lec10 lipid oxid

BiochemistrySixth Edition

Chapter 22:Fatty Acid Metabolism

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

Fatty acid metabolism

An overview TAG are highly concentrated energy source Steps in fa oxidation Additional steps are required for certain fas Fa synthesis Controlling fa metabolism Elongation and unsaturation of fas

Electron Micrograph of Adipocyte

Utilization of fas

3 stages

1. Mobilization‒ TAG fa + glycerol

2. Fa activation and transportation into mitochondria where oxidation takes place.

3. Fa breakdown to Acetyl-CoA

FIGURE 17-3 Mobilization of triacylglycerols stored in adipose tissue. When low levels of glucose in the blood trigger the release of glucagon, 1 the hormone binds its receptor in the adipocyte membrane and thus 2 stimulates adenylyl cyclase, via a G protein, to produce cAMP. This activates PKA, which phosphorylates 3 the hormone-sensitive lipase and 4 perilipin (a family of proteins that restrict access to lipid droplets, preventing untimely lipid mobilization)molecules on the surface of the lipid droplet. Phosphorylation of perilipin permits hormone-sensitive lipase access to the surface of the lipid droplet, where 5 it hydrolyzes triacylglycerols to free fatty acids. 6 Fatty acids leave the adipocyte, bind serum albumin in the blood, and are carried in the blood; they are released from the albumin and 7 enter a myocyte via a specific fatty acid transporter. 8 In the myocyte, fatty acids are oxidized to CO2, and the energy of oxidation is conserved in ATP, which fuels muscle contraction and other energy-requiring metabolism in the myocyte.

Hormones that induce lipolysis Epinephrine Norepinephrine Glucagon Adrenocorticotropic hormones

IN CONTRAST,

INSULIN INHIBITS LIPOLYSIS!

Carnitine carries activated fatty acids into the mitochondrial matrix

Entry of electrons from fatty acid oxidation in the mitochondrial respiratory chain

Fatty Acid Oxidation In one pass through the fatty acid oxidation, one mol. Of

Acetyl CoA, two pairs of electrons, and four hydrogen ions are removed.─ CH3 - - - - COOH

each time it is shorter by 2 C units

The equation for one pass:

(C16) palmitoyl-CoA + CoA + FAD + NAD + H2O

myristoyl CoA + Acetyl-CoA + FADH2 + NADh + H+

Myristoyl CoA enters the -oxidation sequence, another set of four reactions, to give a second Acetyl-CoA.

Fatty Acid Oxidation Continued

The overall reaction is:

palmitoyl CoA + 7CoA + 7NAD + 7FAD + 7H2O

8 Acetyl CoA + 7 FADH2 + 7NADH + 7H+

These four steps are repeated (n/2 – 1) times for even numbered carbon chains.─ FADH2 ETFP (electron transferring flavoprotein),

provides 1.5 ATP─ NADH complex I, provides 2.5ATP─ Thus, four mol. of ATP are formed for each 2-C unit

removed in one pass.

First 3 rounds in the degradationof palmitate

In hibernating animals, fatty acid oxidation provides energy, heat, and water. Fatty acid oxidation is also important for the camel.

I) Palmitoyl-CoA + 7CoA + 7O2 + 28Pi + 28ADP

8Acetyl-CoA + 28 ATP + 7H2O

II) Acetyl-CoA oxidized in the TCA cycle1Acetyl-CoA 10ATP8Acetyl-CoA 80ATP

8Acetyl-CoA + 16O2 + 80Pi + 80ADP

8 CoA + 80ATP + 16H2O + 16CO2

Combine I and II

Palmitoyl-CoA + 23O2 + 108Pi + 108ADP

CoA + 108ATP + 16CO2 + 23H2O

Certain fatty acids require additional steps for degradation

Even numbered are fully saturated and are completely oxidized

Not all fa are simple and even numbered

The oxidation of fa containing double bonds require additional steps

Odd numbered fa yield propionyl CoA at the final thiolysis step. We need to metabolize propionyl CoA.

Oxidation of unsaturated fatty acids

This is somewhat difficult Two additional enzymes are required:

• Isomerase• Reductase

Let’s analyze the oxidation of palmitoleate• Activated• Transported• Undergoes 3 cycles of degradation by the same enzymes as in the

oxidation of saturated fas. However, the cis-d3-enoyl CoA formed in the third round is not a substrate for acylCoA dehydrogenase.

• This should be converted to trans by the ISOMERASE enzyme.

Another problem arises with the oxidation of pufas

What if we have two double bonds?• Linoleate (18:9,12)

Steps• Cis double bond is formed after 3 rounds of beta oxidation• This is coverted to a trans by ISOMERASE• The acylCoA produced by another round of beta oxidation contains

a cis-delta 4 double bond. • Dehydrogenation of this yields a 2,4-dienoyl intermediate, which is

not a substrate for the next enzyme in the b-oxidation pathway. • This problem is solved by 2,4-DIENOYL CoA REDUCTASE!

Thus, 2 extra enzymes are needed for the oxidation of even numbered pufas: isomerase and reductase

Oxidation of odd chain fatty acids

Odd chain fas oxidation is the same except the last step. • The last step produces Acetyl CoA (2C) and Propionyl CoA (3C)

How do we deal with 3C compound?• It is converted into succinyl CoA in a reaction that requires Vit B12.• Succinyl CoA is an intermediate in the TCA cycle.

Fas are also oxidized in peroxisomes Peroxisomes are membrane-enclosed cellular compartments.

• Hydrogen peroxide is produced by fa oxidation and then destroyed enzymatically.

• Peroxisomes have high levels of catalase. The process consists of 4 steps:

1) Dehydrogenation2) Hydration3) Oxidation 4) Thiolytic cleavage

The differences:• In peroxisomes, the flavoprotein dehydrogenase passes electrons

directly to oxygen.• The NADH formed in peroxisomes cannot be reoxidized, and the

peroxisome must export reducing equivalents to the cytosol.

Peroxisomal degradation

Fa oxidation in these organelles stop at octanyl CoA• Meaning, peroxisomes serve to shorten long chains to make

them better substrates for beta oxidation in mitochondria.

Zelweger syndrome, which results from the absence of functional peroxisomes, is characterized by liver, kidney, and muscle abnormalities and usually results in death by age 6.

Ketone bodies are formed from Acetyl CoA when fat breakdown predominates

Acetyl CoA enters the TCA cycle only if fat and ch degradation are balanced.

If oxaloacetate is decreased (if ch is unavailable) then acetyl CoA will not enter TCA cycle.

Also, in fasting and diabetes, oxalocetate is consumed to make glc by the gluconeogenic pathway; therefore, acetyl CoA increases.

Under these conditions increased acetyl CoA makes KETON BODIES• Acetoacetate• Beta hydroxybutyrate• Acetone

Utilization of ketone bodies by extrahepatic tissues

Fatty Acid Synthesis

Production of cytoplasmic Acetyl-CoA Carboxylation of Acetyl-CoA to form

malonyl-CoA Fatty acid synthesis by a multi-enzyme

complex Regulation of fatty acid synthesis Metabolism of unsaturated fatty acids and

eicosanoids

Fatty Acid Synthesis It is not a reversal of fa oxidation. It occurs in the cytoplasm of the cells of

the liver, fat tissue, and mammary gland and, to a lesser extent, in the cytoplasm of the cells of the kidney.

The process incorporates carbons from Acetyl CoA (the immediate substrate) into growing a fa chain, using ATP and NADPH as cofactors. Fa synthesis is increased by insulin.

Production of cytoplasmic acetyl CoA:• Transfer of acetate units from mitochondrial acetylCoA to cytoplasm, forming

cytoplasmic acetyl CoA, since CoA can not cross the mitochondrial membrane.• Carboxylation of AcetylCoA to form malonyl CoA• The irreversible formation of malonyl CoA from acetylCoA is catalyzed by

acetylCoA carboxylase. Biotin, prosthetic group (The 2 step reaction, similar to other biotin-dependent carboxylation reactions. Others: Pyruvate carboxylase, propionylCoA carboxylase)

• The CO2 derived from HCO3 is first transferred to biotin in an ATP-dependent reaction. The biotinyl group serves as a temporary carrier of CO2.

Citrate carries acetyl groups from mitochondria to the cytosol for fa synthesis

The synthesis of palmitate requires 8 mols of acetyl CoA, 14 mols of NADPH, and 7 mols of ATP.

Fa synthesis is in the cytosol. Acetyl CoA is made from pyruvate in the

mitochondria. So, Acetyl CoA must be transferred from the mitochondria to the cytosol.

Solution: acetyl groups are carried as citrate.• When citrate is high, it is transported to the cytosol. In the cytosol,

it is cleaved by ATP-citrate lyase.

Carboxylation of acetyl CoA to form malonyl CoA by Acetyl-CoA carboxylase

Acetyl-CoA carboxylase has 3 functional subunits:1) Biotin carrier protein.

2) Biotin carboxylase, • which activates CO2 by attaching it to a nitrogen in the biotin ring

in an ATP-dependent reaction.

3) Transcarboxylase• which transfers activated CO2 from biotin to acetyl-CoA

producing malonyl CoA. The long-flexible biotin arm makes this.

The formation of Malonyl-CoA

The formation of Malonyl-CoA is the committed step in fa synthesis.

Fa synthesis starts with carboxylation of acetyl-CoA to malonyl-CoA. • Irreversible reaction• Biotin is a cofactor• ATP is also required.

1) Synthesis takes place in the cytosol; oxidation, in the mitochondria.

2) Intermediates in fa synthesis are covalently linked to the ACP (acyl carrier protein), whereas in oxidation, they are bonded to CoA.

3) Fa Synthesis enzymes are joined in a single pp chain called fa synthase. Oxidation is not like that.

4) Growing of the chain is done by adding 2C units obtained from Acetyl-CoA. Activated donor of 2C units is Malonyl-CoA.

5) The reductant in fa synthesis is NADPH.

6) Elongation stops when C16 is made.

Important facts about the synthesis:

Fatty Acid Synthesis

The remaining series of reactions are catalyzed by a multienzyme complex.

• In eukaryotes this enzyme complex, fatty acid synthase, consists of 2 subunits that, together, have 7 enzymatic activities.

• They are polypeptides.

• The proteins act together to catalyze the formation of fatty acids from acetyl coA and malonyl CoA.

• The fundamental reaction order by which the long chains of carbon atoms in fas are assembled consists of 4 steps.1) Condensation of Acetyl group with malonyl group.

2) Reduction of the Carbonyl Group

3) Dehydration

4) Reduction of the Double Bond

Fatty Acid Synthesis This is one passage. With each passage through the

cycle, the fatty acyl chain is extended by 2 carbons. When the chain length is 16, the product (palmitate 16:0) leaves the cycle.

The fa synthase complex has 7 different active sites polypeptides. • Throughout the process, the intermediates remain covalently

attached to one of two thiol groups of the complex. • One point of attachment is the -SH group of a Cys residue in one

of the seven proteins. • Beta-ketoacyl ACP the other is the -SH group of acyl carrier

protein (ACP).

ACP is a small protein (Mr 8860) containing the prosthetic group 4 phosphopantetheine, an intermediate in the synthesis of coenzyme A.

Fatty Acid Synthesis Biosynthesis of fa is a 4-step sequence that lengthens a

growing f. acyl chain by two carbons.1) Condensation

• CO2 is eliminated from malonyl group.

• Net effect is extension of the acyl chain by two carbons. • The beta group is then reduced in three more steps nearly identical

to the reactions of beta oxidation, but in the reverse sequence.

2) The beta-keto group is reduced to an alcohol.3) The elimination of H2O creates a double bond

4) The double bond is reduced to form the corresponding saturated f.acyl group.

The fa chain grows by two-carbon units that are donated by activated malonate, with loss of CO2.

After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of 4, then 6, 8, and so on.

The final product is palmitate (16:0).

Intermediates in fa synthesis are attached to ACP

Intermediates are linked to ACP• Specifically, to the -SH terminus of a phosphopantetheine

group which is linked to a Ser residue of ACP

ACP is a single polypeptide chain of 77 residues.

ACP can be regarded as a giant prosthetic group (macro CoA).

Both ACP and CoA include phosphopantetheine as their reactive units

Fa synthase inhibitors

Fa synthase is overexpressed in some breast cancers. Some inhibitors were tested on mice and a great

weight loss was observed.

Therefore, fa synthase inhibitors are exciting candidates, both as anti-tumor and as anti-obesity drugs!

Fa biosynthesis requirements

The biosynthesis of fa require Acetyl CoA, ATP and NADPH.

• The ATP is required to attach CO2 to acetylCoA to make malonyl CoA

• NADPH is required to reduce the double bonds.1. NADPH supplied from HMP or cytoplasmic

conversion of malate to pyruvate.2. Malate is oxidized and decarboxylated by a

cytoplasmic NADP-dependent malate dehydrogenase or malic enzyme to form pyruvate.

Fatty acid biosynthesis is tightly regulated If we have more energy than our needs, fatty acids are stored. Acetyl coA carboxylase is the rate limiting step in the biosynthesis of fatty

acids (Important site regulation). The reaction is catalyzed by acetyl coA carboxylase

• Switched off by phosphorylation• Activated by dephosphorylation

The carboxylase is regulated by 3 signals:1. Glucagon – inhibits carboxylase activity

2. Epi – inhibits carboxylase activity

3. Insulin – stimulates fas synthesis Control is also done by the levels, within the cell, of

1. Citrate – activates carboxylase• Energy and building blocks are abundant, and we can go ahead and store them

2. Palmitoyl CoA – inhibits carboxylase

3. AMP – inhibits carboxylase

More regulation The proportion of active carboxylase depends on the catalytic

rates of these opposing enzymes.• Protein kinase A inhibits phosphatase by phosphorylating it.

- Carboxylase stays in its inactive form.• The inactive form also predominates when the energy level of the cell

is low.- Phosphorylation is stimulated by high AMP levels.

• Insulin stimulates carboxylase perhaps by activating protein phosphatase 2A.

How about citrate’s role? • Citrate is high when acetyl CoA and ATP are high. • When citrate is high and ATP is available, we can start making fa. • Citrate stimulates carboxylase.• Palmitoyl CoA inhibits this action of citrate on the carboxylase

enzyme.

Dependence of the catalytic activity of acetyl CoA carboxylase on the concentration of citrate

No citrate: Dephosphorylated form is predominant

The presence of citrate partly reverses the inhibition produced by phosphorylation for the enzyme (Acetyl CoA carboxylase)

Citrate facilitates the polymerization of the inactive octamers into active filaments• Acetyl CoA carboxylase exists as an octamer

The level of citrate is high when acetyl CoA and ATP are abundant. Hence, increased level of citrate signifies that 2-C units and ATP are available for the fa synthesis

More regulation

The stimulatory effect of citrate on the carboxylase is antagonized by palmitoyl CoA, which is abundant when there is an excess of fatty acids

Palmitoyl CoA causes the filaments to disassemble into the inactive octamers

Response to diet: In starvation, ffa are increased because Epi, Glucagon stimulate lipase. Insulin, in contrast, inhibits lipolysis

Also, malonyl CoA inhibits carnitine shuttle preventing excess of fatty acyl CoAs to the mitochondrial matrix in times plenty.

Elongation and unsaturation

The major product of fa synthesis is palmitate Longer ones are formed by elongation reactions in ER 2C units are added, the donor is still malonyl CoA ER enzymes also introduce double bonds.

There is an electron-transport chain in the desaturation of fatty acids which includes NADH-cytochrome b5-reductase, cytb5, and desaturase.

pufas

Some pufas can not be synthesized by mammals and are nutritionally essential• C20, 22, and 24 fa may be detected in the tissues. They are derived

from oleic (in plants but not animals) and linoleic acid by chain elongation.

• Palmitoleic and oleic acids are not essential in the diet because animals can make a double bond at the 9 position.

• Linoleic and linolenic acids are essential fas because animals cannot synthesize them.

• Arachidonic acid can be formed from linoleic acid in most animals.

In animals, double bonds can be introduced at the 4,5,6 and 9 positions but never beyond the 9 position.

Plants can introduce double bonds beyond 9 position and can, therefore, synthesize essential fas.

More about pufas

Monounsaturated fatty acids are synthesized by a desaturase system. Many tissues, including liver tissues, can make monounsaturated

forms. The first double bond introduced into a saturated fa is almost always at the 9 position. Enzyme is desaturase.

Synthesis of pufas involves desaturase and elongase systems. In animals, the additional double bonds all introduced between the

existing double bond and the -COOH group, but in plants, they may also be introduced between the 9 and omega carbon. • Linoleate 18:2(9,12) and linolenate 18:3(9,12,15) cannot be synthesized

by mammals, but plants can synthesize both. • The plant desaturases that introduce double bonds at 12 and 15 positions

are located in SER.

Eicosanoids

Eicosanoids are formed from C20 pufas

TXA, LT and PGs are called eicosanoids.

Arachidonate gives rise to Pgs, TX and LT. • A family of very potent biological molecules are made from arachidonate.• They act as short-range messengers, affecting neighboring tissues. • In response to a hormonal or other stimulus, a specific phospholipase affects

membrane phospholipids, releasing arachidonate.

SER enzymes then convert arachidonate into prostaglandins, beginning with the formation of PGG2, very first PGs