Sinteza AG

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Sinteza corpilor cetonici

(cetogeneza)

Principala cale de metabolizare a acetil CoA – includerea în ciclul Krebs (în condiţiile în care scindarealipidelor şi a glucidelor este echilibrată) - “lipidele ard înflacăra glucidelor”

În lipsa glucidelor; inaniţie, diabet - OA se utilizeazăpentru generarea Gl. În lipsa OA, Acetil Co A recurge la formarea corpilor

cetonici: acetoacetatul, β-hidrohibutiratul şiacetona

Sinteza lor are loc în ficat, dar se utilizează de ţesuturileperiferice

Au rol energetic (muşchiul cardiac, stratul cortical alrinichilor)





cetogeneza



Utilizarea corpilor cetonici

Acetoacetatul – 2 mol de acetilCoA, utilizate ulterior în ciclulKrebs (23 ATP)

A doua cale de activare aacetoacetatului poate fi:

Acetona:1. pînă la propandiol (CH3-CHOH-

CH2OH) , scindat la fragmente acetil şiformil

2. Transformată în piruvat (prin hidroxilaredublă)



Cetonemie, cetonurie

Cetonemie- mărirea c% de corpi cetonici însînge Cetonurie – apariţia CC în urină

Diete bogate în lipide, sărace în glucide; inaniţie,diabet, dereglări gastrointestinale la copii saugravide; glucozurie renală

Eliminarea hidroxibutiratului şi acetoacetatuluidin organism (fiind anioni la excreţie) conduce lapierderea de cationi – Na- rezultă cetoacidoza

Pierderea H2O – dehidratarea organismului



Biosinteza lipidelor



Obiectivele: Biosintaza acizilor graşi: 1. saturaţi cu număr par de atomi de carbon; 2. nesaturaţi cu număr par de atomi de carbon; 3. saturaţi cu număr impar de atomi de carbon. Enzimele, coenzimele, reglarea. Biosinteza TAG: substanţele iniţiale, enzimele şi coenzimele,

reglarea. Biosinteza fosfogliceridelor: substratele, reacţiile parţiale ale I

şi a II căi;

Biosinteza sfingolipidelor: precursorii, reacţiile principale,enzimele, reglarea. Metabolismul colesterolului. Biosinteza colesterolului –

substratele, etapele, reacţiile parţiale ale I etape (până la acidulmevalonic), enzimele, coenzimele, reglarea. Căile de utilizare şieliminare ale colesterolului.



Sinteza AG

Sinteza AG şi încorporarea lor în Tg constituiemecanismul principal de stocare a excesului deglucide alimentare (Gl nu se mai transformă înglicogen dar în Tg)

Etapele:

Sinteza de novo cu formarea acidului palmitic

Elongarea acidului palmitic

Introducerea de legături duble în AG



Particularităţile sintezei AG

Are loc în citozol E – acid gras sintetaza – alcătuită din 8 proteine

(domenii)- 7 sunt enzime, a 8-a – proteina( purtătoare ) transportatoare de acil -ACP.

ACP cuprinde 2 grupe SH:1. – SH furnizat de un rest de cisteinil: SH-Cis2. - SH - fosfopanteteina, ataşată prin leg ătura

fosfat-Ser: SH-Pant Ca iniţiator este acetil CoA (rezultat din

glicoliză), pe cînd sursa majoră – malonil CoA rolul reducător îi revine NADPH+H



Sinteza de novo cu formarea acidului

palmitic

Etapele:

1. transferul lui Acetil CoA din mitocondrii încitozol

2. Sinteza de malonil CoA

3. Sinteza acidului palmitic



Transferul lui Acetil CoA din mitocondrii în

citozol



Sinteza de malonil CoA

acetil-CoA + HCO3 + ATP ADP + Pi + malonil-CoA

E- acetil CoA

Carboxilaza

citrat,

Insulina

palmitoil CoA

Glucagonul



Sinteza acidului palmitic







Ciclu de reacţii este reluat: butiril+ACP secondensează cu malonil+ACP- formînd în final C6-acil ACP.

Catena AG creşte pînă la formarea palmitil-S-ACP



R eacţia sumară:

Acetil-ACP+7 malonil-CoA +14 NADPH+H

Palmitat +7CO2+14NADP + + 8HSCoA+6H2O

deoarece malonil CoA se sintetizează din acetilCoA:8 acetil-CoA + 14 NADPH +H + + 7 ATP

palmitate+ 14

NADP

+

+ 8HSCoA + 7

ADP

+

7

Pi



Elongarea AG

Localizată: reticulul endoplasmatic AG este activat

La acidul preexistent (palmitil CoA) se ataşează malonil CoA



Biosinteza AG nesaturaţi

Pot fi sintetizaţi AC mononesaturaţi. Introducerea unei duble legături are locprin acţiunea unei monooxigenaze (introduce gruparea hidroxil), urmată dedeshidratare

Acidul linoleic şi linolenic sunt esenţiali (exogen) Acidul linoleic se transformă în acidul arahidonic conform reacţiilor





Sinteza TAG

2 căi: 1. calea monoacilglicerolului: are loc în peretele intestinal

(enterocite)din produşi absorbiţi (resinteza lipidelor). 2. calea glicerolfosfatului: în toate ţesuturile (activă: ţesutul adipos

şi ficat) AG sunt incorporaţi în TAG sub formă activă de acilCoA: R-COOH + ATP + HS-CoA +H2O R-CO~SCoA + AMP + 2 Pi

E- acil Co A sintetaza



1. calea monoacilglicerolului

TG împreună cu FL,Col, proteine suntincorparate în CM şi secretaţi mai departe în

vasele limfatice.



calea glicerolfosfatului



originea glicerol fosfatului

În ficat:

În ţesut adipos, ficat



Sinteza glicerofosfolipidelor 2 căî de sinteză:

Sinteza de novo - utilizează ca intermediar comunacidul fosfatidic

Calea de rezervă – o sinteză din produse formate

Particularitatea biosintezei FL este participareaprecursorilor în forme active de derivaţi ai citidinfosfatului (CDP) ca CDP-colina, CDP-

etanolamina, CDP-diglicerid.



Sinteza de novo



2 i t di d f t



2. sinteza din produse formate



Sinteza sfingolipidelor

Se formează din palmitoil CoA şi Ser

Sfingozina liberă se formează din ceramidă

Sinteza are loc pe suprafaţa citozolică amembranelor reticulului endoplasmatic





Sinteza sfingolipidelor



Sinteza Colesterolului

Se sintetizează din Acetil-CoA Necesită 18 moli de Acetil-CoA şi 18 de ATP Principalul organ de metabolizare este ficatul, dar are

loc şi în intestin, suprarenale, tegumente Are loc în 3 etape:1. Sinteza acidului mevalonic2. mevalonatul prin mai multe reacţii - 3∆-izopentenil

pirofosfat. 6 molecule de 3∆-izopentenil pirofosfat – scualen

3. Scualenul se supuine ciclizării – lanosterol -- Col







H2C

C

CH3

HO

CH2

C-

O O

CH2 OH

H2C

C

CH2 CH2 O P O P O-

O

O-

O

O-

CH3

H2C

C

CH3HO

CH2

C-

O O

CH2 O P O P O-

O

O-

O

O-

CO2

ATP

ADP + Pi

2 ATP

2 ADP

mevalonate

5-pyrophosphomevalonate

(2 steps)

isopentenyl pyrophosphate



H2C

C

CH2 CH2 O P O P O-

O

O-

O

O-

CH3

H3C

C

CH CH2 O P O P O-

O

O-

O

O-

CH3

isopentenyl pyrophosphate

dimethylallyl pyrophosphate

CH CH2CH3C

CH3

CH CH2CCH2

CH3

CH CH2 O P O P O-

O

O-

O

O-

CCH2

CH3

2

O

NADP+

O2 H2O

HO

H+

NADPH

NADP+ + 2 PPi

NADPH

2 farnesyl pyrophosphate

squalene 2,3-oxidosqualene lanosterol

O

NADP+

O2 H2O

HO

H+NADPH

squalene 2,3-oxidosqualene lanosterol

HO HO

lanosterol cholesterol

19 steps



REGLAREA ŞI

PATOLOGIAMETABOLISMULUI

LIPIDIC



Obiectivele Metabolismul eicosanoizilor. Căile ciclooxigenazică şi lipooxigenazică ale biosintezei lor.

Inactivarea. Metabolismul vitaminelor liposolubile: sursele alimentare, necesităţile diurne, transformările Reglarea metabolismului lipidelor la nivelul celulei. Reglarea neurohormonală a metabolismului lipidelor. Rolul lipotropinelor, ACTH, hormonilor

tiroizi, insulinei, glucagonului, glucocorticoizilor şi catecolaminelor. Relaţiile reciproce dintre metabolismul energetic, glucidic şi lipidic. Dereglările digestiei şi absorbţiei lipidelor. Steatoreea pancreatică, hepatică şi intestinală. Dislipidemiile: a) hipolipoproteinemiile familiale – afecţiunea Tangier, - şi -lipoproteinemia familială; b) hiperlipoproteinemiile primare şi familiale; c) hiperlipoproteinemiile secundare (dobândite) – în diabet zaharat, alcoolism, afecţiuni

ale glandelor endocrine. Cauze, mecanismele dereglării metabolismului lipidelor, manifestările biochimice. 6. Lipidozele tiszlare: a) ereditare – Neimann-Pick, Tay-Sachs, Krabbe, Gaucher, Farber, leucodistrofia

metacromatică, gangliozidoza GM1; b) dobândite – obezitate, ateroscleroză, alcoolism. Cauze, mecanismele dereglării metabolismului lipidelor, manifestările biochimice. 7. A-, hipo- şi hipervitaminozele A, D, E, K – cauze, manifestări metabolice. 8. Rolul eicosanoizilor în procesele inflamatorii, reacţiile alergice, dereglările fluidităţii

sanguine.



Metabolismul eicosanoizilor



ATP-dependent carboxylation provides energy input.

The CO2 is lost later during condensation with thegrowing fatty acid.

The spontaneous decarboxylation drives the condensationreaction.

H3C C SCoA

O

CH2 C SCoA

O

-OOC

acetyl-CoA

malonyl-CoA

The input to fatty acidsynthesis is acetyl-CoA ,

which is carboxylated tomalonyl-CoA .



HCO3 + ATP + acetyl-CoA ADP + Pi + malonyl-CoA

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

O



Biotin is linked to the enzyme by an amide bond between

the terminal carboxyl of the biotin side chain and thee-amino group of a lysine residue. The combined biotin and lysine side chains act as a longflexible arm that allows the biotin ring to translocate

between the 2 active sites.

CHCH

H2C

S

CH

NH

C

N

O

(CH2)4 C NH (CH2)4 CH

CO

NH

O

C

O

O-

Carboxybiotin lysineresidue



Acetyl-CoA Carboxylase, which converts acetyl-CoA tomalonyl-CoA, is the committed step of the fatty acidsynthesis pathway.

The mammalian enzyme is regulated, by

phosphorylation

allosteric control by local metabolites.Conformational changes associated with regulation:

In the active conformation, Acetyl-CoA Carboxylase

associates to form multimeric filamentous complexes. Transition to the inactive conformation is associated

with dissociation to yield the monomeric form of theenzyme (protomer).

Phosphorylated protomer of



The decreased production of malonyl-CoA preventsenergy-utilizing fatty acid synthesis when cellular energystores are depleted. (AMP is abundant only when ATP has

been extensively dephosphorylated.)

AMP-Activated

Kinase catalyzes

phosphorylation

of Acetyl-CoACarboxylase,

causing

inhibition.


Acetyl-CoA Carboxylase (inactive)

Dephosphorylated Polymer ofAcetyl-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

AMP A i d Ki



When AMP is high (ATP low), malonyl-CoA production isdiminished, releasing fatty acid oxidation from inhibition.

This will lead to increased ATP production.

AMP-Activated Kinase has a significant role evenin tissues (e.g., cardiac

muscle) that do notsignificantly synthesize fattyacids.

In such tissues malonyl-CoA , produced via oneisoform of Acetyl-CoACarboxylase, functionsmainly as an inhibitor offatty acid oxidation.

H3C C SCoA

O

CH2 C SCoA

O

-OOC

acetyl-CoA

malonyl-CoA

ATP + HCO3-

ADP + Pi

Acetyl-CoACarboxylase

(inhibited by

AMP-ActivatedKinase)

O



A cAMP cascade, activated by glucagon & epinephrine whenblood glucose is low, may also result in phosphorylation of

Acetyl-CoA Carboxylase via cAMP-Dependent Protein

Kinase. With Acetyl-CoA Carboxylase inhibited, acetyl-CoA remainsavailable for synthesis of ketone bodies, the alternativemetabolic fuel used when blood glucose is low.

H3C C SCoA

O

CH2 C SCoA

O

-OOC

acetyl-CoA

malonyl-CoA



The antagonistic effect of insulin, produced when bloodglucose is high, is attributed to activation of ProteinPhosphatase.




Citrate

Dephosphorylated,

e.g., by insulin-

activated Protein

Phosphatase

Palmitoyl-CoAPhosphorylated, e.g., viaAMP-activated Kinase







Palmitoyl-CoA (product of Fatty Acid Synthase) promotesthe inactive conformation, diminishing production ofmalonyl-CoA, the precursor of fatty acid synthesis.

This is an example of feedback inhibition.

Regulation of

Acetyl-CoA

Carboxylase by

local metabolites:




Citrate

Dephosphorylated,

e.g., by insulin-

activated Protein

Phosphatase

Palmitoyl-CoA

Phosphorylated, e.g., via

AMP-activated Kinase






[Citrate] is high when there is adequate acetyl-CoA enteringKrebs Cycle.

Excess acetyl-CoA is then converted via malonyl-CoA tofatty acids for storage.

Glucose-6-phosphatase

glucose-6-P glucose

Gluconeogenesis Glycolysis

pyruvatefatty acids

acetyl CoA ketone bodies

cholesterol

oxaloacetate citrate

Krebs Cycle

Citrate

allostericallyactivates Acetyl-CoA Carboxylase.



Fatty acid synthesis from acetyl-CoA & malonyl-CoAoccurs by a series of reactions that are:

in bacteria catalyzed by 6 different enzymes plus aseparate acyl carrier protein (ACP)

in mammals catalyzed by individual domains of a very

large polypeptide that includes an ACP domain.Evolution of the mammalian Fatty Acid Synthaseapparently has involved gene fusion.

NADPH serves as electron donor in the two reactionsinvolving substrate reduction.

The NADPH is produced mainly by the Pentose PhosphatePathway.

SH Coenzyme AH



Fatty AcidSynthaseprosthetic groups:

the thiol of the side-chain of a cysteine residue of CondensingEnzyme domain.

the thiol of phosphopantetheine,equivalent in structureto part of coenzyme A.

N

N N

N

NH2

O

OHO

HH

H

CH2

H

OPOPOH2C

O

O O

O

P

O

O

O

C

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

O

-mercaptoethylamine

pantothenate

ADP-3'- phosphate

y

phosphopantetheine

H3N+ C COO

-

CH2

SH

cysteine

SHh h t th i



Phosphopantetheine (Pant) is covalently linked

via a phosphate ester to aserine OH of the acylcarrier protein domainof Fatty Acid Synthase.

The long flexible arm of phosphopantetheinehelps its thiol to move

from one active site toanother within thecomplex. OPOH2C

O

OC

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

O

CH2 CH

NH

C O

-mercaptoethylamine

pantothenate

serineresidue

phosphopantetheine

of acyl carrier protein

phosphate



As each of the substrates acetyl-CoA & malonyl-CoA bindto the complex, the initial attacking group is the oxygen ofa serine hydroxyl group of the Malonyl/acetyl-CoA

Transacylase enzyme domain.

Each acetyl or malonyl moiety is transiently in ester linkageto this serine hydroxyl, before being transferred into

thioester linkage with the phosphopantetheine thiol ofthe acyl carrier protein (ACP) domain.

Acetate is subsequently transferred to a cysteine thiol ofthe Condensing Enzyme domain.

Condensing Malonyl/acetyl-CoA Dehydratase Enoyl -Ketoacyl ACP ThioesteraseEnzyme (Cys) Transacylase (Ser ) Reductase Reductase (Pant)

N- -C

Order of domains in primary structure of mammalian Fatty Acid Synthase



NADPH NADP+NADPH NADP

+ H2O



4. The -ketone is reduced to an alcohol by e- transferfrom NADPH.

5. Dehydration yields a trans double bond.

6. Reduction by NADPH yields a saturated chain.

Pant

S

Cys

SH

C

CH2

C

O

CH3

O

Pant

S

Cys

SH

C

CH2

HC

O

CH3

Pant

S

Cys

SH

Pant

S

Cys

SH

C

CH

HC

O

CH3

C

CH2

CH2

O

CH3

OH

4 5 6

4 -Ketoacyl-ACP Reductase

5 -Hydroxyacyl-ACP Dehydratase

6 Enoyl-ACP Reductase

Malonyl S CoA HS CoA



Following transfer of the growing fatty acid fromphosphopantetheine to the Condensing Enzyme's cysteinesulfhydryl, the cycle begins again, with another malonyl-

CoA.

Pant

S

Cys

SH

C

CH2

CH2

O

CH3

Pant

SH

Cys

S

C

CH2

O

CH2

CH3

Pant

S

Cys

S

C

CH2

O

CH2

CH3

C

CH2

COO-

O

Malonyl-S-CoA HS-CoA

7 2

7 Condensing Enzyme

2 Malonyl/acetyl-CoA-ACP Transacylase (repeat).



Product release:

When the fatty acid is 16 carbon atoms long, a Thioesterase domain catalyzes hydrolysis of the thioesterlinking the fatty acid to phosphopantetheine.

The 16-C saturated fatty acid palmitate is the finalproduct of the Fatty Acid Synthase complex.



Condensing Malonyl/acetyl-CoA Dehydratase Enoyl -Ketoacyl ACP Thioesterase(C ) l (S ) d d ( )

N- -C



The solved structure does not resolve the position of ACP & Thioesterase domains, predicted from primary structureto be near -Ketoacyl Reductase (KR) domains of lateral"arms" of the complex.

Enzyme (Cys) Transacylase (Ser ) Reductase Reductase (Pant) N -C

Order of domains in primary structure of mammalian Fatty Acid Synthase

KR KR

DH DH

KS KS

MAT MAT

ER ER

Arrangement of domains

in Fatty Acid Synthase

“arm”

“leg”

These domains may betoo flexible to be resolved.KR = -Ketoacyl Reductase;ER = Enoyl Reductase;

DH = Dehydratase;KS = -Ketoacyl Synthase(Condensing Enzyme); MAT= Malonyl/Acetyl-CoA

Transacylase.

KR KR



KR KR

DH DH

KS KS

MAT MAT

ER ER

Arrangement of domainsin Fatty Acid Synthase

“arm”

“leg”

Fatty Acid Synthase complex is somewhat asymmetric.

There is evidence for conformational changes relating to

catalysis.Protein flexibility may facilitate transfer of ACP-attachedreaction intermediates among the several active sites in each

half of the complex.

For images see:

website (ETH Zurich)

website (Asturias lab,

Scripps)

article (Maier, Jenni & Ban;

requires subscription to

Science).

http://www.ethlife.ethz.ch/e/articles/sciencelife/banFAShumfung.html

http://www.scripps.edu/cb/asturias/index.php?page=structure_and_molecular_organization_of_mammalian_fatty_acid_synthase

http://dx.doi.org/doi:10.1126/science.1123248

http://dx.doi.org/doi:10.1126/science.1123248

http://www.scripps.edu/cb/asturias/index.php?page=structure_and_molecular_organization_of_mammalian_fatty_acid_synthase

http://www.ethlife.ethz.ch/e/articles/sciencelife/banFAShumfung.html



Explore with Chime the structure of the E. coli -

Ketoacyl-ACP Synthase III, equivalent to thedomains of the mammalian Fatty Acid Synthasethat catalyze the initial acetylation andcondensation reactions.

Oxidation & Fatty Acid Synthesis



-Oxidation & Fatty Acid SynthesisCompared

Oxidation Pathway Fatty Acid Synthesis

pathway location mitochondrial matrix cytosol

acyl carriers(thiols)

Coenzyme-A phosphopantetheine(ACP) & cysteine

e

acceptors/donor FAD & NAD+ NADPH

-OH intermediate L D

2-C product/donor acetyl-CoAmalonyl-CoA

(& acetyl-CoA)

F A id S h i i i ll l d



Fatty Acid Synthase is transcriptionally regulated.In liver:

Insulin, a hormone produced when blood glucose ishigh, stimulates Fatty Acid Synthase expression. Thus excess glucose is stored as fat. Transcription factors that that mediate the stimulatory

effect of insulin include USFs (upstream stimulatoryfactors) and SREBP-1.SREBPs (sterol response element binding proteins)

were first identified for their regulation of cholesterol

synthesis. Polyunsaturated fatty acids diminish transcription of

the Fatty Acid Synthase gene in liver cells, bysuppressing production of SREBPs.



In fat cells:

Expression of SREBP-1 and of Fatty Acid Synthase isinhibited by leptin, a hormone that has a role in regulatingfood intake and fat metabolism.

Leptin is produced by fat cells in response to excess fatstorage.

Leptin regulates body weight by decreasing food intake,

increasing energy expenditure, and inhibiting fatty acidsynthesis.

Elongation beyond the 16-C length of the palmitate product



Elongation beyond the 16-C length of the palmitate productof Fatty Acid Synthase occurs in mitochondria andendoplasmic reticulum (ER).

Fatty acid elongation within mitochondria involves the-oxidation pathway running in reverse, but NADPH

serves as electron donor for the final reduction step.

Polyunsaturated fatty acids esterified to CoA aresubstrates for the ER elongation machinery, which usesmalonyl-CoA as donor of 2-carbon units.

The reaction sequence is similar to Fatty Acid Synthase

but individual steps are catalyzed by separate proteins. A family of enzymes designated Fatty Acid Elongases catalyze the initial condensation step for elongation of

saturated or polyunsaturated fatty acids.



Desaturases introduce double bonds at specific

positions in a fatty acid chain.Mammalian cells are unable to produce double bonds atcertain locations, e.g., D12.

Thus some polyunsaturated fatty acids are dietaryessentials, e.g., linoleic acid, 18:2 cis D9,12 (18 C atomslong, with cis double bonds at carbons 9-10 & 12-13).

C

O

OH

910

oleate 18:1 cis D9

O



Formation of a double bond in a fatty acid involves thefollowing endoplasmic reticulum membrane proteins in

mammalian cells: NADH-cyt b5 Reductase, a flavoprotein with FAD

as prosthetic group.

Cytochrome b5, which may be a separate protein or adomain at one end of the desaturase.

Desaturase, with an active site that contains twoiron atoms complexed by histidine residues.

COH

910

oleate 18:1 cis D9



The desaturase catalyzes a mixed function oxidation reaction.

There is a 4-electron reduction of O2 2 H2O as a fattyacid is oxidized to form a double bond.

2e pass f rom NADH to the desaturase via theFAD-containing reductase & cytochrome b5, the

order of electron transfer being:NADH FAD cyt b5 desaturase

2e are extracted from the fatty acid as the double

bond is formed.E.g., the overall reaction for desaturation of stearate (18:0)to form oleate (18:1 cis D9 ) is:stearate + NADH + H+ + O2 oleate + NAD+ +

2H2O

http://www.lipidsonline.org/slides/slide01.cfm?q=cholesterol+biosynthesis&pg=1


































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