine;products are sphingomyelin and 1,2-diacylglycerol

React ceramide with UDP-galactose to form a galactocerebroside

Additional UDP-sugars or CMP-N-acetyl-neuraminic acid can be added

spingomyelin


Steroid synthesis: overview

Cholesterol is important on its own & as a precursor of steroid hormones, bile salts

Derived formally from isoprene Isoprenoid synthesis based on

mevalonate &isopentenyldiphosphate


Making HMG-CoA Condense 3 molecules of acetyl CoA:

2 acetyl CoA acetoacetyl CoA + CoASH;catalyzed by acetoacetyl CoA synthase

Acetoacetyl CoA + acetyl CoA + H2O 3-hydroxy-3-methylglutaryl CoA + CoASH + H+

catalyzed by HMG CoA synthase These are important intermediates: precursor to

steroids and ketone bodies Not the committed step toward isoprenoids

because we can also make ketone bodies from HMG-CoA


iClicker quiz question Creation of new C-C bonds requires

energy. Where is it coming from in these condensations?

(a) enzymatic catalysis (b) hydrolysis of ATP (c) hydrolysis of thioether bonds (d) hydrolysis of thioester bonds (e) none of the above


Answer: (d)

(a) no. Enzymatic catalysis doesn’t change thermodynamics: it changes kinetics

(b) no. There’s no ATP involved. (c) no. These acyl CoA molecules

contain thioester linkages (d) yes. Hydrolysis of thioester linkages

yields substantial amounts of free energy


HMGCoA tomevalonate

HMGCoA reductase is the first committed step on pathway toward isoprenoids

HMGCoA + 2NADPH + 2H+ mevalonate + 2NADP+ + CoASH

Many drug-discovery projects involve inhibition of this enzyme

PDB 1DQA205 kDa tetramerHuman

PDB 1DQA205 kDa tetramerhuman


Mevalonate to isopentenyl diphosphate Two successive ATP-dependent kinase steps

convert mevalonate to mevalonate 5-diphosphate

ATP-dependent decarboxylation yields isopentenyl diphosphate

This is an isoprene-donating group involved in making non-steroidal isoprenoid compounds as well as steroids


Mevalonate kinase Converts mevalonate to

mevalonate 5-phosphate Secondary control point in

isoprenoid synthetic pathway Human diseases associated

with abnormalities Mevalonic aciduria Hyperimmunoglobulinemia

(Periodic fever syndrome)

PDB 2HFS73 kDa dimer;monomer shownLeishmania major


Isopentenyl diphosphate to squalene Isomerized to dimethylallyl diphosphate That condenses with another molecule of IPDP

to make geranyl diphosphate (C10) Another condensation with IPDP (with the same

enzyme) makes farnesyl diphosphate (C15) Two farnesyl diP fuse head-to-head to make

squalene (C30, no heteroatoms)


Geranyl diphosphate & farnesyl diphosphate

Geranyl diphosphate:C10

Farnesyl diphosphate:C15


Squalene Made via head-to-head synthesis from 2

molecules of farnesyl diphosphate

Squalene: C30


Squalene to cholesterol

Several messy steps move the double bonds around

replace double bonds with ring closures lanosterol

Eliminate 3 methyls, move one double bond, remove another double bond, and voila: cholesterol

lanosterol

lanosterol synthase; converts 2,3-oxidosqualene to lanosterolPDB 1W6K81 kDa monomerhuman


What happens to cholesterol?

Inserted into membranes Assembled into lipoproteins Derivatized to make bile salts Modified into hormones


Other isoprenoids Generally made from isopentenyl

pyrophosphate Pathways to isopentenyl pyroP are

ancient: used in bacteria Pathways to steroids comparatively

recent Cholesterol essential in animal

membranes; plants have other sterols like campesterol (24-methyl-cholesterol)


Lipid catabolism

We’ve been focusing on making lipids Now we’ll look at how they’re broken

down As energy sources In recycling lipid components that have

functional or structural significance


Fatty acid (beta) oxidation Degradation proceeds 2 C at a time

somewhat like synthesis Called -oxidation because in each round the form

that gets shortened is a -ketoacyl CoA Activated form is acyl CoA, not acyl ACP Product is n molecules of acetyl CoA from a

2n-carbon fatty acid Yields n-1 NADH and n-1 QH2

Occurs in the mitochondrion or peroxisome, whereas synthesis occurs in the cytosol


Reactions in -oxidation Proceeds through cycle n times for a 2n-

carbon fatty acid

Diagram courtesy Richard Paselk, Humboldt State U.


Acyl-CoA dehydrogenase

Converts fatty acid saturated at C2,3 to trans-2-Enoyl CoA:—CH2—CH2—COSCoA

Several isozymes for various sizes of FAs

medium-chain acyl CoA dehydrogenasePDB 3MDE169kDa tetramer;dimer shownPig liver


Electron-Transferring Flavoprotein (ETF)

Here, it converts FADH2 created by acyl-CoA dehydrogenase back to FAD via Fe-S protein

Rossmann-fold protein Plays role in other redox

reactions Ultimate acceptor is Q, which

can be re-oxidized in the ETS

PDB 1EFV63 kDa heterodimerhuman


Hydration step

Enzyme is 2-enoyl CoA dehydratase

- roll protein onverts enoyl CoA to L-3-

hydroxyacyl CoA Remember this is the opposite

stereochemistry relative to synthetic intermediate

PDB structure 1S9C: dehydratase domain of human multifunctional enzyme


Second oxidative step

Enzyme is L-3-hydroxyacyl-CoA dehydrogenase

NADH is reduced product NADH can be used in

biosynthesis (via shuttles) or oxidized in the ETSRossmann-fold protein

dehydrogenase domain of multifunctional enzymePDB 1E6W114 kDa tetramerrat


Thiolysis HS-CoA attacks C3-

carbonyl and cleaves off acetyl CoA, resulting in shortening by two carbons

Enzyme is 3-ketoacyl-CoA thiolase

Similar to acetoacyl-CoA thiolase found in isopentenyl diP pathway

Substrate can go through another round

PDB 1QFL171 kDa tetramerZooglea


Formal similarity

… between FA oxidation steps 1-3 and middle reactions of TCA cycle:

–CH2CH2– oxidized to trans-CH=CH—:like succinate to fumarate

Trans-ene hydrated to L-CHOH-CH2—:like fumarate to L-malate

Alcohol oxidized to ketone:like L-malate to oxalacetate


Peroxisomal -oxidation

Very common in many non-mammalian

eukaryotes it’s the only kind In mammals this handles odd cases;

mitochondria are the primary oxidizers Initial reaction doesn’t produce QH2:

it produces hydrogen peroxide as the other product besides trans2enoyl CoA

Reaction catalyzed by acyl-CoA oxidase Peroxisomes don’t have ETS so the reducing

equivalents used in other ways Compartmentation keeps H2O2 away from ETS

PDB 1IS2145 kDa dimerrat liver

15 april 2008 lipid metabolism andy howard introductory biochemistry 15 april 2008

Documents

n acetyl coa

ncarbon fatty acid yields

high humidity

conservation of water

high temperature

atp producedstarting

c16 carboxylic acid

b tropical plants