BIOCHEMISTRY 441

Part A (Bill Parson)

Winter 2007

1. Biosynthesis of fatty acids 2. 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 catabolism 13. DNA & RNA: primary and secondary structure

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

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).

Fatty acids have extended hydrocarbon chains

CO2H

palmitic acid, C16:0

CO2H
1

stearic acid, C18:0

9
10

CO2H
1

palmitoleic acid, C16:1(D9)

9 10

CO2H

oleic acid, C18:1(D9)

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

Fatty acids are components of phospholipids and triacylglycerols

glycerophospholipids
O
HOCH-CH=CH-CH2R O O CH2OCR R-C-NH-CH RCOCH O O+ + CH2OPOCH2CH2N(CH3)3 CH2OPOCH2CH2N(CH3)3 O

sphingolipids

triacylglycerols
O RCOCH O CH2OCR

O CH2OCR

O Sphingolipids on cell surfaces are sites of cell recognition.

Inositol phospholipids participate in intracellular signaling.

Triacylglycerols are stored as energy reserves.

Phospholipid bilayers are the central structural elements of biological membranes.

Fatty acids also are found as cholesterol esters in lipoproteins, and are attached covalently to some proteins.

Triacylglycerols are stored as energy reserves in adipose tissue and other tissues

capillary

lipid droplets

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

Fatty acids are synthesized from acetyl-CoA in adipose tissue & the liver
O CH3-C-S-CoA
ATP, NADPH, CO2 Why is CO2 needed?

O C-S-CoA H3C
palmitic acid (C16:0)
palmitoylCoA

O C-S-CoA H3C
stearic acid (C18:0) 9 oleic acid (C18:1 D9) oleoylCoA stearoylCoA

O C-S-CoA
1

H3C
18 The labeling patterns suggest that the fatty acid chain forms by successive addition of two-carbon units

MalonylCoA serves as the donor of two-carbon units

bound biotin
HN O C ATP
NH HCO3

malonylCoA
O -O C-CH -C-S-CoA 2 2

ADP
Pi HN

O
C

O CH3-C-S-CoA N-CO2
-

O
HN C NH

S O=C

O=C
NH

NH

O=C

NH

carboxylase site

biotin attachment site

transcarboxylase site

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 in separate subunits; in animals, they are on a single, multifunctional polypeptide.

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

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

ACP
CH2 O O=P-OO CH2 CH3-C-CH3 CHOH C=O NH CH2 CH2 C=O NH CH2 CH2 SH
Pantetheine is vitamin B5

O -O C-CH -C-S-CoA 2 2

O -O C-CH -C-S-ACP 2 2 4-phosphopantetheine

HS-ACP

HS-CoA

O CH3-C-S-CoA

O CH3-C-S-ACP

HS-ACP

HS-CoA

Acyl-carrier protein from Bacillus subtilis

4-phosphopantetheine

www.rcsb.org/pdb

from pdb file 1f80.pdb; K. D. Parris et al. Struct. Fold. Design 8: 883 (2000).

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

O
CoA-SH

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

O
MAT

O
MAT

-OH

-O-C-CH2-C-O-

O
ACP--SH

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

malonylACP

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

O

Bacteria have separate malonyl & acetyl transferases CoA-SH

CH3-C-S-CoA

O
MAT

-OH

CH3-C-O-

MAT

O CH3-C-S- ACP Acetyl units then move from ACP to the keto-synthase (KS), which catalyzes the condensation reaction

ACP -SH

KS

-SH

O CH3-C-SKS

The condensation reaction

malonyl-ACP 3-ketoacyl-ACP

O C

HO H+ O-

HCO3 S

O C CH3

ACP
O S-C-CH3

ACP
SH

b-ketoacyl synthase

b-ketoacyl synthase

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

Release of bicarbonate is exothermic and pulls the reaction in the direction of condensation.

O CH3-C-S-

O KS

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

b-ketoacyl synthase

HCO3 O

Reduction by NADPH, dehydration, and a second reduction generates butyryl-ACP

KS -SH

CH3-C-CH2-C-S-ACP
b-ketoacyl reductase

NADPH NADP+ O

O CH3-CH2CH2-C-S-ACP
enoyl reductase

H NADP+ NADPH
dehydrase

CH3-C-CH2-C-S-ACP OH HOH

CH3-C=C-C-S-ACP H

To continue the cycle, the fatty acid chain must move back to the ketoacyl synthase
O CH3-C-SO CH3-CH2CH2-C-SACP-SH H2O O CH3-CH2CH2-C-S-ACP NADP+ NADPH
enoyl reductase b-ketoacyl reductase

O KS

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

b-ketoacyl synthase

KS

CO2 O O

KS -SH

CH3-C-CH2-C-S-ACP NADPH NADP+ O

dehydrase CH3-C-CH2-C-S-ACP

CH3-C=C-C-S-ACP H

OH
HOH

A second turn of the cycle generates hexanoyl-ACP

-O-C-CH2-C-S-ACP O CH3-CH2CH2-C-S-

b-ketoacyl synthase

KS

CO2 O O

KS -SH

CH3CH2CH2-C-CH2-C-S-ACP O CH3CH2CH2-CH2CH2-C-S-ACP NADP+ NADPH

enoyl reductase b-ketoacyl reductase

NADPH NADP+ O

dehydrase

CH3CH2 CH2-C-CH2-C-S-ACP OH HOH

CH3CH2CH2-C=C-C-S-ACP H

The cycle stops when the fatty acid chain reaches 16 carbons
Thioesterase hydrolyzes palmitoyl-ACP, releasing palmitate (C16:0)
C14:0

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

O KS

b-ketoacyl synthase

CH3-(CH2)11CH2-C-SACP-SH palmitate

CO2 O O

KS -SH
thioesterase

CH3-(CH2)11CH2-C-CH2-C-S-ACP NADPH NADP+ H OH HOH O

O CH3-(CH2)11CH2-CH2CH2-C-S-ACP
C16:0

H2O

NADP+ H O NADPH CH3-(CH2)11CH2-C=C-C-S-ACP

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

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

Thioesterase

QuickTime and a TIFF (Un compressed) decompre ssor are neede d to see this picture.

C14:0

C10:0

malonyl

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

C12:0

C16:0

C4:0

C6:0

C2:0

C8:0

Ketoacyl synthase internal acyltransferase

Malonyl/acetyltransferase

Vmax (mmol/min/mg)

The enzymes of fatty acid synthesis have fused into a single protein during evolution
+H N 3

MT
OH CO2-

CO2+H N 3

+H N 3

KR

CO2+H N 3

+H N 3

KS
SH
+H N 3

CO2-

ER DH

ACP

CO2CO2+H N 3

TE
OH

CO2-

AT
OH

+H N 3

CO2-

O-phosphopantetheine

E. coli: eight separate proteins

Approx. number of amino acid residues in each domain 400
+H N 3

320

140

600

220

230

75

300

KS
SH

MAT
OH

DH

core

ER

KR

ACP

TE

CO2-

Animals: one multifunctional protein

OH O-phosphopantetheine

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

Architecture of the mammalian fatty acid synthase

The active form of the enzyme is a dimer with a total MW of ~250,000.
QuickTime and a TIFF (Un compressed) decompressor are neede d to see this picture.

The ACP & TE domains are not resolved in the crystal structure, probably because they are very mobile.

The white and blue spheres indicate the active sites. Hollow spheres in the domain colors represent the length of phosphopantheteine, showing how closely ACP must approach each site during the catalytic cycle.
T. Maier et al. Science 311: 1258 (2006) 2cf2.pdb

Differences between fatty acid synthesis and oxidation

oxidation
CO-S-CoA
FAD FADH2 NADP+ NADPH

synthesis
CO-S-ACP

ACP-SH

CO-S-CoA CO-S-ACP H2O HO H CO-S-CoA

NAD+ NADH

mitochondrion cytosol H OH

H2O

CO-S-ACP
NADP+ NADPH

O CO-S-CoA CoA-SH CH3-CO-S-CoA CO-S-CoA

O CO-S-ACP

CO2
CO-S-ACP CO2CO-S-ACP

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

CO-S-CoA - O C-CH CO-S-CoA 2 2 CoA-SH, CO2 NADPH NADP H2O NADPH NADP

palmitoylCoA (C16:0)

These reactions occur in mitochondria & the smooth ER.

Different enzymes are involved, and CoA is used in place of ACP, but the reactions are otherwise formally the same as in synthesis of palmitate.

CO-S-CoA stearoylCoA (C18:0)

Citrate carries 2-carbon units from mitochondria to the cytosol

citrate synthase Mitrochondrion Cytosol

acetyl-CoA

CH3CO-S-CoA
O=C-CO2oxaloacetate CH2 CO2-

CoA-SH CO2CH2 HO-C-CO2CH2 CO2citrate CO2CH2 HO-C-CO2CH2 CO2-

citrate transporter

CoA-SH + ATP citrate lyase CH3CO-S-CoA

ADP + Pi O=C-CO2-

CH2 CO2-

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

Integration of fatty acid synthesis with carbohydrate metabolism

Mitochondrion
citrate pyruvate CoA-SH acetylCoA amino acids TCA cycle oxaloacetate ox. phos. ATP ADP + Pi NADH

Cytosol
citrate ATP
citrate lyase

fatty acids CoA-SH

NADPH ATP acetylCoA

ADP + Pi

oxaloacetate
malate dehydrogenase

NADH

NAD+
malate malate
malic enzyme

NAD+

pyruvate carboxylase

NADP+ NADPH + CO2 glucose

ATP + CO2 pyruvate

pyruvate

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

the enzyme is regulated by both allosteric effects and phosphorylation

acetylCoA
acetyl-CoA carboxylase

insulin stimulates dephosphorylation (activation)

the phosphorylated enzyme is inactive acetyl-CoA -O--P carboxylase

malonylCoA

cAMP-dependent protein kinase glucagon, epinephrine, and adiponectin stimulate phosphorylation (inactivation)

palmitoylCoA

X
carnitine-acyltransferase I

malonylCoA inhibits carnitine-acyltransferase I, blocking transport of palmitoylCoA into mitochondria for oxidation

The active (unphosphorylated) form of acetylCoA carboxylase forms long filaments

400

An imbalance between energy input and output can lead to obesity

Food
adipose tissue

Work or Growth

ADP ATP

fatty acids & triacylglcerols

Obesity

Heat CO2 + H2O

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

Leptin and adiponectin convey signals of nutritional excess

sympathetic nervous system neuronal signals other parts of the brain

hypothalamus

leptin
Increase catabolism & thermogenesis (express gene for uncoupling protein) adipose tissue blood

Increase blood pressure & heart rate

leptin, adiponectin

Decrease fatty acid synthesis; switch on catabolism (phosphorylate acetylCoA carboxylase)

heart, muscle, liver

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

Defects in leptin or its receptor can cause obesity

weight 67 g

weight 35 g

These mice are the same age. Both are homozygous for a defective variant of leptin. The mouse on the right received daily injections of purified leptin; the mouse on the left was not treated. But most obese humans do not have a deficiency in leptin.

Ghrelin and PYY convey short-term signals of hunger or satiety

other parts of the brain

Youre hungry! Eat!

hypothalamus

Youre full! Stop eating!

ghrelin

PYY3-36
intestine

stomach

Suggested reading on obesity and regulation of energy balance: J. Marx, Cellular warriers at the battle of the bulge Science 299: 846 (2003) E.D. Rosen & B.M. Spiegelman, Adipocytes as regulators of energy balance and glucose homeostasis Nature 444: 847 (2006).