Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

FIGURE 24.6

α

γ

H

2

H

2

H

2

H

2

H

2

H

2

H

2

–O C

C

C

C

C

C

C

CH3

C

C

C

C

C

C

C

C

H2

H2

H2

H2

H2

H2

H2

O

β

This bond

Oxidation

is cleaved.

α

γ

H

2

H

2

H

2

H

2

H

2

H

2

H

2 CH3

–O C

C

C

C

C

C

C

C

Cβ

C

C

C

C

C

C

O

H2

H2

H2

H2

H2

H2

O

γ

H

2

H

2

H

2

H

2

H

2

H

2 CH3

–O

–O C

C

C

C

C

C

C

CH3

C

C

C

C

C

C

C

O

H2

H2

H2

H2

H2

H2

O

24.2 ● -Oxidation of Fatty Acids

781

● Fatty acids are degraded by repeated cycles of oxidation at the -carbon and cleavage of the C OC bond to yield acetate units.

is acetyl-CoA, not free acetate. Because the entire process begins with oxidation of the carbon that is “ ” to the carboxyl carbon, the process has come to be known as -oxidation.

Coenzyme A Activates Fatty Acids for Degradation

The process of -oxidation begins with the formation of a thiol ester bond between the fatty acid and the thiol group of coenzyme A. This reaction, shown in Figure 24.7, is catalyzed by acyl-CoA synthetase, which is also called acyl-CoA ligase or fatty acid thiokinase. This condensation with CoA activates the fatty acid for reaction in the -oxidation pathway. For long-chain fatty acids, this reaction normally occurs at the outer mitochondrial membrane, prior to entry of the fatty acid into the mitochondrion, but it may also occur at the surface of the endoplasmic reticulum. Shortand medium-length fatty acids undergo this activating reaction in the mitochondria. In all cases, the reaction is accompanied by the hydrolysis of ATP to form AMP and pyrophosphate. As shown in Figure 24.7, the two combined reactions have a net G° of about 0.8 kJ/mol, so that the reaction is favorable but easily reversible. However, there is more to the story. As we have seen in several similar cases, the pyrophosphate produced in this reaction is rapidly hydrolyzed by inorganic pyrophosphatase to two molecules of phosphate, with a net G° of about 33.6 kJ/mol.

COO– +

CoASH

+

ATP

∆ G ' for ATP

AMP + P P = –32.3

kJ

mol

∆ G ' for acyl-CoA synthesis

= +31.5

kJ

mol

Net ∆

G '

=

–0.8

kJ

mol

FIGURE 24.7 ● The acyl-CoA synthetase reaction activates fatty acids for -oxidation. The reaction is driven by hydrolysis of ATP to AMP and pyrophosphate and by the subsequent hydrolysis of pyrophosphate.

C SCoA +	AMP	+ P P
O	H2O
		P P
∆	G o' =	kJ
		–33.6
		mol

782 Chapter 24 ● Fatty Acid Catabolism

O

O

O

–O

P

ATP

P

O

P

O

O

Adenosine

O–

O–

O–

O–

C

Fatty acid

O

O

O

–O

O–

P

O

P

O–

O–

Pyrophosphate

R

O

Enzyme-bound

CoA S

C

O

P

O

Adenosine

acyl-adenylate

H

O

intermediate

O–

FIGURE 24.8 ● The mechanism of the acyl-CoA synthetase reaction involves fatty acid carboxylate attack on ATP to form an acyladenylate intermediate. The fatty acyl CoA thioester product is formed by CoA attack on this intermediate.

R

O

CoA

Transient

S

C

O

P

O

Adenosine

tetrahedral

O–

intermediate

O–

R

O

CoA

C + –O

O

S

P

Adenosine

O

O–

Fatty acyl-CoA

AMP

Thus, pyrophosphate is maintained at a low concentration in the cell (usually less than 1 mM) and the synthetase reaction is strongly promoted. The mechanism of the acyl-CoA synthetase reaction is shown in Figure 24.8 and involves attack of the fatty acid carboxylate on ATP to form an acyladenylate intermediate, which is subsequently attacked by CoA, forming a fatty acyl-CoA thioester.

Carnitine Carries Fatty Acyl Groups Across the

Inner Mitochondrial Membrane

All of the other enzymes of the -oxidation pathway are located in the mitochondrial matrix. Short-chain fatty acids, as already mentioned, are transported into the matrix as free acids and form the acyl-CoA derivatives there. However, long-chain fatty acyl-CoA derivatives cannot be transported into the matrix directly. These long-chain derivatives must first be converted to acylcarnitine derivatives, as shown in Figure 24.9. Carnitine acyltransferase I, located on the outer side of the inner mitochondrial membrane, catalyzes the formation of

FIGURE 24.9

24.2 ● -Oxidation of Fatty Acids

783

	CH3
CH3	N + CH3				O-acylcarnitine
	CH2
						CH3
HO	C H
						+
	CH2	C	O		CH3	N CH3
			SH
	C	S				CH2
			CoA
O	O–	CoA		C	O	CH
L-Carnitine				O		CH2
Intermembrane						COO–	Carnitine: acylcarnitine
		Carnitine					translocase
space
		acyltrans-
		ferase I
							Carnitine
Mitochondrial							acyltrans-
							ferase II
matrix
						CH3				CH3
					H3C	N + CH3	CoA	CoA H3C		N + CH3
						CH2	SH	S		CH2
				C	O	CH	O	C	HO	C H
				O		CH2				CH2
				O-acylcarnitine		COO–				COO–

● The formation of acylcarnitines and their transport across the inner mitochondrial membrane. The process involves the coordinated actions of carnitine acyltransferases on both sides of the membrane and of a translocase that shuttles O-acylcarnitines across the membrane.

L-Carnitine

the O-acylcarnitine, which is then transported across the inner membrane by a translocase. At this point, the acylcarnitine is passed to carnitine acyltransferase II on the matrix side of the inner membrane, which transfers the fatty acyl group back to CoA to re-form the fatty acyl-CoA, leaving free carnitine, which can return across the membrane via the translocase.

Several additional points should be made. First, although oxygen esters usually have lower group-transfer potentials than thiol esters, the OOacyl bonds in acylcarnitines have high group-transfer potentials, and the transesterification reactions mediated by the acyltransferases have equilibrium constants close to 1. Second, note that eukaryotic cells maintain separate pools of CoA in the mitochondria and in the cytosol. The cytosolic pool is utilized principally in fatty acid biosynthesis (Chapter 25), and the mitochondrial pool is important in the oxidation of fatty acids and pyruvate, as well as some amino acids.

-Oxidation Involves a Repeated Sequence of Four Reactions

For saturated fatty acids, the process of -oxidation involves a recurring cycle of four steps, as shown in Figure 24.10. The overall strategy in the first three steps is to create a carbonyl group on the -carbon by oxidizing the C OC bond to form an olefin, with subsequent hydration and oxidation. In essence, this cycle is directly analogous to the sequence of reactions converting succi-

784 Chapter 24 ● Fatty Acid Catabolism

H

H

O

R

CH2

Cβ

Cα

C

S

CoA

H

H

H

O

Fatty acyl-CoA

FAD

O

H

C

C

S

CoA

α

CoA

Acyl-CoA

Acetyl-CoA

R

CH2

C

β

S

H

dehydrogenase

Fatty acyl-CoA

4

shortened by two carbons

FADH2

1

Thiolase

CoASH

Cleavage

Oxidation

Successive cycles

O

H

O

H

O

CoA

CoA

R

CH2

C

C

C

S

R

CH2

C

C

C

S

β

α

β

α

H

H

β -Ketoacyl-CoA

trans-∆ 2-Enoyl-CoA

3

NADH + H+

	L-Hydroxyacyl-CoA
	dehydrogenase
FIGURE 24.10 ● The -oxidation of satu-	NAD+

rated fatty acids involves a cycle of four enzyme-catalyzed reactions. Each cycle produces single molecules of FADH2, NADH, and acetyl-CoA and yields a fatty acid shortened by two carbons. (The delta [ ] symbol connotes a double bond, and its superscript indicates the lower-numbered carbon involved.)

Oxidation			Hydration	H2O
				Enoyl-CoA
			2	hydratase
H		H	O
R CH2 C		C	C S CoA
	β	α
HO		H
L-β -Hydroxyacyl-CoA

nate to oxaloacetate in the TCA cycle. The fourth reaction of the cycle cleaves the -keto ester in a reverse Claisen condensation, producing an acetate unit and leaving a fatty acid chain that is two carbons shorter than it began. (Recall from Chapter 20 that Claisen condensations involve attack by a nucleophilic agent on a carbonyl carbon to yield a -keto acid.)

A c y l - C o A Dehydrogenase—The First Reaction of -Oxidation

The first reaction, the oxidation of the C OC bond, is catalyzed by acyl-CoA dehydrogenases, a family of three soluble matrix enzymes (with molecular weights of 170 to 180 kD), which differ in their specificity for either long-, medium-, or short-chain acyl-CoAs. They carry noncovalently (but tightly) bound FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure 24.11, FADH2 transfers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is reoxidized by a specific oxidoreductase (an iron–sulfur protein), which in turn sends the electrons on to the electron transport chain at the level of coenzyme Q. As always, mitochondrial oxidation of FAD in this way eventually results in the net formation of about 1.5 ATP. The mechanism of the acyl-CoA dehydrogenase (Figure 24.12) involves deprotonation of the fatty acid chain at the -carbon, followed by hydride transfer from the -carbon to FAD. The structure of the medium-chain dehydrogenase from pig liver places an FAD molecule in an extended conformation between a bundle of -helices and a distorted -barrel (Figure 24.13).

FIGURE 24.12

24.2 ● -Oxidation of Fatty Acids

785

H H O

R CH2 Cβ Cα C SCoA

H H

Fatty acyl-CoA

FAD	ETFred	ETF: UQ	UQH2
Acyl-CoA		oxidoreductaseox
dehydrogenase
FADH2	ETFox	ETF: UQ	UQ
		oxidoreductasered
H O

R CH2 C C C SCoA

H

trans-∆ 2-Enoyl-CoA

FIGURE 24.11 ● The acyl-CoA dehydrogenase reaction. The two electrons removed in this oxidation reaction are delivered to the electron transport chain in the form of reduced coenzyme Q (UQH2).

FAD

H

H

O

H

H

O

R

CH2

C

C

C

SCoA

R

CH2

C

C

C

SCoA

H

H

H

B

E

R

CH2

H

O

C

C

SCoA

H

C

Mitochondrial			H2O
electron
transport
chain		1	O2
		2
1.5 ADP	1.5	ATP
+ 1.5 P

● The mechanism of acyl-CoA dehydrogenase. Removal of a proton from the-C is followed by hydride transfer from the-carbon to FAD.

C

			FIGURE 24.13 ● The subunit structure of
		N	medium chain acyl-CoA dehydrogenase from
			pig liver mitochondria. Note the location of
H		L
			the bound FAD (red). (Adapted from Kim, J.-T., and
			Wu, J., 1988. Structure of the medium-chain acyl-CoA dehydro-
			genase from pig liver mitochondria at 3-Å resolution.
		I	Proceedings of the National Academy of Sciences, USA
			85:6671–6681.)
G
		P
J
	K	P
		D
	A	E

B

C F

786 Chapter 24 ● Fatty Acid Catabolism

A D E E P E R L O O K

The Akee Tree

The akee (also spelled ackee) tree is native to West Africa and was brought to the Caribbean by African slaves. It was introduced to science by William Bligh, captain of the infamous sailing ship the Bounty, and its botanical name is (appropriately) Blighia sapida (the latter name from the Latin sapidus meaning “tasty”). A popular dish in the Caribbean consists of akee and salt fish.

“Akee, rice, salt fish are nice,

And the rum is fine any time of year.”

—From the song Jamaica Farewell

(R.R. Head/Earth Scenes/Animals, Animals)

A Metabolite of Hypoglycin from Akee Fruit

Inhibits Acyl-CoA Dehydrogenase

The unripened fruit of the akee tree contains hypoglycin, a rare amino acid (Figure 24.14). Metabolism of hypoglycin yields methylenecyclopropylacetyl-CoA (MCPA-CoA). Acyl-CoA dehydrogenase will accept MCPA-CoA as a substrate,

FIGURE 24.14 ● The conversion of hypoglycin from akee fruit to a form that inhibits acyl-CoA dehydrogenase.

CH2 H

H2C C CH CH2 C COO–

NH+3

Hypoglycin A

CoASH

O

CH2

H2C C CH CH2 C SCoA

Methylenecyclopropylacetyl-CoA

(MCPA-CoA)

H+

O

O

H2C

CH2

–

– C

CH

CH

C

SCoA

H2C

C

CH

CH

C

SCoA

H2C

Reactive intermediate

24.2 ● -Oxidation of Fatty Acids

787

removing a proton from the -carbon to yield an intermediate that irreversibly inactivates acyl-CoA dehydrogenase by reacting covalently with FAD on the enzyme. For this reason, consumption of unripened akee fruit can lead to vomiting and, in severe cases, convulsions, coma, and death. The condition is most severe in individuals with low levels of acyl-CoA dehydrogenase.

E n o y l - C o A Hydratase Adds Water Across the Double Bond

The next step in -oxidation is the addition of the elements of H2O across the new double bond in a stereospecific manner, yielding the corresponding hydroxyacyl-CoA (Figure 24.15). The reaction is catalyzed by enoyl-CoA hydratase. At least three different enoyl-CoA hydratase activities have been detected in various tissues. Also called crotonases, these enzymes specifically convert trans-enoyl-CoA derivatives to L- -hydroxyacyl-CoA. As shown in Figure 24.15, these enzymes will also metabolize cis-enoyl-CoA (at slower rates) to give specifically D- -hydroxyacyl-CoA. Recently, a novel enoyl-CoA hydratase was discovered, which converts trans-enoyl-CoA to D- -hydroxyacyl-CoA, as shown in Figure 24.15.

L-Hydroxyacyl - CoA Dehydrogenase Oxidizes the -Hydroxyl Group

The third reaction of this cycle is the oxidation of the hydroxyl group at the-position to produce a -ketoacyl-CoA derivative. This second oxidation reaction is catalyzed by L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD as a coenzyme. NADH produced in this reaction represents metabolic energy. Each NADH produced in mitochondria by this reaction drives the synthesis of 2.5 molecules of ATP in the electron transport pathway. L-Hydroxyacyl-

H2O

H

O

R

CH2

H

SCoA

C

C

O

Crotonase

R

CH2

C

CH2

C

H

C

OH

SCoA

trans-Enoyl-CoA

L-β -Hydroxyacyl-CoA

H2O

O

H

H

OH

SCoA

C

C

O

R

CH2

C

CH2

C

Crotonase

C

R

CH2

SCoA

H

cis-Enoyl-CoA

D-β -Hydroxyacyl-CoA

H2O

O

OH

R

CH2

H

O

SCoA

C

C

R

CH2

C

CH2

C

H

C

SCoA

H

trans-Enoyl-CoA

D-β -Hydroxyacyl-CoA

FIGURE 24.15 ● The conversion of trans- and cis-enoyl CoA derivatives to L- and D- - hydroxyacyl CoA, respectively. These reactions are catalyzed by enoyl-CoA hydratases (also called crotonases), enzymes that vary in their acyl-chain length specificity. A recently discovered enzyme converts trans-enoyl-CoA directly to D- -hydroxyacyl-CoA.

788 Chapter 24 ● Fatty Acid Catabolism

FIGURE 24.16 ● The L- -hydroxyacyl-CoA dehydrogenase reaction.

CoA dehydrogenase shows absolute specificity for the L-hydroxyacyl isomer of the substrate (Figure 24.16). (D-Hydroxyacyl isomers, which arise mainly from oxidation of unsaturated fatty acids, are handled differently.)

- K e t o a c y l - C o A Intermediates Are Cleaved in the Thiolase Reaction

The final step in the -oxidation cycle is the cleavage of the -ketoacyl-CoA. This reaction, catalyzed by thiolase (also known as -ketothiolase), involves the attack of a cysteine thiolate from the enzyme on the -carbonyl carbon, followed by cleavage to give the enolate of acetyl-CoA and an enzyme-thioester intermediate (Figure 24.17). Subsequent attack by the thiol group of a second CoA and departure of the cysteine thiolate yields a new (shorter) acyl-CoA. If the reaction in Figure 24.17 is read in reverse, it is easy to see that it is a Claisen condensation—an attack of the enolate anion of acetyl-CoA on a thioester. Despite the formation of a second thioester, this reaction has a very favorable Keq, and it drives the three previous reactions of -oxidation.

O

S

C

R

+

O

E

BH

O

O

O

–

O

–CH2

O

H3C

C

SCoA

SCoA

+ R

SCoA

C

C

CH2

C

E

S

C

CH2

C

SCoA

S–

β -Ketoacyl-CoA

R

E

+

O–

BH

CH2

O

O–

C

SCoA

O

S

C

R

S

C

R

R

C

SCoA

SCoA

E

B..

CoA

E

+

Acyl-CoA

H

S

BH

FIGURE 24.17 ● The mechanism of the thiolase reaction. Attack by an enzyme cysteine thiolate group at the -carbonyl carbon produces a tetrahedral intermediate, which decomposes with departure of acetyl-CoA, leaving an enzyme thioester intermediate. Attack by the thiol group of a second CoA yields a new (shortened) acyl-CoA.

24.2 ● -Oxidation of Fatty Acids

789

Repetition of the -Oxidation Cycle Yields a

Succession of Acetate Units

In essence, this series of four reactions has yielded a fatty acid (as a CoA ester) that has been shortened by two carbons, and one molecule of acetyl-CoA. The shortened fatty acyl-CoA can now go through another -oxidation cycle, as shown in Figure 24.10. Repetition of this cycle with a fatty acid with an even number of carbons eventually yields two molecules of acetyl-CoA in the final step. As noted in the first reaction in Table 24.2, complete -oxidation of palmitic acid yields eight molecules of acetyl-CoA as well as seven molecules of FADH2 and seven molecules of NADH. The acetyl-CoA can be further metabolized in the TCA cycle (as we have already seen). Alternatively, acetyl-CoA can also be used as a substrate in amino acid biosynthesis (Chapter 26). As noted in Chapter 23, however, acetyl-CoA cannot be used as a substrate for gluconeogenesis.

Complete -Oxidation of One Palmitic Acid

Yields 106 Molecules of ATP

If the acetyl-CoA is directed entirely to the TCA cycle in mitochondria, it can eventually generate approximately 10 high-energy phosphate bonds—that is, 10 molecules of ATP synthesized from ADP (Table 24.2). Including the ATP formed from FADH2 and NADH, complete -oxidation of a molecule of palmi- toyl-CoA in mitochondria yields 108 molecules of ATP. Subtracting the two high-energy bonds needed to form palmitoyl-CoA, the substrate for -oxida- tion, one concludes that -oxidation of a molecule of palmitic acid yields 106 molecules of ATP. The G° for complete combustion of palmitate to CO2 is9790 kJ/mol. The hydrolytic energy embodied in 106 ATPs is 106 30.5 kJ/mol 3233 kJ/mol, so the overall efficiency of -oxidation under standardstate conditions is approximately 33%. The large energy yield from fatty acid oxidation is a reflection of the highly reduced state of the carbon in fatty acids. Sugars, in which the carbon is already partially oxidized, produce much less energy, carbon for carbon, than do fatty acids. The breakdown of fatty acids is regulated by a variety of metabolites and hormones. Details of this regulation are described in Chapter 25, following a discussion of fatty acid synthesis.

Table 24.2

Equations for the Complete Oxidation of Palmitoyl-CoA to CO2 and H2O

		ATP	Free Energy
Equation		Yield	(kJ/mol) Yield
CH3(CH2)14COOCoA 7 [FAD] 7 H2O 7 NAD 7 CoA 88n
8	CH3CO-CoA 7 [FADH2] 7 NADH 7 H
7	[FADH2] 10.5 Pi 10.5 ADP 3.5 O2 88n
	7 [FAD] 17.5 H2O 10.5 ATP	10.5	320
7	NADH 7 H 17.5 Pi 17.5 ADP 3.5 O2 88n
	7 NAD 24.5 H2O 17.5 ATP	17.5	534
8-Acetyl-CoA 16 O2 80 ADP 80 Pi 88n
	8 CoA 88 H2O 16 CO2 80 ATP	80	2440
CH3(CH2)14COOCoA 108 Pi 108 ADP 23 O2 88n
108 ATP 16 CO2 130 H2O CoA		108	3294
Energetic “cost” of forming palmitoyl-CoA from palmitate and CoA		2		61
		106	3233

790 Chapter 24 ● Fatty Acid Catabolism

Migratory Birds Travel Long Distances on

Energy from Fatty Acid Oxidation

Because they represent the most highly concentrated form of stored biological energy, fatty acids are the metabolic fuel of choice for sustaining the incredibly long flights of many migratory birds. Although some birds migrate over land masses and dine frequently, other species fly long distances without stopping to eat. The American golden plover flies directly from Alaska to Hawaii, a 3300-kilometer flight requiring 35 hours (at an average speed of nearly 60 miles/hr) and more than 250,000 wing beats! The ruby-throated hummingbird, which winters in Central America and nests in southern Canada, often flies nonstop across the Gulf of Mexico. These and similar birds accomplish these prodigious feats by storing large amounts of fatty acids (as triacylglycerols) in the days before their migratory flights. The percentage of dry-weight body fat in these birds may be as high as 70% when migration begins (compared with values of 30% and less for nonmigratory birds).

(a) Gerbil

Fatty Acid Oxidation Is an Important Source of

Metabolic Water for Some Animals

Large amounts of metabolic water are generated by -oxidation (130 H2O per palmitoyl-CoA). For certain animals—including desert animals, such as gerbils, and killer whales (which do not drink seawater)—the oxidation of fatty acids can be a significant source of dietary water. A striking example is the camel (Figure 24.18), whose hump is essentially a large deposit of fat. Metabolism of fatty acids from this store provides needed water (as well as metabolic energy) during periods when drinking water is not available. It might well be said that “the ship of the desert” sails on its own metabolic water!

(b) Ruby-throated hummingbird

(e) Camels

(c) Golden plover

(d) Orca

FIGURE 24.18 ● Animals whose existence is strongly dependent on fatty acid oxidation:

(a) gerbil, (b) ruby-throated hummingbird, (c) golden plover, (d) orca (killer whale), and

(e) camels. (a, Photo Researchers, Inc.; b, Tom J. Ulrich/Visuals Unlimited; c, S. J. Krasemann/Photo Researchers, Inc.; d, © Francois Gohier/Photo Researchers, Inc.; e, © George Holton/Photo Researchers, Inc.)