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

25.3 ● Eicosanoid Biosynthesis and Function

831

A D E E P E R L O O K

The Discovery of Prostaglandins

The name prostaglandin was given to this class of compounds by Ulf von Euler, their discoverer, in Sweden in the 1930s. He extracted fluids containing these components from human semen. Because he thought they originated in the prostate gland, he named them prostaglandins. Actually, they were synthesized in the seminal vesicles, and it is now known that similar substances are synthesized in most animal tissues (both male and female). Von Euler observed that injection of these substances into animals caused smooth muscle contraction and dramatic lowering of blood pressure.

Von Euler (and others) soon found that it is difficult to analyze and characterize these obviously interesting compounds because they are present at extremely low levels. Prostaglandin

E2 , or PGE2 , is present in human serum at a level of less than 10 14 M! In addition, they often have half-lives of only 30 seconds to a few minutes, not lasting long enough to be easily identified. Moreover, most animal tissues upon dissection and homogenization rapidly synthesize and degrade a variety of these substances, so the amounts obtained in isolation procedures are extremely sensitive to the methods used and highly variable even when procedures are carefully controlled.

Sune Bergstrom and his colleagues described the first structural determinations of prostaglandins in the late 1950s. In the early 1960s, dramatic advances in laboratory techniques such as NMR spectroscopy and mass spectrometry made further characterization possible.

8	5	COOH
H	H
11	14

O2

5, 8,11,14-Eicosatetraenoic acid (arachidonic acid)

	. ...........	........
	O
	O	H

H	H
O
O
H
H H
O
O
H

COOH

...

O2

Peroxide radical

COOH

O H

OH

PGG2

COOH

HO H

PGH2

FIGURE 25.28 ● Prostaglandin endoperoxide synthase, the enzyme that converts arachidonic acid to prostaglandin PGH2, possesses two distinct activities: cyclooxygenase (steps 1 and 2) and glutathione (GSSG)–dependent hydroperoxidase (step 3). Cyclooxygenase is the site of action of aspirin and many other analgesic agents.

FIGURE 25.29

832 Chapter 25 ● Lipid Biosynthesis

(a)

O

HO

NH C CH3

Acetaminophen

	H3C	CH3
		CH COOH
	CH CH2

H3C

Ibuprofen

(b)

COO–

O

O

C

CH3

Acetylsalicylate (aspirin)

O

Ser

OH

Ser

O

C

CH3

COO–

Active

Inactive

cyclooxygenase

OH

cyclooxygenase

Salicylate

● (a) The structures of several common analgesic agents. Acetaminophen is marketed under the tradename Tylenol®. Ibuprofen is sold as Motrin®, Nuprin®, and Advil®. (b) Acetylsalicylate (aspirin) inhibits the cyclooxygenase activity of endoperoxide synthase via acetylation (covalent modification) of Ser530.

“Take Two Aspirin and . . .” Inhibit Your Prostaglandin Synthesis

In 1971, biochemist John Vane was the first to show that aspirin (acetylsalicylate; Figure 25.29) exerts most of its effects by inhibiting the biosynthesis of prostaglandins. Its site of action is prostaglandin endoperoxide synthase. Cyclooxygenase activity is destroyed when aspirin O-acetylates Ser530 on the enzyme. From this you may begin to infer something about how prostaglandins (and aspirin) function. Prostaglandins are known to enhance inflammation in animal tissues. Aspirin exerts its powerful anti-inflammatory effect by inhibiting this first step in their synthesis. Aspirin does not have any measurable effect on the peroxidase activity of the synthase. Other nonsteroidal anti-inflamma- tory agents, such as ibuprofen (Figure 25.29) and phenylbutazone, inhibit the cyclooxygenase by competing at the active site with arachidonate or with the peroxyacid intermediate (PGG2, Figure 25.28). See A Deeper Look, page 834.

25.4 ● Cholesterol Biosynthesis

The most prevalent steroid in animal cells is cholesterol (Figure 25.30). Plants contain no cholesterol, but they do contain other steroids very similar to cholesterol in structure (see page 256). Cholesterol serves as a crucial component of cell membranes and as a precursor to bile acids (e.g., cholate, glycocholate,

(a)			21	H 22		24	H
			H3C					27
			H3C	C		23	C			CH3
				20				25
			18
	19	11	12	17			CH3
			13
	CH3		C	D 16			26
			14	15
	1	9
2			8
	10	B
3	A		7
	5
HO	4	6

							21		H 22	24	H
(b)								H3C					27
									C	23	C				CH3
			19				18		20				25
								CH3
	2			CH3	11	12					CH3
HO			1					13	16		26
	A		10 B		9	C		D 17
4					8
3		5	6		7	14	15

FIGURE 25.30 ● The structure of cholesterol, drawn (a) in the traditional planar motif and (b) in a form that more accurately describes the conformation of the ring system.

FIGURE 25.32

taurocholate) and steroid hormones (e.g., testosterone, estradiol, progesterone). Also, vitamin D3 is derived from 7-dehydrocholesterol, the immediate precursor of cholesterol. Liver is the primary site of cholesterol biosynthesis.

Mevalonate Is Synthesized from Acetyl-CoA via HMG-CoA Synthase

The cholesterol biosynthetic pathway begins in the cytosol with the synthesis of mevalonate from acetyl-CoA (Figure 25.31). The first step is the -ketothi- olase-catalyzed Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. In the next reaction, acetyl-CoA and acetoacetyl-CoA join to form 3-hydroxy-3-methylglutaryl-CoA, which is abbreviated HMG-CoA. The reac- tion—a second Claisen condensation—is catalyzed by HMG-CoA synthase. The third step in the pathway is the rate-limiting step in cholesterol biosynthesis. Here, HMG-CoA undergoes two NADPH-dependent reductions to produce 3Rmevalonate (Figure 25.32). The reaction is catalyzed by HMG-CoA reductase, a 97-kD glycoprotein that traverses the endoplasmic reticulum membrane with its active site facing the cytosol. As the rate-limiting step, HMG-CoA reductase is the principal site of regulation in cholesterol synthesis.

–OOC CH2

3-Hydroxy-3- methylglutarylCoA (HMG-CoA)

–OOC CH2

Enzyme-bound intermediate

H+

CH3 O

C CH2 C S CoA

OH

O

H H

C

NH2

N First

NADPH

R

H

CH3 O

C CH2 C S CoA

OH H

O

H H

C

NH2

N Second

NADPH

R

CoASH

CH3 H

–OOC CH2 C CH2 C OH

OH H

3R -Mevalonate

25.4 ● Cholesterol Biosynthesis

833

O

CH3 C SCoA

Acetyl-CoA

O

CH3 C SCoA

Acetyl-CoA

Thiolase

CoASH

O O

CH3 C CH2 C SCoA

Acetoacetyl-CoA

O

CH3 C SCoA

Acetyl-CoA HMG-CoA

synthase

CoASH

CH3 O

–OOC CH2 C CH2 C SCoA

OH

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)

2 NADPH + 2 H+

2NADP+

HMG-CoA reductase

CoASH

CH3

H

–OOC

CH2

C

CH2

C

OH

OH

H

3R -Mevalonate

FIGURE 25.31 ● The biosynthesis of 3R-meva- lonate from acetyl-CoA.

● A reaction mechanism for HMG-CoA reductase. Two successive NADPHdependent reductions convert the thioester, HMG-CoA, to a primary alcohol.

834 Chapter 25 ● Lipid Biosynthesis

A D E E P E R L O O K

The Molecular Basis for the Action of Nonsteroidal Anti-inflammatory Drugs

Prostaglandins are potent mediators of inflammation. The first and committed step in the production of prostaglandins from arachidonic acid is the bis-oxygenation of arachindonate to prostaglandin PGG2. This is followed by reduction to PGH2 in a peroxidase reaction. Both these reactions are catalyzed by prostaglandin endoperoxide synthase, also known as PGH2 synthase or cyclooxygenase, thus abbreviated COX. This enzyme is inhibited by the family of drugs known as nonsteroidal anti-inflam- matory drugs, or NSAIDs. Aspirin, ibuprofen, flurbiprofen, and acetaminophen (trade name Tylenol ®) are all NSAIDs.

There are two isoforms of COX in animals: COX-1 (figure a), which carries out normal, physiological production of prostaglandins, and COX-2 (figure b), which is induced by cytokines, mitogens, and endotoxins in inflammatory cells and is responsible for the production of prostaglandins in inflammation.

The enzyme structure shown here is that of residues 33 to

583 of COX-1 from sheep, inactivated by bromoaspirin. These 551

(a)

residues comprise three distinct domains. The first of these, residues 33 to 72 (purple), form a small compact module that is similar to epidermal growth factor. The second domain, composed of residues 73 to 116 (yellow), forms a right-handed spiral of four -helical segments along the base of the protein. These-helical segments form a membrane-binding motif. The helical segments are amphipathic, with most of the hydrophobic residues (shown in green) facing away from the protein, where they can interact with a lipid bilayer. The third domain of the COX enzyme, the catalytic domain (in blue), is a globular structure that contains both the cyclooxygenase and peroxidase active sites.

The cyclooxygenase active site lies at the end of a long, narrow, hydrophobic tunnel or channel. Three of the -helices of the membrane-binding domain lie at the entrance to this tunnel. The

(b)

Three different regulatory mechanisms are involved:

1.Phosphorylation by cAMP-dependent protein kinases inactivates the reductase. This inactivation can be reversed by two specific phosphatases (Figure 25.33).

2.Degradation of HMG-CoA reductase. This enzyme has a half-life of only three hours, and the half-life itself depends on cholesterol levels: high [cholesterol] means a short half-life for HMG-CoA reductase.

3.Gene expression—cholesterol levels control the amount of mRNA. If [cholesterol] is high, levels of mRNA coding for the reductase are reduced. If [cholesterol] is low, more mRNA is made. (Regulation of gene expression is discussed in Chapter 31.)

25.4 ● Cholesterol Biosynthesis

835

walls of the tunnel are defined by four -helices, formed by residues 106 to 123, 325 to 353, 379 to 384, and 520 to 535 (shown in orange).

In this bromoaspirin-inactivated structure, Ser530, which lies along the wall of the tunnel, is bromoacetylated, and a molecule of salicylate is also bound in the tunnel. Deep in the tunnel, at the far end, lies Tyr385, a catalytically important residue. Hemedependent peroxidase activity is implicated in the formation of a proposed Tyr385 radical, which is required for cyclooxygenase activity. Aspirin and other NSAIDs block the synthesis of prostaglandins by filling and blocking the tunnel, preventing the migration of arachidonic acid to Tyr385 in the active site at the back of the tunnel.

There are thought to be at least four different mechanisms of action for NSAIDs. Aspirin (and also bromoaspirin) covalently

modifies a residue in the tunnel, thus irreversibly inactivating both COX-1 and COX-2. Ibuprofen acts instead by competing in a reversible fashion for the substrate-binding site in the tunnel.

Flurbiprofen and indomethacin, which comprise the third class of inhibitors, cause a slow, time-dependent inhibition of COX-1 and COX-2, apparently via formation of a salt bridge between a carboxylate on the drug and Arg120, which lies in the tunnel.

The drug SC-558 acts by a fourth mechanism, specifically inhibiting COX-2. It is a weak competitive inhibitor of COX-1 but inhibits COX-2 in a slow, time-dependent process. Specific COX- 2 inhibitors will likely be the drugs of the future because they selectively block the inflammation mediated by COX-2, without the potential for stomach lesions and renal toxicity that arise from COX-1 inhibition.

COOH		COOH
O		O
O	CH3	O	CH2Br
Aspirin		Bromoaspirin
		Cl

F

O

N

	OH
	H3C	H3CO
	O
		COOH
	Flurbiprofen	Indomethacin

O

H2N S O

N

N

CF3

COOH

Br

SC-558

Ibuprofen

A Thiolase Brainteaser

If acetate units can be condensed by the thiolase reaction to yield acetoacetate in the first step of cholesterol synthesis, why couldn’t this same reaction also be used in fatty acid synthesis, avoiding all the complexity of the fatty acyl synthase? The answer is that the thiolase reaction is more or less reversible but slightly favors the cleavage reaction. In the cholesterol synthesis pathway, subsequent reactions, including HMG-CoA reductase and the following kinase reactions, pull the thiolase-catalyzed condensation forward. However, in the case of fatty acid synthesis, a succession of eight thiolase condensations would be distinctly unfavorable from an energetic perspective. Given the necessity of repeated reactions in fatty acid synthesis, it makes better energetic sense to use a reaction that is favorable in the desired direction.

836 Chapter 25 ● Lipid Biosynthesis

HMG-CoA reductase kinase (inactive)

ATP	HPO24–
HMG-CoA	HMG-CoA reductase
reductase	kinase phosphatase
kinase kinase
ADP	H2O

HMG-CoA reductase kinase P

(active)

HMG-CoA reductase (active)

ATP

HPO24–

HMG-CoA reductase phosphatase

ADP

H2O

HMG-CoA reductase P (inactive)

FIGURE 25.33 ● HMG-CoA reductase activity is modulated by a cycle of phosphorylation and dephosphorylation.

Squalene Is Synthesized from Mevalonate

The biosynthesis of squalene involves conversion of mevalonate to two key 5- carbon intermediates, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which join to yield farnesyl pyrophosphate and then squalene. A series of four reactions converts mevalonate to isopentenyl pyrophosphate and then to dimethylallyl pyrophosphate (Figure 25.34). The first three steps each consume an ATP, two for the purpose of forming a pyrophosphate at the 5-position and the third to drive the decarboxylation and double bond formation in the third step. Pyrophosphomevalonate decarboxylase phosphorylates the 3-hydroxyl group, and this is followed by trans elimination of the phosphate and carboxyl groups to form the double bond in isopentenyl pyrophosphate. Isomerization of the double bond yields the dimethylallyl pyrophosphate. Condensation of these two 5-carbon intermediates produces geranyl pyrophosphate; addition of another 5-carbon isopentenyl group gives farnesyl pyrophosphate. Both steps in the production of farnesyl pyrophosphate occur with release of pyrophosphate, hydrolysis of which drives these reactions forward. Note too that the linkage of isoprene units to form farnesyl pyrophosphate occurs in a head-to-

25.4 ● Cholesterol Biosynthesis

837

H3C

OH

–OOC

C

CH2OH

CH2

CH2

Mevalonate

Mevalonate

ATP

kinase

ADP

Phosphomevalonate

ATP

kinase

ADP

H3C

OH

–OOC

C

CH2O

P P

CH2

CH2

5-Pyrophosphomevalonate

Pyrophosphomevalonate

ATP

decarboxylase

ADP + P

+

CO2

H3C

CH2O

P P

C

CH2

Isopentenyl pyrophosphate

H2C

Isopentenyl

pyrophosphate isomerase

H C

CH2O

P P

3

C

C

Dimethylallyl pyrophosphate

H3C

H

Isopentenyl pyrophosphate

P P

Isopentenyl pyrophosphate

P P

H3C

CH2O

P P

H3C

H3C

CH2

C

C

CH2

C

C

CH2

H

C

C

CH2

H

H3C

H

Farnesyl pyrophosphate

NADPH +

H+

NADP+ + 2 P P

Squalene

FIGURE 25.34 ● The conversion of mevalonate to squalene.

C R I T I C A L D E V E L O P M E N T S I N B I O C H E M I S T R Y

The Long Search for the Route of Cholesterol Biosynthesis

Heilbron, Kamm, and Owens suggested as early as 1926 that squalene is a precursor of cholesterol. That same year, H. J. Channon demonstrated that animals fed squalene from shark oil produced more cholesterol in their tissues. Bloch and Rittenberg showed in the 1940s that a significant amount of the carbon in the tetracyclic moiety and in the aliphatic side chain of cholesterol was derived from acetate. In 1934, Sir Robert Robinson suggested a scheme for the cyclization of squalene to form cholesterol before the biosynthetic link between acetate and squalene was understood. Squalene is actually a polymer of isoprene units, and Bonner and Arreguin suggested in 1949 that three acetate units could join to form 5-carbon isoprene units (see figure, part a).

In 1952, Konrad Bloch and Robert Langdon showed conclusively that labeled squalene is synthesized rapidly from labeled acetate and also that cholesterol is derived from squalene. Langdon, a graduate student of Bloch’s, performed the critical experiments in Bloch’s laboratory at the University of Chicago, while Bloch spent the summer in Bermuda attempting to demonstrate that radioactively labeled squalene would be converted to cholesterol in shark livers. As Bloch himself admitted, “All I was able to learn was that sharks of manageable length are very difficult to catch and their oily livers impossible to slice” (Bloch, 1987).

In 1953, Bloch, together with the eminent organic chemist R. B. Woodward, proposed a new scheme (see figure, part b) for the cyclization of squalene. (Together with Fyodor Lynen, Bloch received the Nobel Prize in medicine or physiology in 1964 for his work.) The picture was nearly complete, but one crucial question remained: How could isoprene be the intermediate in the

transformation of acetate into squalene? In 1956, Karl Folkers and his colleagues at Merck, Sharpe and Dohme isolated mevalonic acid and also showed that mevalonate was the precursor of isoprene units. The search for the remaining details (described in the text) made the biosynthesis of cholesterol one of the most enduring and challenging bioorganic problems of the forties, fifties, and sixties. Even today, several of the enzyme mechanisms remain poorly understood.

(b)

Squalene

(a)		OH
		Lanosterol
	CH3
CH2	C C CH2	(Many steps)
	H
	Isoprene
		Cholesterol

(a) An isoprene unit and a scheme for head-to-tail linking of isoprene units. (b) The cyclization of squalene to form lanosterol, as proposed by Bloch and Woodward.

tail fashion. This is the general rule in biosynthesis of molecules involving isoprene linkages. The next step—the joining of two farnesyl pyrophosphates to produce squalene—is a “tail-to-tail” condensation and represents an important exception to the general rule.

Squalene monooxygenase, an enzyme bound to the endoplasmic reticulum, converts squalene to squalene-2,3-epoxide (Figure 25.35). This reaction employs FAD and NADPH as coenzymes and requires O2 as well as a cytosolic protein called soluble protein activator. A second ER membrane enzyme, 2,3- oxidosqualene lanosterol cyclase, catalyzes the second reaction, which involves a succession of 1,2 shifts of hydride ions and methyl groups.

838

25.4 ● Cholesterol Biosynthesis

839

FIGURE 25.35 ● Cholesterol is synthesized from squalene via lanosterol. The primary route from lanosterol involves 20 steps, the last of which converts 7-dehydrocholesterol to cholesterol. An alternative route produces desmosterol as the penultimate intermediate.

Squalene

Squalene monooxygenase

O	Squalene-2,3-epoxide
H+	2,3-Oxidosqualene:
H+
	lanosterol cyclase
	H3C
H3C
	CH3
HO	Lanosterol
H3C CH3	Many steps (alternative route)
Many steps
		H3C
H3C	H3C
H3C
	HO	Desmosterol
7-Dehydrocholesterol

O

H3C

H3C

Cholesterol

HO

O

R C CoA

Acyl-CoA cholesterol acyltransferase (ACAT)

CoA

H3C

H3C

O

R C O

Cholesterol esters

840 Chapter 25 ● Lipid Biosynthesis

H U M A N B I O C H E M I S T R Y

Lovastatin Lowers Serum Cholesterol Levels

Chemists and biochemists have long sought a means of reducing serum cholesterol levels to reduce the risk of heart attack and cardiovascular disease. Because HMG-CoA reductase is the ratelimiting step in cholesterol biosynthesis, this enzyme is a likely drug target. Mevinolin, also known as lovastatin (see figure), was isolated from a strain of Aspergillus terreus and developed at Merck, Sharpe and Dohme for this purpose. It is now a widely prescribed cholesterol-lowering drug. Dramatic reductions of serum cholesterol are observed at doses of 20 to 80 mg per day.

Lovastatin is administered as an inactive lactone. After oral ingestion, it is hydrolyzed to the active mevinolinic acid, a competitive inhibitor of the reductase with a KI of 0.6 nM. Mevinolinic acid is thought to behave as a transition-state analog (Chapter 16) of the tetrahedral intermediate formed in the HMG-CoA reductase reaction (see figure).

Derivatives of lovastatin have been found to be even more potent in cholesterol-lowering trials. Synvinolin lowers serum cholesterol levels at much lower doses than lovastatin.

		HO	O			HO	COO–	HO
								COO–
			O				OH	H3C
		O	H			O	H	OH
		2				2
CH3	R	O		CH3	R	O
		CH3 H	CH3			CH3 H	CH3
								Mevalonate
		CH3				CH3
1 R=H Mevinolin (Lovastatin, MEVACOR )						Mevinolinic acid
2 R=CH3 Synvinolin (Simnastatin, ZOCOR				)

The structures of (inactive) lovastatin, (active) mevinolinic acid, mevalonate, and synvinolin.

HO COO–

H3C

OH

H

S–CoA

Tetrahedral intermediate in HMG-CoA reductase mechanism

Conversion of Lanosterol to Cholesterol

Requires 20 Additional Steps

Although lanosterol may appear similar to cholesterol in structure, another 20 steps are required to convert lanosterol to cholesterol (Figure 25.35). The enzymes responsible for this are all associated with the endoplasmic reticulum. The primary pathway involves 7-dehydrocholesterol as the penultimate intermediate. An alternative pathway, also composed of many steps, produces the intermediate desmosterol. Reduction of the double bond at C-24 yields cholesterol. Cholesterol esters—a principal form of circulating cholesterol—are synthesized by acyl-CoA:cholesterol acyltransferases (ACAT) on the cytoplasmic face of the endoplasmic reticulum.

25.5 ● Transport of Many Lipids Occurs via

Lipoprotein Complexes

When most lipids circulate in the body, they do so in the form of lipoprotein complexes. Simple, unesterified fatty acids are merely bound to serum albumin and other proteins in blood plasma, but phospholipids, triacylglycerols, cholesterol, and cholesterol esters are all transported in the form of lipoproteins. At various sites in the body, lipoproteins interact with specific receptors and enzymes that transfer or modify their lipid cargoes. It is now customary to classify lipoproteins according to their densities (Table 25.1). The densities are