1.FattyAcids

7/29/2019 1.FattyAcids

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BIOCHEMISTRY 441 Winter 2007

Part A (Bill Parson)

1. Biosynthesis of fatty acids2. Triacylglycerols, phospholipids & complex lipids

3. Cholesterol & lipoproteins

4. Photosynthesis: antennas & reaction centers

5. Photosynthesis: electron transfer & photophosphorylation

6. Photosynthesis: carbon fixation by C3 and C4 pathways

7. Amino acid metabolism: transamination and NH3 transport

8. Urea cycle, amino acid catabolism & biosynthesis

9. Aromatic amino acids & neurotransmitters

10. One-carbon metabolism

11. Biosynthesis of pyrimidines & purines

12. Deoxyribonucleotide biosynthesis & nucleotide catabolism13. DNA & RNA: primary and secondary structure

Part B (Ted Young)DNA replication, repair and transcription (schedule to

follow)


2/29

Announcements

Lecture slides are posted on the web at the same address

as for Biochemistry 440, but with 441 in the URL:

http://courses.washington.edu.bioc441

User name: bioc441

Password: DNA

Check the web site for updates.

Videotapes of the lectures will be available in the library(usually within a day after each lecture).
http://courses.washington.edu.bioc441/http://courses.washington.edu.bioc441/


3/29

Fatty acids have extended hydrocarbon chains

Most natural fatty acids have an even number of carbons.Unsaturated fatty acids usually have cisdouble bonds.

stearic acid, C18:0CO2H

CO2H palmitic acid, C16:0

oleic acid, C18:1(D9)CO2H10 9

109 CO2H palmitoleic acid, C16:1(D9)1

1


4/29

Fatty acids are components of phospholipids and triacylglycerols

glycerophospholipids

CH2OCRO

RCOCHO

CH2OPOCH2CH2N(CH3)3+O-

O

Phospholipid bilayers are the centralstructural elements of biologicalmembranes.

triacylglycerols

RCOCHCH2OCR

O

CH2OCRO

O

Triacylglycerols arestored as energyreserves.

Fatty acids also are found ascholesterol esters in lipoproteins,and are attached covalently tosome proteins.

HOCH-CH=CH-CH2R

CH2OPOCH2CH2N(CH3)3+O-

O

R-C-NH-CHO

sphingolipids

Sphingolipids on cell surfacesare sites of cell recognition.

Inositol phospholipidsparticipate in intracellularsignaling.


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Triacylglycerols are stored as energy reserves inadipose tissue and other tissues

Cross section of four adipocytes from a guinea pig.Lipid droplets, consisting mainly of triacylglycerols,fill most of the volume of the cells.

lipid droplets

capillary


6/29

Fatty acids are synthesized from acetyl-CoA in adipose tissue & the liver

CH3

-C-S-CoAO ATP, NADPH, CO2

palmitic acid (C16:0) palmitoylCoA

H3C

C-S-CoAO

stearic acid (C18:0) stearoylCoA

H3CC-S-CoAO

The labeling patterns suggest that the fatty acid chain

forms by successive addition of two-carbon units

9

1

H3C

oleic acid (C18:1 D9)oleoylCoA

C-S-CoA

O

18

Why is CO2

needed?


7/29

CH3-C-S-CoA

O-O2C-CH2-C-S-CoA

O

malonylCoA

ATP PiADP

HCO3-

boundbiotin O

NHHN

S

C

O=CNH

O

NHHN

S

C

O=CNH

O

N-CO2-HN

S

C

O=CNH

carboxylasesite

transcarboxylasesitebiotin

attachmentsite

MalonylCoA serves as thedonor of two-carbon units

MalonylCoA is formed from acetylCoA and CO2 by a multifunctional enzyme,

acetylCoA carboxylase (biotin carboxylase-transcarboxylase). Biotin is attached

covalently to a Lys residue of the enzyme. In bacteria, the three domains are inseparate subunits; in animals, they are on a single, multifunctional polypeptide.


8/29

ACP has 4-phosphopantetheine linked to a Ser residue.Malonyl and acetyl groups are transferred from CoA to thesulfur atom of the 4-phosphopantetheine.

The growing fatty acid chain isattached to acyl-carrier protein (ACP)

HS-ACP HS-CoA

CH3-C-S-CoAO CH3-C-S-ACP

O

HS-ACP HS-CoA

-

O2C-CH2-C-S-CoA

O-

O2C-CH2-C-S-ACP

O

4-phospho-pantetheine

SH

CH2

OO=P-O-

CH2O

CH3-C-CH3

CHOHC=ONHCH2CH2

C=ONHCH2CH2

ACP

Pantetheine is vitamin B5


9/29

Acyl-carrier protein from Bacillus subtilis

from pdb file 1f80.pdb;

K. D. Parris et al. Struct.Fold. Design8: 883 (2000).

4-phosphopantetheine

www.rcsb.org/pdb


10/29

Malonyl-/Acetyl transferase (MAT) catalyzestransfer of malonyl units from CoA to ACP

-O-C-CH2-C-S--ACPO O

ACP--SH

-O-C-CH2-C-S-CoA

O OCoA-SH

O-O-C-CH2-C-O-

OMATMAT -OH

malonylCoA

malonylACP


11/29

Malonyl-/Acetyl transferase (MAT)also transfers acetyl units to ACP

Bacteria have

separate malonyl &

acetyl transferases

Acetyl units thenmove from ACP to theketo-synthase (KS),which catalyzes thecondensation reaction

CH3-C-S-CoAO CoA-SH

MAT -OH

CH3-C-S- ACP

OACP -SH

CH3-C-S-

O

KSKS -SH

CH3-C-O- MAT

O


12/29

The condensation reaction

Release of bicarbonate isexothermic and pulls thereaction in the direction ofcondensation.

3-ketoacyl-ACPmalonyl-ACP

The acetyl group first moves fromACP to a Cys residue of the synthase,then combines with malonyl-ACP togive a 3-ketoacyl-ACP.

S

ACP

OO

CH3C

SH

b-ketoacylsynthase

HCO3-

O

S-C-CH3

b-ketoacylsynthase

S

ACP

OO

O-C

HO-

H+


13/29

Reduction by NADPH,dehydration, and asecond reduction

generates butyryl-ACP

dehydrase

CH3-C-CH2-C-S-ACP

O O

O

CH3-CH2CH2-C-S-ACP

HOH

CH3-C-CH2-C-S-ACP

H O

OH

CH3-C=C-C-S-ACP

OH

H

b-ketoacylreductase

NADPH

NADP+

enoyl reductase

NADPH

NADP+

b-ketoacyl synthase

O

-O-C-CH2-C-S-ACP

OO

CH3-C-S- KS

KS -SH

HCO3-


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CH3-C-CH2-C-S-ACP

H O

OH

To continue the cycle, the fatty acid chain must move backto the ketoacyl synthase

b-ketoacyl synthase

b-ketoacylreductase

dehydrase

enoyl reductase

CH3-C-CH2-C-S-ACPO O

CH3-CH2CH2-C-S-ACP

O

CO2

O

-O-C-CH2-C-S-ACP

OO

CH3-C-S- KS

ACP-SH

KSCH3-CH2CH2-C-S-

O

KS -SH

HOH

H2O

CH3-C=C-C-S-ACP

OH

H

NADPH

NADP+

NADPH

NADP+


15/29

A second turn of the cycle generateshexanoyl-ACP

b-ketoacyl synthase

b-ketoacylreductase

dehydraseenoyl reductase

CO2

O

-O-C-CH2-C-S-ACP

O

KSCH3-CH2CH2-C-S-

O

KS -SH

HOH

NADPH

NADP+

NADPH

NADP+

CH2-C-CH2-C-S-ACPO OCH3CH2

CH2-C=C-C-S-ACP

OH

HCH3CH2

CH2-CH2CH2-C-S-ACP

O

CH3CH2

CH3CH2CH2-C-CH2-C-S-ACP

H O

OH


16/29

The cycle stops when the fatty acid chain reaches 16 carbons

b-ketoacyl synthase

thioesterase

CH3-(CH2)11CH2-CH2CH2-C-S-ACP

O

CO2

O

-O-C-CH2-C-S-ACP

O

KSCH3-(CH2)11CH2-C-S-

O

KS -SH

HOH

palmitate

CH3-(CH2)11CH2-C-CH2-C-S-ACP

H O

OHCH3-(CH2)11CH2-C=C-C-S-ACP

OH

H

NADPH

NADP+

NADPH

NADP+

Thioesterase hydrolyzes palmitoyl-ACP,

releasing palmitate (C16:0)

CH3-(CH2)11CH2-C-CH2-C-S-ACPOO

H2O

ACP-SH

C16:0

C14:0


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Chain-length specificity of the substrate-loading,chain-elongation and chain-termination activitiesof mammalian fatty-acid synthase

S. Smith et al. Prog. Lipid Res.42: 289 (2003)

Vmax

(mmol/min/mg)

QuickTime and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Malonyl/acetyltrans

ferase

Thioesterase

Keto

acylsynthase

internal

acyltransferase

malonyl

C2:0

C4:0

C6:0

C8:0

C10:0

C12:0

C14:0

C16:0


18/29

+H3N MAT KRDH core ER TEKS CO2-

400 320 140 600 220 230 75 300

SH OHO-phospho-pantetheine

ACP

Approx. number of amino acidresidues in each domain

Animals: one multifunctional protein

OH

The enzymes of fatty acid synthesis have fused into a singleprotein during evolution

OH

TE CO2-+H3NER+H3N CO2-

DH CO2-+H3N

OH

AT CO2-+H3N

KR CO2-

+

H3N

SH

KS CO2-+H3N OHMT

CO2

-+H3N

CO2-+H3N

O-phosphopantetheine

ACP

E. coli: eight separate proteins

How does the growing fatty acid chain bound to ACP reach all the active sites?


19/29

Architecture of the mammalian fatty acid synthase

T. Maier et al. Science311:1258 (2006) 2cf2.pdb

QuickTime and a

TIFF (Uncompressed) decompressorare needed to see this picture.

The white and blue spheres indicate the active sites. Hollow spheres inthe domain colors represent the length of phosphopantheteine, showinghow closely ACP must approach each site during the catalytic cycle.

The ACP & TEdomains are notresolved in thecrystal structure,probably becausethey are verymobile.

The active form ofthe enzyme is adimer with a totalMW of ~250,000.


20/29

Differences between fatty acid synthesis and oxidation

FAD

FADH2

NAD+

NADH

H2O mitochondrion

CO-S-CoA

CO-S-CoA

CO-S-CoAHO H

CO-S-CoAO

CO-S-CoA

CH3-CO-S-CoA

CoA-SH

NADP+

NADPH

H2O

cytosol

CO-S-ACP

ACP-SHNADP+

NADPH

CO-S-ACP

CO-S-ACP

CO2

CO-S-ACP

CO2-

CO-S-ACPO

CO-S-ACPOHH

synthesisoxidation


21/29

Fatty acids with longer chains (C18:0 & C20:0) are synthesizedfrom palmitoylCoA & malonylCoA by an elongation mechanism

CO-S-CoA palmitoylCoA (C16:0)

stearoylCoA (C18:0)

CO-S-CoA

Different enzymes areinvolved, and CoA is usedin place of ACP, but the

reactions are otherwiseformally the same as insynthesis of palmitate.

These reactions occur

in mitochondria & thesmooth ER.

H2O

CoA-SH, CO2

O2C-CH2CO-S-CoA-

NADPH

NADP

NADPH

NADP


22/29

citratetransporter

Citrate carries 2-carbon units from mitochondria to the cytosol

CH3CO-S-CoACO2-CH2

HO-C-CO2-

CH2CO2-

CH2

CO2-

O=C-CO2-

CoA-SH + ATP

ADP + Pi

CoA-SH

Mitrochondrion Cytosol

CH3CO-S-CoA

CO2-CH2

HO-C-CO2-

CH2CO2-

+ CH2CO2-

O=C-CO2-

oxaloacetate

citrate

citrate

synthaseacetyl-CoA

citratelyase

Citrate lyase uses ATP to drive the breakdown ofcitrate to acetylCoA & oxaloacetate in the cytosol


23/29

ox. phos.

citratelyase

malatedehydro-genase

malicenzyme

pyruvatecarboxylase

Integration of fatty acid synthesis with carbohydrate metabolism

glucose

pyruvate

pyruvate

acetylCoA

CoA-SH

aminoacids oxaloacetate

citrate

malateNAD+

NADH

ATP + CO2

ADP + Pi

acetylCoA

CoA-SH

fattyacids

ATP

ADP+ Pi

citrate

oxaloacetate

NAD+

NADH

malate

pyruvate

Mitochondrion Cytosol

NADP+

NADPH + CO2

NADPH

ATP

TCAcycle

ATP


24/29

AcetylCoA carboxylase (biotin carboxylase/transcarboxylase)is the main control point for fatty acid synthesis in animals

the enzyme is regulated by bothallosteric effects and phosphorylation

acetyl-CoAcarboxylase

acetylCoA

malonylCoA

citrate

palmitoylCoA

glucagon, epinephrine, andadiponectin stimulatephosphorylation (inactivation)

insulin stimulatesdephosphorylation(activation)

X

X malonylCoA inhibits carnitine-acyltransferase I,blocking transport of palmitoylCoA into

mitochondria for oxidation

citratelyase

cAMP-dependentprotein kinase

acetyl-CoAcarboxylase -O--P

carnitine-acyltransferase I

the phosphorylatedenzyme is inactive


25/29

The active (unphosphorylated)form of acetylCoA carboxylaseforms long filaments

400

o


26/29

An imbalance between energy input and output can lead to obesity

~65% of the adult U.S. population are considered to be overweight (BMI* > 25); ~35%are obese (BMI > 30). More than 10% of U.S. children aged 2 to 5 are overweight.

Obesity raises the risk of heart disease, stroke, type-II diabetes and cancer.

adiposetissue

ADP

ATP

CO2 + H2O

Heat

Work orGrowth

Food

fatty acids& triacyl-glcerols

Obesity

*BMI = (weight in kg)/(height in m)2 = 703x(weight in pounds)/(height in inches)2


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Leptin and adiponectin convey signals of nutritional excess

blood

leptin

leptin,adiponectin

neuronalsignals

Increasebloodpressure &

heart rate

Increase catabolism

& thermogenesis

sympatheticnervous

system

Decrease fatty acid synthesis;switch on catabolism

(phosphorylate acetylCoA carboxylase)

(express gene foruncoupling protein)

heart, muscle, liver

adiposetissue

other partsof the brain

hypothalamus

In obesity, leptin decreases synthesis of insulin, which can lead to diabetes.


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Defects in leptin or its receptor can cause obesity

These mice are the same age. Both are homozygous for adefective variant of leptin. The mouse on the right received dailyinjections of purified leptin; the mouse on the left was not treated.

But most obese humans do not have a deficiency in leptin.

weight35 gweight

67 g


29/29

Ghrelin and PYY convey short-termsignals of hunger or satiety

other parts

of the brain

hypothalamus

Youre hungry!

Eat!

ghrelin

stomach

Youre full!

Stop eating!

PYY3-36

intestine

Suggested reading on obesity and regulation of energy balance:

J. Marx, Cellular warriers at the battle of the bulge Science299: 846 (2003)E.D. Rosen & B.M. Spiegelman, Adipocytes as regulators of energy balance

and glucose homeostasis Nature444: 847 (2006).

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