Voet D., Voet Ju.G. Biochemistry

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(a)

(b)

erol digestion therefore depends on the surface area of the

interface, a quantity that is greatly increased by the churn-

ing peristaltic movements of the intestine combined with

the emulsifying action of bile salts. These are powerful di-

gestive detergents that, as we shall see in Section 25-6C, are

synthesized by the liver and secreted via the gallbladder

into the small intestine where lipid digestion and absorp-

tion mainly take place.

b. Pancreatic Lipase Requires Activation and

Has a Catalytic Triad

Pancreatic lipase (triacylglycerol lipase) catalyzes the

hydrolysis of triacylglycerols at their 1 and 3 positions to

form sequentially 1,2-diacylglycerols and 2-acylglycerols,

together with the Na

⫹

and K

⫹

salts of fatty acids (soaps).

These soaps, being amphipathic, aid in the lipid emulsifica-

tion process.

The enzymatic activity of pancreatic lipase greatly in-

creases when it contacts the lipid–water interface, a phe-

nomenon known as interfacial activation. Binding to the

lipid–water interface requires the presence of mixed mi-

celles of phosphatidylcholine (Fig. 12-4) and bile salts, as

well as the pancreatically produced protein named coli-

pase, which forms a 1:1 complex with lipase. This complex

aids in the adsorption of the enzyme to emulsified oil

droplets as well as stabilizes the enzyme in an active con-

formation. The X-ray structures, determined by Christian

Cambillau, of pancreatic lipase–colipase complexes, alone

and cocrystallized with mixed micelles of phosphatidyl-

choline and bile salts, have revealed the structural basis of

lipase activation as well as how colipase and micelles aid li-

pase in binding to the lipid–water interface (Fig. 25-1).

The active site of the 449-residue pancreatic lipase,

which is contained in the enzyme’s N-terminal domain, has

a catalytic triad that closely resembles that in the serine

proteases (Section 15-3B; recall that ester hydrolysis is

mechanistically similar to peptide hydrolysis). In aqueous

solution (Fig. 25-1a), the lipase’s active site is covered by a

26-residue helical lid. However, in the presence of the

mixed micelles (Fig. 25-1b), the lid undergoes a complex

structural reorganization that exposes the active site;

causes a contacting 10-residue loop, the ␤5 loop, to change

conformation in a way that forms the active enzyme’s

oxyanion hole; and generates a hydrophobic surface about

the entrance to the active site. Indeed, the active site of the

mixed micelle–containing complex contains a long rod of

electron density that contacts the catalytic triad’s Ser

residue and appears to be a phosphatidylcholine molecule.

Colipase binds to the C-terminal domain of lipase such

that the hydrophobic tips of the three loops that comprise

much of this 90-residue protein extend from the complex on

the same face as lipase’s active site.A continuous hydropho-

bic plateau is thereby created that extends over a distance

of ⬎50 Å past the active site (bottom of Fig.25-1b) and that,

presumably, helps bind the complex to the lipid surface. In

the presence of the mixed micelles, colipase changes confor-

mation so as to form three hydrogen bonds to the opened

lid, thereby stabilizing it in this conformation.

The mixed micelles are not visible in the X-ray

structure. However, neutron diffraction studies, by Juan

Fontecilla-Camps, of crystals of a lipase–colipase–micelle

complex in which the lipase is in its active conformation

reveal that the activating micelle interacts, not with the

substrate site, but with the concave face of colipase and the

adjacent tip of the lipase’s C-terminal domain (left side of

Section 25-1. Lipid Digestion, Absorption, and Transport 941

Table 25-1 Energy Content of Food Constituents

Constituent ⌬H (kJ ⴢ g

⫺1

dry weight)

Carbohydrate 16

Fat 37

Protein 17

Source: Newsholme, E.A. and Leech, A.R., Biochemistry for the Medical

Sciences, p. 16, Wiley (1983).

Figure 25-1 X-ray structures of pancreatic lipase in complex

with colipase. (a) In aqueous solution, and (b) cocrystallized with

mixed micelles of phosphatidylcholine and bile salts.The lipase is

drawn in ribbon form with its N-terminal domain (residues

1–336) cyan, its C-terminal domain (residues 337–449) green, the

lid (residues 237–262) magenta, and the ␤5 loop (residues 76–85)

orange. The colipase is yellow. A phosphatidylcholine molecule

that is bound in the lipase active site in Part b is shown in stick

form with C green, O red, and P orange.The micelles, which have

irregular structures (e.g., Fig. 2-9), are not visible. [Based on

X-ray structures by Christian Cambillau, LCCMB-CNRS,

Marseille, France. PDBids 1N8S and 1LPB.]

JWCL281_c25_940-1018.qxd 8/9/10 9:43 AM Page 941

Fig. 25-1b).Apparently, micelle binding and substrate bind-

ing involve different regions of the lipase–colipase com-

plex. Hence, strictly speaking, lipase activation appears not

to be interfacial but, instead, occurs in the aqueous phase

and requires the binding of colipase and a micelle.

c. Pancreatic Phospholipase A

Has a

Modified Catalytic Triad

Phospholipids are degraded by pancreatic phospholipase

, which hydrolytically excises the fatty acid residue at C2

to yield the corresponding lysophospholipids (Fig. 25-2),

which are also powerful detergents. Indeed, the phospho-

lipid lecithin (phosphatidylcholine) is secreted in the bile,

presumably to aid in lipid digestion.

Phospholipase A

, as does triacylglycerol lipase, prefer-

entially catalyzes reactions at interfaces. However, as Paul

Sigler’s determinations of the X-ray structures of the

phospholipases A

from cobra venom and bee venom re-

vealed, its mechanism of interfacial activation differs from

that of triacylglycerol lipase in that it does not change its

conformation. Instead, phospholipase A

contains a hy-

drophobic channel that provides the substrate with direct

access from the phospholipid aggregate (micelle or mem-

brane) surface to the bound enzyme’s active site. Hence,

on leaving its micelle to bind to the enzyme, the substrate

need not become solvated and then desolvated (Fig. 25-3).

In contrast, soluble and dispersed phospholipids must first

surmount these significant kinetic barriers in order to bind

to the enzyme.

The catalytic mechanism of phospholipase A

also dif-

fers substantially from that of triacylglycerol lipase. Al-

though the phospholipase A

active site contains the His

and Asp components of a catalytic triad, an enzyme-bound

water molecule occupies the position expected for an ac-

tive site Ser. Moreover, the active site contains a bound

2

ion and does not form an acyl–enzyme intermediate.

Sigler therefore proposed that phospholipase A

catalyzes

the direct hydrolysis of phospholipid with a His–Asp “cat-

alytic dyad” activating an active site water molecule for

nucleophilic attack on the ester, and with the Ca

2

ion sta-

bilizing the oxyanion transition state. However, the subse-

quently determined X-ray structure, by Mahendra Jain and

942 Chapter 25. Lipid Metabolism

Figure 25-2 Catalytic action of phospholipase A

. Phospholipase

hydrolytically excises the C2 fatty acid residue from a

phospholipid to yield the corresponding lysophospholipid.The

Figure 25-3 Substrate binding to phospholipase A

. (a) A

hypothetical model of phospholipase A

in complex with a micelle

of lysophosphatidylethanolamine as shown in cross section.The

protein is drawn in cyan, the phospholipid head groups are yellow,

and their hydrocarbon tails are blue. The calculated atomic

–

COH

phospholipase C

phospholipase A

phospholipase D

Phospholipid Lysophospholipid

CHOH

–

bonds hydrolyzed by other types of phospholipases, which are

named according to their specificities, are also indicated.

Lipid micelle

(b)

Phospholipase A

motions of the assembly are indicated through a series of

superimposed images taken at 5-ps intervals. [Courtesy of

Raymond Salemme, E.I. du Pont de Nemours & Company.]

(

b) Schematic diagram of a productive interaction between

phospholipase A

and a phospholipid contained in a micelle.

(a)

JWCL281_c25_940-1018.qxd 4/20/10 1:58 PM Page 942

d. Bile Salts and Fatty Acid–Binding Protein

Facilitate the Intestinal Absorption of Lipids

The mixture of fatty acids and mono- and diacylglycerols

produced by lipid digestion is absorbed by the cells lining

the small intestine (the intestinal mucosa) in a process facil-

itated by bile salts.The micelles formed by the bile salts take

up the nonpolar lipid degradation products so as to permit

their transport across the unstirred aqueous boundary layer

at the intestinal wall. The importance of this process is

demonstrated in individuals with obstructed bile ducts:

They absorb little of their dietary lipids but, rather, elimi-

nate them in hydrolyzed form in their feces (steatorrhea).

Evidently, bile salts are not only an aid to lipid digestion but

are essential for the absorption of lipid digestion products.

Bile salts are likewise required for the efficient intestinal

absorption of the lipid-soluble vitamins A, D, E, and K.

Inside the intestinal cells, fatty acids form complexes

with intestinal fatty acid–binding protein (I-FABP), a cyto-

Section 25-1. Lipid Digestion, Absorption, and Transport 943

Brian Bahnson, of phospholipase A

in complex with the

tetrahedral intermediate mimic MJ33

suggests that a second, previously unobserved water mole-

cule, which is liganded by the Ca

2

ion, is the attacking nu-

cleophile (Fig. 25-4a). This has led to the formulation of a

reaction mechanism (Fig. 25-4b) in which the Asp–His–

water catalytic triad and the Ca

2

ion both activate the sec-

ond water molecule, with the Ca

2

ion also stabilizing the

resulting tetrahedral intermediate.

MJ33 [1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-

2-phosphomethanol]

O O

–

(CH

)

O CH

Figure 25-4 Structure and mechanism of phospholipase A

(a) The X-ray structure of the 124-residue monomeric porcine

phospholipase A

(lavender) in complex with the tetrahedral

intermediate mimic MJ33.The enzyme’s active site contains a

catalytic triad similar to those of the serine proteases (Fig. 15-20)

with a water molecule replacing the catalytic Ser.The His 48 and

Asp 99 side chains of the catalytic triad together with MJ33 are

drawn in stick form colored according to atom type (C gray, N

blue, O red, F green, and P yellow). The water molecule of the

catalytic triad and the catalytically important Ca

2

ion are

represented by red and cyan spheres. Catalytically important

hydrogen bonds and Ca

2

liganding interactions are represented

by thin black lines.The tetrahedral phosphoryl group of MJ33

presumably occupies the site of the unobserved H

O. Residues

65 to 74 of the protein have been deleted for clarity. [Based on

an X-ray structure by Mahendra Jain and Brian Bahnson,

University of Delaware. PDBid 1FXF.] (b) The catalytic

mechanism of phospholipase A

. (1) The catalytic triad activates

a second water molecule to attack the scissile carbonyl carbon

with Ca

2

coordinating the activated water molecule as well as

electrostatically stabilizing the resulting tetrahedral intermediate

(rather than doing so via nucleophilic catalysis as occurs in the

serine proteases; Fig. 15-23). (2) The tetrahedral intermediate

decomposes to yield products. [After Berg, O.G., Gelb, M.H.,

Tsai, M.-D., and Jain, M.K., Chem. Rev. 101, 2638 (2001).]

His

Catalytic triad

(b)

–

Asp

...

His

Tetrahedral

intermediate

H R

RCH

CHR

OXCH

–

Asp

...

His

–

Asp

...

(a)

JWCL281_c25_940-1018.qxd 6/8/10 8:59 AM Page 943

cles called chylomicrons. These, in turn, are released into

the bloodstream via the lymph system for delivery to the

tissues. Similarly, triacylglycerols synthesized by the liver

are packaged into very low density lipoproteins (VLDL)

and released directly into the blood. These lipoproteins,

whose origins, structures, and functions are discussed in

Section 12-5, maintain their otherwise insoluble lipid com-

ponents in aqueous solution.

The triacylglycerol components of chylomicrons and

VLDL are hydrolyzed to free fatty acids and glycerol in the

capillaries of adipose tissue and skeletal muscle by lipopro-

tein lipase (Section 12-5Ba). The resulting free fatty acids

are taken up by these tissues while the glycerol is trans-

ported to the liver or kidneys. There it is converted to the

glycolytic intermediate dihydroxyacetone phosphate by

the sequential actions of glycerol kinase and glycerol-3-

phosphate dehydrogenase (Fig. 25-6).

Mobilization of triacylglycerols stored in adipose tissue

involves their hydrolysis to glycerol and free fatty acids by

hormone-sensitive triacylglycerol lipase (or just hormone-

sensitive lipase). The free fatty acids are released into the

bloodstream, where they bind to serum albumin (or just al-

bumin), a soluble 585-residue monomeric protein that com-

prises about half of the blood serum protein. In the absence

of albumin, the maximum solubility of free fatty acids is

⬃10

6

M.Above this concentration, free fatty acids form mi-

celles that act as detergents to disrupt protein and mem-

brane structure and would therefore be toxic.However,the ef-

fective solubility of fatty acids in fatty acid–albumin complexes

is as much as 2 mM. Nevertheless, those rare individuals with

analbuminemia (severely depressed levels of albumin) suf-

fer no apparent adverse symptoms; evidently, their fatty

acids are transported in complex with other serum proteins.

The X-ray structure of human serum albumin in its com-

plexes with a variety of common fatty acids, determined by

Stephen Curry, reveals that each albumin molecule can bind

up to seven fatty acid molecules (Fig. 25-7). However, these

binding sites have different fatty acid–binding affinities so

that,under normal physiological conditions, albumin carries

between 0.1 and 2 fatty acid molecules per protein mole-

cule. Albumin also binds an extraordinarily broad range of

drugs and is thereby a major and usually unpredictable in-

fluence on their pharmacokinetics (Section 15-4Ba). In-

deed, the large amounts of fatty acids in the blood after

meals can significantly affect the pharmacokinetics of a

drug through competitive and/or cooperative interactions.

944 Chapter 25. Lipid Metabolism

plasmic protein, which serves to increase the effective sol-

ubility of these water-insoluble substances and also to

protect the cell from their detergent-like effects (recall

that soaps are fatty acid salts).The X-ray structures of rat

I-FABP, both alone and in complex with a single molecule

of palmitate, were determined by James Sacchettini. This

monomeric, 131-residue protein consists largely of 10 an-

tiparallel  strands organized into a stack of two approxi-

mately orthogonal  sheets (Fig. 25-5).The palmitate occu-

pies a gap between two of the  strands such that it lies

between the  sheets with an orientation that, over much of

its length, is more or less parallel to the gapped  strands

(this structure has therefore been described as forming a

“-clam”). The palmitate’s carboxyl group interacts with

Arg 106, Gln 115, and two bound water molecules, whereas

the methylene chain is encased by the side chains of several

hydrophobic, mostly aromatic, residues.

e. Lipids Are Transported in Lipoprotein Complexes

The lipid digestion products absorbed by the intestinal

mucosa are converted by these tissues to triacylglycerols

(Section 25-4F) and then packaged into lipoprotein parti-

NAD

NADH + H

–

CHHO O

glycerol-3-

phosphate

dehydrogenase

glycerol

kinase

L-Glycerol-

3-phosphate

Dihydroxy-

acetone

phosphate

L-Glycerol

2–

HHO

ATP ADP

Figure 25-6 Conversion of glycerol to the glycolytic intermediate dihydroxyacetone phosphate.

Figure 25-5 X-ray structure of rat intestinal fatty acid–binding

protein in complex with palmitate. The protein is drawn in ribbon

form colored in rainbow order from its N-terminus (blue) to its

C-terminus (red).The palmitate is shown in space-filling form

with C green and O red. [Based on an X-ray structure by James

Sacchettini,Albert Einstein College of Medicine. PDBid 2IFB.]

JWCL281_c25_940-1018.qxd 4/20/10 1:58 PM Page 944

labels to trace metabolic pathways, fed dogs fatty acids

labeled at their  (last) carbon atom by a benzene ring

and isolated the phenyl-containing metabolic products

from their urine. Dogs fed labeled odd-chain fatty acids ex-

creted hippuric acid, the glycine amide of benzoic acid,

whereas those fed labeled even-chain fatty acids excreted

phenylaceturic acid, the glycine amide of phenylacetic acid

(Fig. 25-8). Knoop therefore deduced that the oxidation of

the carbon atom  to the carboxyl group is involved in fatty

acid breakdown.Otherwise,the phenylacetic acid would be

further oxidized to benzoic acid. Knoop proposed that this

breakdown occurs by a mechanism known as  oxidation

in which the fatty acid’s C



atom is oxidized. It was not un-

til after 1950, following the discovery of coenzyme A, that

the enzymes of fatty acid oxidation were isolated and their

reaction mechanisms elucidated. This work confirmed

Knoop’s hypothesis.

A. Fatty Acid Activation

Before fatty acids can be oxidized, they must be “primed”

for reaction in an ATP-dependent acylation reaction to

form fatty acyl-CoA.This activation process is catalyzed by

a family of at least three acyl-CoA synthetases (also called

thiokinases) that differ according to their chain-length

specificities. These enzymes, which are associated with

either the endoplasmic reticulum (ER) or the outer mito-

chondrial membrane, all catalyze the reaction

In the activation of

O-labeled palmitate by a long-

chain acyl-CoA synthetase, both the AMP and the acyl-

CoA products become

O labeled. This observation

fatty acyl-CoA  AMP  PP

Fatty acid  CoA  ATP Δ

2 FATTY ACID OXIDATION

The biochemical strategy of fatty acid oxidation was under-

stood long before the advent of biochemical techniques

involving enzyme purification or the use of radioactive

tracers. In 1904, Franz Knoop, in the first use of chemical

Section 25-2. Fatty Acid Oxidation 945

Figure 25-7 X-ray structure of human serum albumin in

complex with 7 molecules of palmitic acid. The protein is drawn

in semitransparent ribbon form colored in rainbow order from

its N-terminus (blue) to its C-terminus (red).The fatty acids are

shown in space-filling form with C green and O red. [Based on

an X-ray structure by Stephen Curry, Imperial College of Science,

Technology, and Medicine, London, U.K. PDBid 1E7H.]

Figure 25-8 Franz Knoop’s classic experiment indicating that

fatty acids are metabolically oxidized at their -carbon atom.

-Phenyl-labeled fatty acids containing an odd number of carbon

atoms are oxidized to the phenyl-labeled C

product, benzoic

acid, whereas those with an even number of carbon atoms are

oxidized to the phenyl-labeled C

product, phenylacetic acid.

Glycine residue

Hippuric acidBenzoic acidOdd-chain fatty acid

Fatty acid fed

Breakdown product Excretion product

COOHCH

C C

(CH

)

(n + 1) C

Glycine residue

Phenylaceturic acidPhenylacetic acidEven-chain fatty acid

COOHCH

C C

(CH

)

(n + 1) C

These products are excreted as their respective glycine amides,

hippuric and phenylaceturic acids.The vertical arrows indicate

the deduced sites of carbon oxidation.The intermediate C

products are oxidized to CO

and H

O and were therefore not

isolated.

JWCL281_c25_940-1018.qxd 6/8/10 8:59 AM Page 945

indicates that the reaction has an acyladenylate mixed an-

hydride intermediate that is attacked by the sulfhydryl group

of CoA to form the thioester product (Fig. 25-9).The reac-

tion involves both the cleavage and the synthesis of bonds

with large negative free energies of hydrolysis so that the

free energy change associated with the overall reaction is

close to zero. The reaction is driven to completion in the

cell by the highly exergonic hydrolysis of the product py-

rophosphate (PP

) catalyzed by the ubiquitous inorganic

pyrophosphatase. Thus, as commonly occurs in metabolic

pathways, a reaction forming a “high-energy” bond through

the hydrolysis of one of ATP’s phosphoanhydride bonds is

driven to completion by the hydrolysis of its second such

bond.

B. Transport Across the Mitochondrial Membrane

Although fatty acids are activated for oxidation in the cy-

tosol, they are oxidized in the mitochondrion as Eugene

Kennedy and Albert Lehninger established in 1950. We

must therefore consider how fatty acyl-CoA is transported

across the inner mitochondrial membrane. A long-chain

fatty acyl-CoA cannot directly cross the inner mitochon-

drial membrane. Rather, its acyl portion is first transferred to

carnitine (Fig. 25-10), a compound that occurs in both plant

and animal tissues. This transesterification reaction has an

equilibrium constant close to 1, which indicates that the

O-acyl bond of acyl-carnitine has a free energy of hydrolysis

similar to that of the thioester. Carnitine palmitoyltrans-

ferases I and II, which can transfer a variety of acyl groups,

are located,respectively, on the external and internal surfaces

of the inner mitochondrial membrane. The translocation

process itself is mediated by a specific carrier protein that

transports acyl-carnitine into the mitochondrion while trans-

porting free carnitine in the opposite direction. Acyl-CoA

transport therefore occurs via four reactions (Fig. 25-11):

1. The acyl group of a cytosolic acyl-CoA is transferred

to carnitine, thereby releasing the CoA to its cytosolic pool.

2. The resulting acyl-carnitine is transported into the

mitochondrial matrix by the transport system.

3. The acyl group is transferred to a CoA molecule

from the mitochondrial pool.

4. The product carnitine is returned to the cytosol.

946 Chapter 25. Lipid Metabolism

–

H SCoA

SCoA

–

P O

Adenosine

–

Adenosine

–

Adenosine

inorganic

pyrophosphatase

ATP

AMPAcyl-CoA

Acyladenylate

mixed anhydride

Fatty acid

–

(CH

)

SCo

R+

COO

–

Carnitine (4-trimethylamino-

3-hydroxybutyrate)

(CH

)

CH CH

COO

–

Acyl-carnitine

R O

+ H SCoA

carnitine palmitoyltransferase

Figure 25-9 Mechanism of fatty acid activation catalyzed by

acyl-CoA synthetase. Experiments utilizing

O-labeled fatty

acids (*) demonstrate that the formation of acyl-CoA involves an

intermediate acyladenylate mixed anhydride.

Figure 25-10 Acylation of carnitine catalyzed by carnitine

palmitoyltransferase.

Figure 25-11 Transport of fatty acids into the mitochondrion.

Cytosol Matrix

Inner

mitochondrial

membrane

Carnitine

carrier protein

carnitine

palmitoyl

transferase I

carnitine

palmitoyl

transferase II

Carnitine Carnitine

Carnitine

SCoA

SCo

SCoAHSCoAH

R CarnitineC

JWCL281_c25_940-1018.qxd 6/8/10 8:59 AM Page 946

2. Hydration of the double bond by enoyl-CoA hydratase

(EH) to form a 3-

L-hydroxyacyl-CoA.

3. NAD

⫹

-dependent dehydrogenation of this ␤-

hydroxyacyl-CoA by 3-

L-hydroxyacyl-CoA dehydroge-

nase (HAD) to form the corresponding ␤-ketoacyl-CoA.

4. C

␣

¬C

␤

cleavage in a thiolysis reaction with CoA as

catalyzed by ␤-ketoacyl-CoA thiolase (KT; also called just

thiolase) to form acetyl-CoA and a new acyl-CoA contain-

ing two less C atoms than the original one.

The first three steps of this process chemically resemble the

citric acid cycle reactions that convert succinate to oxalo-

acetate (Sections 21-3F–H):

–

Succinate

FAD FADH

succinate

dehydrogenase

–

HO H

–

Fumarate

fumarase

L-Malate

malate

dehydrogenase

NAD

NADH

+ H

Oxaloacetate

The cell thereby maintains separate cytosolic and mito-

chondrial pools of CoA. The mitochondrial pool functions

in the oxidative degradation of pyruvate (Section 21-2A)

and certain amino acids (Sections 26-3E–G) as well as fatty

acids, whereas the cytosolic pool supplies fatty acid biosyn-

thesis (Section 25-4). The cell similarly maintains separate

cytosolic and mitochondrial pools of ATP and NAD

⫹

C. ␤ Oxidation

Fatty acids are dismembered through the ␤ oxidation of fatty

acyl-CoA, a process that occurs in four reactions (Fig. 25-12):

1. Formation of a trans-␣,␤ double bond through dehydro-

genation by the flavoenzyme acyl-CoA dehydrogenase (AD).

Section 25-2. Fatty Acid Oxidation 947

Figure 25-12 The ␤-oxidation pathway of fatty acyl-CoA.

See the Animated Figures

SCoA

Fatty acyl-CoA

H H

FADH

FAD

ETF

red

ETF:ubiquinone

oxidoreductase

ETF:ubiquinone

oxidoreductase

red

Mitochondrial

electron-

transport

chain

SCoA

trans-

-Enoyl-CoA

enoyl-CoA hydratase (EH)

SCoA

3-L-Hydroxyacyl-CoA

acyl-CoA

dehydrogenase (AD)

NADH

NAD

3-L-hydroxyacyl-CoA

dehydrogenase (HAD)

+ H

SCoA

-Ketoacyl-CoA

CoASH

-ketoacyl-CoA thiolase (KT)β

(CH

)

(CH

)

(CH

)

(CH

)

(CH

)

SCoA

Fatty acyl-CoA

(2 C atoms shorter)

SCoA

Acetyl-CoA

2ADP

+ 2P

2ATP

15678

JWCL281_c25_940-1018.qxd 8/9/10 9:43 AM Page 947

Mitochondria contain four acyl-CoA dehydrogenases,

with specificities for short- (C

to C

), medium- (C

to C

long- (between medium and very long), and very long-

chain (C

to C

) fatty acyl-CoAs. The reaction catalyzed

by these enzymes is thought to involve removal of a proton

at C

␣

and transfer of a hydride ion equivalent from C

␤

FAD (Fig. 25-12, Reaction 1). The X-ray structure of the

medium-chain acyl-CoA dehydrogenase (MCAD) in com-

plex with octanoyl-CoA, determined by Jung-Ja Kim,

clearly shows how the enzyme orients the enzyme’s base

(Glu 376), the substrate C

␣

¬C

␤

bond, and the FAD pros-

thetic group for reaction (Fig. 25-13).

a. Acyl-CoA Dehydrogenase Is Reoxidized

via the Electron-Transport Chain

The FADH

resulting from the oxidation of the fatty

acyl-CoA substrate is reoxidized by the mitochondrial

electron-transport chain through the intermediacy of a se-

ries of electron-transfer reactions. Electron-transfer flavo-

protein (ETF) transfers two electrons from FADH

to the

flavoiron–sulfur protein ETF:ubiquinone oxidoreductase,

which in turn transfers two electrons to the mitochondrial

electron-transport chain by reducing coenzyme Q (CoQ;

Fig. 25-12, Reactions 5–8). Reduction of O

to H

O by the

electron-transport chain beginning at the CoQ stage re-

sults in the synthesis of 1.5 ATPs per two electrons trans-

ferred (Section 22-2Bc).

b. Acyl-CoA Dehydrogenase Deficiency

Has Fatal Consequences

The unexpected death of an apparently healthy infant,

often overnight, has been, for lack of any real explanation,

termed sudden infant death syndrome (SIDS). MCAD has

been shown to be deficient in up to 10% of these infants,

making this genetic disease more prevalent than phenylke-

tonuria (PKU) (Section 26-3Hd), a genetic defect in

phenylalanine degradation for which babies born in the

United States are routinely tested. Glucose is the principal

energy metabolism substrate just after eating,but when the

glucose level later decreases, the rate of fatty acid oxida-

tion must correspondingly increase. The sudden death of

infants lacking MCAD may be caused by the imbalance

between glucose and fatty acid oxidation.

Lys 304, which becomes Glu in the most prevalent mu-

tation among individuals with MCAD deficiency, is ⬃20 Å

distant from the enzyme’s active site and hence cannot par-

ticipate in binding substrate or FAD. However, since the

side chains of Asp 300 and Asp 346 lie within 6 Å of Glu

304, near a subunit–subunit interface, it seems likely that

the high concentration of negative charges resulting from

the Lys 304 S Glu mutation structurally destabilizes the

enzyme.

Deficiency of acyl-CoA dehydrogenase has also been

implicated in Jamaican vomiting sickness, whose victims

suffer violent vomiting followed by convulsions, coma, and

death. Severe hypoglycemia is observed in most cases. This

condition results from eating unripe ackee fruit, which con-

tains hypoglycin A, an unusual amino acid, which is metab-

olized to methylenecyclopropylacetyl-CoA (MCPA-CoA;

Fig. 25-14). MCPA-CoA, a substrate for acyl-CoA dehy-

drogenase, is thought to undergo the first step of the reac-

tion that this enzyme catalyzes, removal of a proton from

␣

, to form a reactive intermediate that covalently modi-

fies the enzyme’s FAD prosthetic group (Fig. 25-14). Since

a normal step in the enzyme’s reaction mechanism gener-

ates the reactive intermediate, MCPA-CoA is said to be a

mechanism-based inhibitor.

c. Long-Chain Enoyl-CoAs Are Converted to

Acetyl-CoA and a Shorter Acyl-CoA by

Mitochondrial Trifunctional Protein

The products of acyl-CoA dehydrogenases are 2-enoyl-

CoAs. Depending on their chain lengths their processing is

continued by one of three systems (Fig. 25-12): the short-

chain, medium-chain, or long-chain 2-enoyl-CoA hydratases

(EHs), hydroxyacyl-CoA dehydrogenases (HADs), and

␤-ketoacyl-CoA thiolases (KTs). The long-chain (LC) ver-

sions of these enzymes are contained on one ␣

␤

octameric

protein, mitochondrial trifunctional protein, located in the

inner mitochondrial membrane. LCEH and LCHAD are

contained on the ␣ subunits while LCKT is located on the

␤ subunits. The protein is therefore a combination multi-

functional protein (more than one enzyme activity on a sin-

948 Chapter 25. Lipid Metabolism

Figure 25-13 Ribbon diagram of the active site region in a

subunit of medium-chain acyl-CoA dehydrogenase from pig liver

mitochondria in complex with octanoyl-CoA. The enzyme is a

tetramer of identical 385-residue subunits, each of which binds an

FAD prosthetic group (green) and its octanoyl-CoA substrate

(whose octanoyl and CoA moieties are blue and white) in largely

extended conformations.The octanoyl-CoA binds such that its

␣

¬C

␤

bond is sandwiched between the carboxylate group of

Glu 376 (red) and the flavin ring (green), consistent with the

proposal that Glu 376 is the general base that abstracts the ␣

proton in the ␣,␤ dehydrogenation reaction catalyzed by the

enzyme. [Based on an X-ray structure by Jung-Ja Kim, Medical

College of Wisconsin. PDBid 3MDE.]

See Interactive

Exercise 23

JWCL281_c25_940-1018.qxd 10/19/10 9:41 AM Page 948

gle polypeptide chain)–multienzyme complex (a complex

of polypeptides catalyzing more than one reaction). The

advantage of such a trifunctional enzyme is the ability to

channel the intermediates toward the final product. In-

deed, no long-chain hydroxyacyl-CoA or ketoacyl-CoA in-

termediates are released into solution by this system.

d. The Thiolase Reaction Occurs via

Claisen Ester Cleavage

The final stage of the fatty acid ␤-oxidation process, the

thiolase reaction, forms acetyl-CoA and a new acyl-CoA

which is two carbon atoms shorter than the one that began

the cycle.This occurs in five reaction steps (Fig. 25-15):

1. An active site thiol is added to the substrate -keto

group.

2. Carbon–carbon bond cleavage forms an acetyl-CoA

carbanion intermediate that is stabilized by electron with-

drawal into this thioester’s carbonyl group. Such a reaction

is known as a Claisen ester cleavage (the reverse of a

Claisen condensation). The citric acid cycle enzyme citrate

synthase also catalyzes a reaction that involves a stabilized

acetyl-CoA carbanion intermediate (Section 21-3A).

3. The acetyl-CoA carbanion intermediate is proto-

nated by an enzyme acid group, yielding acetyl-CoA.

4 and 5. Finally, CoA displaces the enzyme thiol group

from the enzyme–thioester intermediate, yielding acyl-CoA.

The formation of an enzyme–thioester intermediate in-

volving an active site thiol group is based on the observa-

tion that incubation of the enzyme with [

C]acetyl-CoA

Section 25-2. Fatty Acid Oxidation 949

Figure 25-14 Metabolic conversions of hypoglycin A to yield a

product that inactivates acyl-CoA dehydrogenase. Spectral

Figure 25-15 Mechanism of action of -ketoacyl-CoA thiolase. An active site

Cys residue participates in the formation of an enzyme–thioester intermediate.

COO

–

CCH

SCoACH

CCH

metabolism

Possible reactive intermediate that reacts with the FAD of acyl-CoA dehydrogenase

Hypoglycin A Methylenecyclopropylacetyl-Co

(MCPA-CoA)

CCH

SCoACCH CCH

SCoAC

acyl-CoA

dehydrogenase

changes suggest that the enzyme’s FAD prosthetic group has

been modified.

SCoA

-Ketoacyl-CoA

–

SCoA

–

SCoA

Enzyme–thioester

intermediate

Acetyl-CoA

–

SCoA

–

SCoA

Acyl-CoA

CoAS H

JWCL281_c25_940-1018.qxd 4/20/10 1:58 PM Page 949

The resulting cis-, double bond–containing enoyl-CoA

is not a substrate for enoyl-CoA hydratase. Enoyl-CoA iso-

merase, however, mediates conversion of the cis-

double

bond to the more stable, ester-conjugated trans-

form:

Such compounds are normal substrates of enoyl-CoA

hydratase so that  oxidation can then continue.

Problem 2: A 

Double Bond Inhibits

Hydratase Action

The next difficulty arises on the left-hand pathway in

Fig. 25-17 in the fifth round of  oxidation.The presence of

a double bond at an even-numbered carbon atom results in

the formation of 2,4-dienoyl-CoA, which is a poor substrate

for enoyl-CoA hydratase. However, NADPH-dependent

SCoA

Enz

enoyl-CoA

isomerase

...

SCoA

Enz

950 Chapter 25. Lipid Metabolism

yields a specifically labeled enzyme Cys residue (the re-

verse of steps 4 and 5):

e. Fatty Acid Oxidation Is Highly Exergonic

The function of fatty acid oxidation is, of course, to gen-

erate metabolic energy. Each round of  oxidation pro-

duces one NADH, one FADH

, and one acetyl-CoA. Oxi-

dation of acetyl-CoA via the citric acid cycle generates

additional FADH

and NADH, which are reoxidized

through oxidative phosphorylation to form ATP. Complete

oxidation of a fatty acid molecule is therefore a highly ex-

ergonic process, which yields numerous ATPs. For exam-

ple, oxidation of palmitoyl-CoA (which has a C

fatty acyl

group) involves seven rounds of  oxidation, yielding 7

FADH

, 7 NADH, and 8 acetyl-CoA. Oxidation of the 8

acetyl-CoA, in turn, yields 8 GTP, 24 NADH, and 8

FADH

. Since oxidative phosphorylation of the 31 NADH

molecules yields 77.5 ATP and that of the 15 FADH

yields

22.5 ATPs, subtracting the 2 ATP equivalents required for

fatty acyl-CoA formation (Section 25-2A), the oxidation of

one palmitate molecule has a net yield of 106 ATP.

D. Oxidation of Unsaturated Fatty Acids

Almost all unsaturated fatty acids of biological origin (Sec-

tion 12-1A) contain only cis double bonds, which most often

begin between C9 and C10 (referred to as a ⌬

or 9-double

bond; Table 12-1). Additional double bonds, if any, occur at

three-carbon intervals and are therefore never conjugated.

Two examples of unsaturated fatty acids are oleic acid and

linoleic acid (Fig. 25-16). Note that one of the double bonds

in linoleic acid is at an odd-numbered carbon atom and the

other is at an even-numbered carbon atom. Double bonds

at these positions in fatty acids pose three problems for the

-oxidation pathway that are solved through the actions of

four additional enzymes (Fig. 25-17):

Problem 1: A ␤,␥ Double Bond

The first enzymatic difficulty occurs on the left-hand

pathway in Fig. 25-17 after the third round of  oxidation:

Acetyl-CoAThiolase

SCoA

trypsin degradation

CoASH

Cys Ala Ser

Gly MetVal Lys

Figure 25-16 Structures of two common unsaturated fatty

acids. Most unsaturated fatty acids contain unconjugated cis

double bonds.

Figure 25-17 (Opposite) Problems in the oxidation of

unsaturated fatty acids and their solutions. Linoleic acid is used

as an example. The first problem, the presence of a , double

bond seen in the left-hand pathway, is solved by the bond’s

enoyl-CoA isomerase–catalyzed conversion to a trans-,

double bond.The second problem in the left-hand pathway, that

a 2,4-dienoyl-CoA is not a substrate for enoyl-CoA hydratase, is

eliminated by the NADPH-dependent reduction of the 

bond

by 2,4-dienoyl-CoA reductase to yield the -oxidation substrate

trans-2-enoyl-CoA in E. coli but trans-3-enoyl-CoA in mammals.

Mammals therefore also have 3,2-enoyl-CoA isomerase, which

converts the trans-3-enoyl-CoA to trans-2-enoyl-CoA.The third

problem, the isomerization of 2,5-dienoyl-CoA (originating from

the oxidation of unsaturated fatty acids with double bonds at

odd-numbered C atoms) to 3,5-dienoyl-CoA by 3,2-enoyl-CoA

isomerase, is solved by 3,5–2,4-dienoyl-CoA isomerase, which

converts the 3,5-dienoyl-CoA to 2,4-dienoyl-CoA, a substrate

for 2,4-dienoyl-CoA reductase.

918

112

Oleic acid

(9-cis-Octadecenoic acid)

Linoleic acid

(9,12-cis-Octadecadienoic acid)

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