Biosynthesis Fatty acids

BIOSYNTHESIS

OF FATTY ACIDS

doc. Ing. Zenbia Chavkov, CSc.

The pathway for the of FAs is

not the reversal of the oxidation pathwayBoth pathways are separated within

different cellular compartments

In humansthe pathway for FA synthesis occurs primarily

in the cytoplasmof the liver and adipose tissue,to a lesser extend in lactating mammary glands,

brain, lungs,

and kidneys

whereas,

oxidation occurs in the mitochondria

The other major difference

is the use of nucleotide co-factors

Oxidation of fats involves the reduction of FAD, NAD+

Synthesis of fats

involves the oxidation of NADPH

Both oxidation and synthesis of fatsutilize an activated 2C intermediate,

acetyl-CoA

Acetyl-CoAmust be first transported

out of mitochondria

using

citrate shuttle transport system

The total energy requirementfor converting mitochondrial acetyl-CoA

into cytoplasmic acetyl-CoA is

1 ATP

Origin of Cytoplasmic Acetyl-CoA

Acetyl-CoAis generated in the mitochondria primarily from the sources:

The pyruvate dehydrogenase (PDH) reaction(glycolysis glucose pyruvic acid acetyl-CoA)

Fatty acid oxidation AAs degradation and ketone bodies

In order to be utilized for fatty acid synthesis

they must be present in the cytoplasm

The shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energy diminishes

Source of reducing equivalents

The origin of NADPH for FA biosynthesis

depends on cell type

In liver, the 2 NADPHs come from the pentose phosphate pathway

In adipose tissue,NADPH is generated by malic enzyme

-OOC-CH2-CH-COO- CH3-C-COO

-

| ||

OH O

The pentose phosphate pathway

Another source for NADPH

for these reactions isthe isocitrate shuttle

The synthesis of malonyl-CoAis the first committed step of FAs synthesis

Acetyl-CoA carboxylase (ACC),is the major site of regulation of FAs synthesis

ACC requires a biotin co-factor

First, CO2 is covalently bound to biotin using energy from hydrolysis of ATP

Then, the CO2 is transferred to acetyl-CoA producing malonyl-CoA

The biotinyl group serves as a temporary carrier of CO2

The carboxylation of

acetyl-CoAto form

malonyl-CoA

catalyzed by

acetyl-CoA

carboxylase

is the rate-limiting step of FA biosynthesis

ll

Enzyme-biotin HCO3

- + ATP

ADP + Pi Enzyme-biotin-CO2

-

O

CH3-C-SCoA

acetyl-CoA O

-O2C-CH2-C-SCoA

malonyl-CoA

ll

Enzyme-biotin

1

2

The overall reaction may be summarized as:

Acetyl-CoA Carboxylase activity,

in the mammals is regulated by

phosphorylation

allosteric regulation by local metabolites

The active conformation of the enzyme

associates in

multimeric filamentous complexes

The inactive conformation of the enzyme

exists as

individual protomers

The rate of fatty acid synthesis is controlled by the equilibrium between

monomeric and polymeric acetyl-CoA carboxylase

(ACC)The activity of ACC requires polymerization

This conformational change is controlled

by local metabolites(citrate, palmitoyl-CoA and other long-chain

fatty acyl-CoAs)

Regulation by local metabolites

The equilibrium between monomeric and polymeric

acetyl-CoA carboxylase is

inhibited bypalmitoyl- CoA

(product of FA synthase) other long-chain

fatty acyl-CoAs

enhanced by citrate

(promoting enzyme polymerization)

Regulation of Acetyl-CoA carboxylase activitythrough

hormone mediated phosphorylation

Glucagon and epinephrinepromote phosphorylation

anddecrease the enzymatic

activity ( )

Insulin promotes dephosphorylation and increases the activity ( )

With Acetyl-CoA Carboxylase inhibited,

acetyl-CoA remains available

for ketone bodies synthesis

the alternative metabolic fuelused when blood glucose is low

Changes in dietaffect the amount of fatty acid biosynthesis

by affecting the amount of acetyl-CoA carboxylase

A dietrich in carbohydrate or low in fatincreases the biosynthesis of the enzyme

by affecting the rate of transcription

Starvation or diet high in fathas the opposite effect and

reduces the rate of synthesis of acetyl-CoA carboxylase

Synthesis of the Acyl chain

The reactions of FA biosynthesis take place on

a multifunctional protein,called fatty acid synthase (fatty acid synthase complex)

A polyprotein is a single protein with more then 1 activity,

and fatty acid synthase is formed from 2 chains of this protein

The active enzyme is a dimer of identical subunits

There is some evidence

that the 2 copies of the multi-domain enzyme

are aligned antiparallel, as below

Pant-SH HS-Cys

Cys-SH HS-Pant

Fatty Acid Synthase dimer

Fatty AcidSynthase

prosthetic groups:

The thiol of the side-chain of a cysteineresidue of condensing enzyme domain

The thiol

of phosphopantetheine,equivalent in structure to part of coenzyme A

H3N+ C COO

CH2

SH

H

cysteine

The fatty acid synthase complexcontains 2 types thiol groups

The central thiol,made up of

4phosphopantetheinea derivative of coenzyme A, covalently linked

by a phosphodiester bond to

serine residue of acyl carrier protein, or ACP

The peripheral thiol,belongs to a cysteinyl residue

on ketoacyl-ACP synthase

Like fat oxidation,

fat synthesis involves 4 enzymatic activities

-keto-ACP synthase,

-keto-ACP reductase,

3-OH acyl-ACP dehydratase

Enoyl-CoA reductase

The two reduction reactions

require NADPH oxidation to NADP+

The acetyl-CoA are transferred to ACP+ malonyl CoA by the action of:

Acetyl-CoA transacylase Malonyl-CoA transacylase

The attachment of these carbon atoms to ACP

allows them to enter the fatty acid synthesis cycle

During the sequence of reaction, the growing FA takesthe form of a thioester attached to the:

Peripheral SH group of a cysteine residue of the protein

or to the central SH groupof a protein-bound phosphopantetheine

The biosynthetic intermediatesdo not diffuse awayfrom the polyprotein

but are passed

from one enzyme active site to the next active site

by acyl carrier protein (ACP)

Individual steps

of the Fatty Acid Synthase

reaction pathway

In the first reaction, (to initiate biosynthesis)

acetyl-CoA is transferred:

From CoA

To the central SH (thiol) groupof phosphopantetheine

to form a covalent bond with release of CoA

Then,

the acetyl groupis transferred

to the peripheral SH(thiol) group of a cysteine

Next,

the malonyl group

is transferred to the pantetheine central SH group of ACP, just vacated by the acetyl group

Now the reactants are poised for the first condensation reaction

Peripheral thiol

Central thiol

In the first step,

the acetyl group and

malonyl groupsare condensed,

with the release of CO2

This forms CONDENSATIONacetoacetate attachedto the pantetheine (central) SH group

The condensation reaction of fatty acid biosynthesis is

catalyzed by -ketoacyl-ACP synthase

REDUCTION, step 2.

Using NADPH,

acetoacetyl-ACP undergoes a reduction,

yielding -hydroxybutyryl-ACP and NADP+ in reaction

The ketone is reduced to a hydroxyl group,

mediated

by -ketoacyl-ACP reductase

DEHYDRATION, step 3.

Then the compound is dehydrated

to 2,3-trans-butenoyl-ACP(crotonyl-ACP) catalyzed by

-hydroxyacyl-ACP-dehydratase

REDUCTION, step 4.

The double bond is reduced by NADPH + H+

in reaction catalyzed by

2,3 trans-enoyl-ACP reductase

to form butyryl-S-ACP

HLengthened fatty acid chain is then translocated

to the peripheral SH (thiol) group of a cysteineketoacyl ACP synthase

Another malonyl group is added to the central SH (thiol) group

of ACP

This series of reactions form

a 4-carbon acyl group still attached to the phosphopantetheine (central -SH)

In the next reaction:

The growing fatty acyl chain is transferred to the

cysteine (peripheral thiol),

Another malonyl group is added to the pantetheine

-SH (central thiol) and cycle begins again

This cycle of condensation,

reduction, dehydration, reduction

and transfer of the acyl group continues

until chain of 16 carbons has been created

The resulting palmitoyl group - palmitate is released from

the fatty acid synthase complexby an exergonic hydrolysis reaction

Palmitate,a 16-C saturated fatty acid,

is the final product of the FA synthase reactions

Therefore:

Acetate group is added at the beginning

Then need 1 malonate to extend the chain by 2 carbons

3C = malonate

2C = acetate

1C = CO2

3C2C

1C

FAS FAS

3C

1C

FAS2C

2C

2C

2C

2C

3C

1C

2C

That fatty acid synthesis by multienzyme complex

stops at palmitate is probably

due to limitation in the size of an active siteof fatty acid synthase

Palmitatecan then undergo separate

elongation and/or unsaturationto yield other fatty acid molecules

Fatty acid biosynthesis

is energetically expensive

however, occurs when is

abundant precursor

to provide both

the massthe energy

BIOENERGETICS OF FA BIOSYNTHESIS

1 mole ATP is required for the generation of 1 mole of acetyl-CoA from citrate

7 moles of ATP are required for the transport of acetyl-CoA from mitochondria into cytosol, as a substrate

for the synthesis of malonyl-CoA

7 additional moles of ATP are required for the synthesis of 7 moles of malonyl-CoA

from acetyl-CoA and CO2

A total of 15 ATPequivalents are required

for the synthesis of palmitate from citrate

14 moles of NADPHare required for the biosynthesis of 1 mole of palmitate

THE REGULATION OF FAT METABOLISM

Occurs via two distinct mechanisms

One is short term regulation which is regulation effected by events such as

substrate availability, allosteric effectors and/or enzyme modification

Control of a given pathways' regulatory enzymes can

also occur by

alteration of enzyme synthesis and turn-over rates of synthesis

These changes are long term regulatory effects

Insulin stimulates lipogenesis

by several mechanisms

It increases the transport of glucose into cell(in adipose tissue)

and thereby increases the availability of both: pyruvate for FAs synthesis glycerol-3-P for esterification of the newly formed FAs

Insulis converts the inactive form of pyruvatedehydrogenase to the active form(in adipose tissue but not in liver!)

Insulin activates acetylCo-A carboxylase. It involves dephosphorylation by a protein phosphatase

accompanied by change in aggregation of monomers to a more polymeric state

Insulin by its ability

to depress the level of intracellular cAMP, inhibits lipolysis in adipose tissue

and thereby reduces the concentrationof plasma free FAs and long-chain acyl-CoA,

an inhibitor of lipogenesis

By this same mechanism

insulin antagonizes the action of glucagon and epinephrine,

which inhibit acetyl-CoA carboxylase and therefore lipogenesis,

by increasing cAMP, allowing cAMP dependent protein kinase to inactivate the enzyme by phosphorylation

Regulation of fat metabolism also occurs through

malonyl-CoA induced inhibition of carnitine acyltransferase I.

This functions

to prevent the newly synthesized FAsfrom entering the mitochondria

and being oxidized

ELONGATION AND DESATURATION

Stearic are major constituent of FAs

Oleic acids found in human cells

The fatty acid product released

from fatty acid synthase (FAS)

is palmitate

which is a 16:0 fatty acid,

(16 carbons and no sites of unsaturation)

Although

the FA synthase complex stops at 16 C atoms,

human cells:

Can extend the length of the FA chainPosseses the machinery for converting

saturated to unsaturated FAs

2-carbon units can be added: To endogenously synthesized or dietary fatty acids

by elongation reactions

ELONGATION AND UNSATURATIONof fatty acids occurs after palmitate (16C)

in both the mitochondriaand endoplasmic reticulum

(microsomal membranes)

The endoplasmic reticulum pathway

is quantitatively more important

This strategy agrees with the role of mitochondria

functioning as a catabolic organell

The substrate for the elongation reactionsis fatty acyl-CoA

and not fatty acyl-ACP

The endoplasmic reticulum

contains the enzyme activities found in the FA synthase

complex, that succesively

reduce, dehydrate, and reducethe compound

to produce fatty acyl-CoA containing 2 additional carbon atoms

The reactions are analogous to the condensation reactions

that occurs during conventional FA biosynthesis

The resultant product is 2C longer

The fatty acyl-CoA substratefor the elongation reaction

is malonyl-CoA

More then 1 elongation reaction can occur,

and fatty acids up to 26 C atoms can be synthesized

The reduction reactions of elongation

require NADPH as co-factorjust as for the similar reactions

catalyzed by FAS

Mitochondrial elongation

Involves acetyl-CoA unitsis a reversal of oxidation

Except that the final reductionutilizes NADPH instead of FADH2

as co-factor

Acetyl-CoA, not malonyl-CoAdonates the 2C units in mitochondria

C-C-C-C-C-C=C-C-C=C-C-C=C-C-C=C-C-C-C-COOH

C-C-C-C-C-C=C-C-C=C-C-C-C-C-C-C-C-COOH

125818

Animals cannot

put double

bonds in this

part of the

molecule,

plants can!

Essential fatty acids:

Linoleate 18:2(9,12)

Arachidonate

20:4(5,8,11,14)

n : = ( x,y..)1114

191218

CH3

COOH

17 15 12 9 7 5 3

(cis 9,12 octadecadienoic acid)Linoleic acid

Since these enzymes cannot introduce sites of unsaturation beyond C9

they cannot synthesize either

linoleate (18:2D9, 12 )linolenate (18:3D9, 12, 15)

These fatty acids must be acquired from the diet and are, therefore, referred to as

essential fatty acids

These essential FAs are

necessary for normal membrane structure

Linoleic acidespecially important, serves as a precursor

for the synthesis of arachidonic acid,from which

the eicosanoids(the prostaglandins, thromboxanes)

are formed

is also a constituent of epidermal cell

sphingolipidsthat function as

the skins water permeability barrier

It is this role of FAs in eicosanoid synthesis that leads to

poor growth, wound healingdermatitis

in persons on fat free diets

Desaturation

occurs in the ER membranes

in mammalian cells involves

4 broad specificity fatty acyl-CoA desaturases

(non-heme iron containing enzymes)

These mixed-function oxidase requireNADPH and molecular oxygen

to add a hydroxyl group to the fatty acid

Arachidonic acidis produced by

elongation and the addition of 2 double bondsas shown in Fig.

desaturation

Common inhealthy diet

Some availablefrom meat and eggs

-6 Pathway

Anti-inflammatorymetabolites

Pro-inflammatorymetabolites

Linoleic acid (18:2)

-Linolenic acid (18:3)

Dihomo- -linolenic acid (20:3)

Arachidonic acid (20:4)

Released from stores

(Slow)

Meat andeggs

Acetyl CoA carboxylase

Irreversible two-step reaction

CO2

BIOTIN

Biotin carrier protein

-O C

O

Lys

C=O

H-N

Biotin carboxylase

C=O

H-N Lys

BIOTIN

TranscarboxylaseO = C

-O

AMP-Activated Kinase

catalyzes

phosphorylation of

Acetyl-CoA

Carboxylase

causing

inhibition ( )

Phosphorylated protomer of

Acetyl-CoA Carboxylase (inactive)

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active)

Citrate

Dephosphorylated,

e.g., by insulin-

activated Protein

Phosphatase

Palmitoyl-CoA

Phosphorylated, e.g., via

AMP-activated Kinase

when cellular stress or

exercise depletes ATP.

Regulation of Acetyl-CoA Carboxylase

The primary phosphorylation of ACC occurs through the action of AMP-activated protein kinase, AMPK

This is not the same as cAMP- dependent protein kinase, PKA!

Phosphorylation

causes the filamentous enzyme to dissociate into inactive mononomers