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

Table 25.1

Composition and Properties of Human Lipoproteins

				Composition (% dry weight)
Lipoprotein	Density	Diameter
Class	(g/mL)	(nm)	Protein	Cholesterol	Phospholipid	Triacylglycerol
HDL	1.063–1.21	5–15	33	30	29	8
LDL	1.019–1.063	18–28	25	50	21	4
IDL	1.006–1.019	25–50	18	29	22	31
VLDL	0.95–1.006	30–80	10	22	18	50
Chylomicrons	0.95	100–500	1–2	8	7	84

Adapted from Brown, M., and Goldstein, J., 1987. In Braunwald, E., et al., eds., Harrison’s Principles of Internal Medicine, 11th ed. New York: McGraw-Hill; and Vance, D., and Vance, J., eds., 1985. Biochemistry of Lipids and Membranes. Menlo Park, CA: Benjamin/Cummings.

related to the relative amounts of lipid and protein in the complexes. Because most proteins have densities of about 1.3 to 1.4 g/mL, and lipid aggregates usually possess densities of about 0.8 g/mL, the more protein and the less lipid in a complex, the denser the lipoprotein. Thus, there are high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL), very low density lipoproteins (VLDL), and also chylomicrons. Chylomicrons have the lowest protein-to-lipid ratio and thus are the lowest-density lipoproteins. They are also the largest.

The Structure and Synthesis of the Lipoproteins

HDL and VLDL are assembled primarily in the endoplasmic reticulum of the liver (with smaller amounts produced in the intestine), whereas chylomicrons form in the intestine. LDL is not synthesized directly, but is made from VLDL. LDL appears to be the major circulatory complex for cholesterol and cholesterol esters. The primary task of chylomicrons is to transport triacylglycerols. Despite all this, it is extremely important to note that each of these lipoprotein classes contains some of each type of lipid. The relative amounts of HDL and LDL are important in the disposition of cholesterol in the body and in the development of arterial plaques (Figure 25.36). The structures of the various

FIGURE 25.36 ● Photograph of an arterial plaque. (Science Photo Library/Photo Researchers, Inc.)

841

842 Chapter 25 ● Lipid Biosynthesis

lipoproteins are approximately similar, and they consist of a core of mobile triacylglycerols or cholesterol esters surrounded by a single layer of phospholipid, into which is inserted a mixture of cholesterol and proteins (Figure 25.37). Note that the phospholipids are oriented with their polar head groups facing outward to interact with solvent water, and that the phospholipids thus shield the hydrophobic lipids inside from the solvent water outside. The proteins also function as recognition sites for the various lipoprotein receptors throughout the body. A number of different apoproteins have been identified in lipoproteins (Table 25.2), and others may exist as well. The apoproteins are abundant in hydrophobic amino acid residues, as is appropriate for interactions with lipids. A cholesterol ester transfer protein also associates with lipoproteins.

Lipoproteins in Circulation Are Progressively

Degraded by Lipoprotein Lipase

The livers and intestines of animals are the primary sources of circulating lipids. Chylomicrons carry triacylglycerol and cholesterol esters from the intestines to other tissues, and VLDLs carry lipid from liver, as shown in Figure 25.38. At

( a )

( b )

FIGURE 25.37 ● A model for the structure of a typical lipoprotein. (a) A core of cholesterol and cholesteryl esters is surrounded by a phospholipid (monolayer) membrane. Apolipoprotein A-I is modeled here as a long amphipathic -helix, with the nonpolar face of the helix embedded in the hydrophobic core of the lipid particle and the polar face of the helix exposed to solvent. (b) A ribbon diagram of apolipoprotein A-I. (Adapted from Borhani, D. W., Rogers, D. P., Engler, J. A., and Brouillette, C. G., 1997. Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proceedings of the National Academy of Sciences 94:12291–12296.)

FIGURE 25.38

Table 25.2

Apoproteins of Human Lipoproteins

		Concentration
		in Plasma
Apoprotein	Mr	(mg/100 mL)	Distribution
A-1	28,300	90–120	Principal protein in HDL
A-2	8,700	30–50	Occurs as dimer mainly in HDL
B-48	240,000	5	Found only in chylomicrons
B100	500,000	80–100	Principal protein in LDL
C-1	7,000	4–7	Found in chylomicrons, VLDL,
			HDL
C-2	8,800	3–8	Found in chylomicrons, VLDL,
			HDL
C-3	8,800	8–15	Found in chylomicrons, VLDL,
			IDL, HDL
D	32,500	8–10	Found in HDL
E	34,100	3–6	Found in chylomicrons, VLDL,
			IDL, HDL

Adapted from Brown, M., and Goldstein, J., 1987. In Braunwald, E., et al., eds., Harrison’s Principles of Internal Medicine, 11th ed. New York: McGraw-Hill; and Vance, D., and Vance, J., eds., 1985. Biochemistry of Lipids and Membranes, Menlo Park, CA: Benjamin/Cummings.

Endoplasmic reticulum

1 Synthesis of apoproteins, phosphatidylcholine, triacylglycerol, cholesterol, cholesterol esters occurs in the endoplasmic reticulum

2 Assembly of components into prelipoprotein particles in the ER, then transfer to Golgi

3 Golgi processes particles with additional phospholipids and perhaps also cholesterol and cholesterol esters added

Secretory vesicle

Golgi

4Formation of secretory vesicle containing lipoprotein particles

VLDL

Liver cell

5 The VLDL is released into the circulation

Extracellular space

● Lipoprotein components are synthesized predominantly in the ER of liver cells. Following assembly of lipoprotein particles (red dots) in the ER and processing in the Golgi, lipoproteins are packaged in secretory vesicles for export from the cell (via exocytosis) and released into the circulatory system.

843

FIGURE 25.39

844 Chapter 25 ● Lipid Biosynthesis

● Endocytosis and degradation of lipoprotein particles. (ACAT is acyl-CoA cholesterol acyltransferase.)

Apoprotein B

LDL

LDL

LDL receptor

Recycling

vesicle

Vesicle loses its coating and forms endosome

LDL receptors bud off and form a small recycling vesicle

		Endosome formation
		may or may not include
	Lysosome	fusion with another
		vesicle

Protease

Apoprotein B is degraded by lysosomal protease and released as amino acids

Amino acids

Cholesterol esters in core are hybridized by ACAT and stored in cell

Remaining vesicle fuses with lysosome

Synthesis of

LDL receptors

Inhibits

Synthesis of

cholesterol

HMG-CoA

reductase

Free cholesterol released

Free cholesterol

Oversupply of

cholesterol

ACAT +

Activates

FIGURE 25.40

25.5 ● Transport of Many Lipids Occurs via Lipoprotein Complexes

845

various target sites, particularly in the capillaries of muscle and adipose cells, these particles are degraded by lipoprotein lipase, which hydrolyzes triacylglycerols. Lipase action causes progressive loss of triacylglycerol (and apoprotein) and makes the lipoproteins smaller. This process gradually converts VLDL particles to IDL and then LDL particles, which are either returned to the liver for reprocessing or redirected to adipose tissues and adrenal glands. Every 24 hours, nearly half of all circulating LDL is removed from circulation in this way. The LDL binds to specific LDL receptors, which cluster in domains of the plasma membrane known as coated pits (discussed in subsequent paragraphs). These domains eventually invaginate to form coated vesicles (Figure 25.39). Within the cell, these vesicles fuse with lysosomes, and the LDLs are degraded by lysosomal acid lipases.

High-density lipoproteins (HDL) have much longer life spans in the body (5 to 6 days) than other lipoproteins. Newly formed HDL contains virtually no cholesterol ester. However, over time, cholesterol esters are accumulated through the action of lecithin:cholesterol acyltransferase (LCAT), a 59-kD glycoprotein associated with HDLs. Another associated protein, cholesterol ester transfer protein, transfers some of these esters to VLDL and LDL. Alternatively, HDLs function to return cholesterol and cholesterol esters to the liver. This latter process apparently explains the correlation between high HDL levels and reduced risk of cardiovascular disease. (High LDL levels, on the other hand, are correlated with an increased risk of coronary artery and cardiovascular disease.)

Structure of the LDL Receptor

The LDL receptor in plasma membranes (Figure 25.40) consists of 839 amino acid residues and is composed of five domains. These domains include an LDLbinding domain of 292 residues, a segment of about 350 to 400 residues containing N-linked oligosaccharides, a 58-residue segment of O-linked oligosaccharides, a 22-residue membrane-spanning segment, and a 50-residue segment extending into the cytosol. The clustering of receptors prior to the formation of coated vesicles requires the presence of this cytosolic segment.

Defects in Lipoprotein Metabolism Can Lead to

Elevated Serum Cholesterol

The mechanism of LDL metabolism and the various defects that can occur therein have been studied extensively by Michael Brown and Joseph Goldstein, who received the Nobel Prize in medicine or physiology in 1985. Familial hypercholesterolemia is the term given to a variety of inherited metabolic defects that lead to greatly elevated levels of serum cholesterol—much of it in the form of LDL particles. The general genetic defect responsible for familial hypercholesterolemia is the absence or dysfunction of LDL receptors in the body. Only about half the normal level of LDL receptors is found in heterozygous individuals (persons carrying one normal gene and one defective gene). Homozygotes (with two copies of the defective gene) have few if any functional LDL receptors. In such cases, LDLs (and cholesterol) cannot be absorbed, and plasma levels of LDL (and cholesterol) are very high. Typical heterozygotes display serum cholesterol levels of 300 to 400 mg/dL, but homozygotes carry serum cholesterol levels of 600 to 800 mg/dL or even higher. There are two possible causes of an absence of LDL receptors. Either receptor synthesis does not occur at all, or the newly synthesized protein does not successfully reach the plasma membrane, due to faulty processing in the Golgi or faulty transport

LDL-binding domain

292 residues

N

N-linked oligosaccharide domain 350–400 residues

O-linked oligosaccharide domain

58 residues

Transmembrane domain

22 residues

Cytosolic domain 50 residues

C

● The structure of the LDL receptor. The amino-terminal binding domain is responsible for recognition and binding of LDL apoprotein. The O-linked oligosacchariderich domain may act as a molecular spacer, raising the binding domain above the glycocalyx. The cytosolic domain is required for aggregation of LDL receptors during endocytosis.

846 Chapter 25 ● Lipid Biosynthesis

to the plasma membrane. Even when LDL receptors are made and reach the plasma membrane, they may fail to function for two reasons. They may be unable to form clusters competent in coated pit formation because of folding or sequence anomalies in the carboxy-terminal domain, or they may be unable to bind LDL because of sequence or folding anomalies in the LDL-binding domain.

25.6 ● Biosynthesis of Bile Acids

Bile acids, which exist mainly as bile salts, are polar carboxylic acid derivatives of cholesterol that are important in the digestion of food, especially the solubilization of ingested fats. The Na and K salts of glycocholic acid and taurocholic acid are the principal bile salts (Figure 25.41). Glycocholate and taurocholate are conjugates of cholic acid with glycine and taurine, respectively.

H3C
CH3
		H3C
7α -Hydroxylase							COO–
HO	CH3				OH
Cholesterol
			Many steps		CH3
HO		OH
					CH3
	7α -Hydroxycholesterol
			HO		OH
					H
			Cholic acid
+	SO3–						+	COO–
H3N CH2 CH2							H3N CH2
Taurine							Glycine
		O					O
H	C	C	SO	–	H	C	C
3				3	3			COO–
HO		N			HO		N
	CH3	H				CH3	H
CH3					CH3
HO	OH		HO		H	OH
H
Taurocholic acid					Glycocholic acid

FIGURE 25.41 ● Cholic acid, a bile salt, is synthesized from cholesterol via 7 -hydroxy- cholesterol. Conjugation with taurine or glycine produces taurocholic acid and glycocholic acid, respectively. Taurocholate and glycocholate are freely water-soluble and are highly effective detergents.

25.7 ● Synthesis and Metabolism of Steroid Hormones

847

NADPH + H+

NADP+

Cytochrome P450		Cytochrome P450
reductase (Flavin)		reductase (Flavin-H2)

FIGURE 25.42 ● The mixed-function oxidase activity of 7 -hydroxylase.

7α -Hydroxylase		7α -Hydroxylase
(Cytochrome P450)		(Cytochrome P450)
Fe2+		Fe3+

2 H+ + O2 H2O

Cholesterol

7α -Hydroxycholesterol

Because they contain both nonpolar and polar domains, these bile salt conjugates are highly effective as detergents. These substances are made in the liver, stored in the gallbladder, and secreted as needed into the intestines.

The formation of bile salts represents the major pathway for cholesterol degradation. The first step involves hydroxylation at C-7 (Figure 25.41). 7 - Hydroxylase, which catalyzes the reaction, is a mixed-function oxidase involving cytochrome P450. Mixed-function oxidases use O2 as substrate. One oxygen atom goes to hydroxylate the substrate, while the other is reduced to water (Figure 25.42). The function of cytochrome P450 is to activate O2 for the hydroxylation reaction. Such hydroxylations are quite common in the synthetic routes for cholesterol, bile acids, and steroid hormones and also in detoxification pathways for aromatic compounds. Several of these are considered in the next section. 7 -Hydroxycholesterol is the precursor for cholic acid.

25.7 ● Synthesis and Metabolism of Steroid Hormones

Steroid hormones are crucial signal molecules in mammals. (The details of their physiological effects are described in Chapter 34.) Their biosynthesis begins with the desmolase reaction, which converts cholesterol to preg-

848 Chapter 25 ● Lipid Biosynthesis

O

C H

		Isocaproic aldehyde
		O		O
H3C		H3C	(Endoplasmic reticulum)	H3C
H3C	Desmolase	H3C	H3C
	(Mitochondria)
HO	HO		O
Cholesterol		Pregnenolone		Progesterone

O CH2OH

C

O CH

				HO
	OH			H3C
	H3C	O	CH2OH
	H3C		C
		H3C	OH	O
		HO		Aldosterone
		H3C
	O
	Testosterone
		O
	OH	Cortisol
	H3C

HO

β -Estradiol

FIGURE 25.43 ● The steroid hormones are synthesized from cholesterol, with intermediate formation of pregnenolone and progesterone. Testosterone, the principal male sex hormone steroid, is a precursor to - estradiol. Cortisol, a glucocorticoid, and aldosterone, a mineralocorticoid, are also derived from progesterone.

nenolone (Figure 25.43). Desmolase is found in the mitochondria of tissues that synthesize steroids (mainly the adrenal glands and gonads). Desmolase activity includes two hydroxylases and utilizes cytochrome P450.

Pregnenolone and Progesterone Are the Precursors

of All Other Steroid Hormones

Pregnenolone is transported from the mitochondria to the ER, where a hydroxyl oxidation and migration of the double bond yield progesterone. Pregnenolone synthesis in the adrenal cortex is activated by adrenocorticotropic hormone (ACTH), a peptide of 39 amino acid residues secreted by the anterior pituitary gland.

25.7 ● Synthesis and Metabolism of Steroid Hormones

849

Progesterone is secreted from the corpus luteum during the latter half of the menstrual cycle and prepares the lining of the uterus for attachment of a fertilized ovum. If an ovum attaches, progesterone secretion continues to ensure the successful maintenance of a pregnancy. Progesterone is also the precursor for synthesis of the sex hormone steroids and the corticosteroids. Male sex hormone steroids are called androgens, and female hormones, estrogens. Testosterone is an androgen synthesized in males primarily in the testes (and in much smaller amounts in the adrenal cortex). Androgens are necessary for sperm maturation. Even nonreproductive tissue (liver, brain, and skeletal muscle) is susceptible to the effects of androgens.

Testosterone is also produced primarily in the ovaries (and in much smaller amounts in the adrenal glands) of females as a precursor for the estrogens.-Estradiol is the most important estrogen (Figure 25.43).

Steroid Hormones Modulate Transcription in the Nucleus

Steroid hormones act in a different manner from most hormones we have considered. In many cases, they do not bind to plasma membrane receptors, but rather pass easily across the plasma membrane. Steroids may bind directly to receptors in the nucleus or may bind to cytosolic steroid hormone receptors, which then enter the nucleus. In the nucleus, the hormone-receptor complex binds directly to specific nucleotide sequences in DNA, increasing transcription of DNA to RNA (Chapters 31 and 34).

Cortisol and Other Corticosteroids Regulate a

Variety of Body Processes

Corticosteroids, including the glucocorticoids and mineralocorticoids, are made by the cortex of the adrenal glands on top of the kidneys. Cortisol (Figure 25.43) is representative of the glucocorticoids, a class of compounds that (1) stimulate gluconeogenesis and glycogen synthesis in liver (by signaling the synthesis of PEP carboxykinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, and glycogen synthase); (2) inhibit protein synthesis and stimulate protein degradation in peripheral tissues such as muscle; (3) inhibit allergic and inflammatory responses; (4) exert an immunosuppressive effect, inhibiting DNA replication and mitosis and repressing the formation of antibodies and lymphocytes; and (5) inhibit formation of fibroblasts involved in healing wounds and slow the healing of broken bones.

Aldosterone, the most potent of the mineralocorticoids (Figure 25.43), is involved in the regulation of sodium and potassium balances in tissues. Aldosterone increases the kidney’s capacity to absorb Na , Cl , and H2O from the glomerular filtrate in the kidney tubules.

Anabolic Steroids Have Been Used Illegally To

Enhance Athletic Performance

The dramatic effects of androgens on protein biosynthesis have led many athletes to the use of synthetic androgens, which go by the blanket term anabolic steroids. Despite numerous warnings from the medical community about side effects, which include kidney and liver disorders, sterility, and heart disease, abuse of such substances is epidemic. Stanozolol (Figure 25.44) was one of the agents found in the blood and urine of Ben Johnson following his recordsetting performance in the 100-meter dash in the 1988 Olympic Games. Because use of such substances is disallowed, Johnson lost his gold medal, and Carl Lewis was declared the official winner.

OH

H3C

CH3

H3C

HN

N

H

Stanozolol

FIGURE 25.44 ● The structure of stanozolol, an anabolic steroid.

850 Chapter 25 ● Lipid Biosynthesis

PROBLEMS

1.Carefully count and account for each of the atoms and charges in the equations for the synthesis of palmitoyl-CoA, the synthesis of malonyl-CoA, and the overall reaction for the synthesis of palmi- toyl-CoA from acetyl-CoA.

2.Use the relationships shown in Figure 25.1 to determine which carbons of glucose will be incorporated into palmitic acid. Consider the cases of both citrate that is immediately exported to the cytosol following its synthesis and citrate that enters the TCA cycle.

3.Based on the information presented in the text and in Figures 25.4 and 25.5, suggest a model for the regulation of acetyl-CoA carboxylase. Consider the possible roles of subunit interactions, phosphorylation, and conformation changes in your model.

4.Consider the role of the pantothenic acid groups in animal fatty acyl synthase and the size of the pantothenic acid group itself, and estimate a maximal separation between the malonyl transferase and the ketoacyl-ACP synthase active sites.

5.Carefully study the reaction mechanism for the stearoyl-CoA desaturase in Figure 25.14, and account for all of the electrons flowing through the reactions shown. Also account for all of the hydrogen and oxygen atoms involved in this reaction, and convince yourself that the stoichiometry is correct as shown.

FURTHER READING

Athappilly, F. K., and Hendrickson, W. A., 1995. Structure of the biotinyl domain of acetyl-CoA carboxylase determined by MAD phasing. Structure 3:1407.

Bloch, K., 1965. The biological synthesis of cholesterol. Science 150:19–28.

Bloch, K., 1987. Summing up. Annual Review of Biochemistry 56:1–19.

Borhani, D. W., Rogers, D. P., Engler, J. A., and Brouillette, C. G., 1997. Crystal structure of truncated human apolipoprotein A-I suggests a lipidbound conformation. Proceedings of the National Academy of Sciences 94:12291– 12296.

Boyer, P. D., ed., 1983. The Enzymes, 3rd ed., Vol. 16. New York: Academic Press.

Carman, G. M., and Henry, S. A., 1989. Phospholipid biosynthesis in yeast.

Annual Review of Biochemistry 58:635–669.

Carman, G. M., and Zeimetz, G. M., 1996. Regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. Journal of Biological Chemistry 271:13292–13296.

Chang, S. I., and Hammes, G. G., 1990. Structure and mechanism of action of a multifunctional enzyme: Fatty acid synthase. Accounts of Chemical Research 23:363–369.

Dunne, S. J., Cornell, R. B., Johnson, J. E., et al., 1996. Structure of the membrane-binding domain of CTP phosphocholine cytidylyltransferase.

Biochemistry 35:1975.

Friedman, J. M., 1997. The alphabet of weight control. Nature 385:119– 120.

Hansen, H. S., 1985. The essential nature of linoleic acid in mammals.

Trends in Biochemical Sciences 11:263–265.

6.Write a balanced, stoichiometric reaction for the synthesis of phosphatidylethanolamine from glycerol, fatty acyl-CoA, and ethanolamine. Make an estimate of the G° for the overall process.

7.Write a balanced, stoichiometric reaction for the synthesis of cholesterol from acetyl-CoA.

8.Trace each of the carbon atoms of mevalonate through the synthesis of cholesterol, and determine the source (i.e., the position in the mevalonate structure) of each carbon in the final structure.

9.Suggest a structural or functional role for the O-linked saccharide domain in the LDL receptor (Figure 25.40).

10.Identify the lipid synthetic pathways that would be affected by abnormally low levels of CTP.

11.Determine the number of ATP equivalents needed to form palmitic acid from acetyl-CoA. (Assume for this calculation that each NADPH is worth 3.5 ATP.)

12.Write a reasonable mechanism for the 3-ketosphinganine synthase reaction, remembering that it is a pyridoxal phosphatedependent reaction.

Jackowski, S., 1996. Cell cycle regulation of membrane phospholipid metabolism. Journal of Biological Chemistry 271:20219–20222.

Jeffcoat, R., 1979. The biosynthesis of unsaturated fatty acids and its control in mammalian liver. Essays in Biochemistry 15:1–36.

Kent, C., 1995. Eukaryotic phospholipid biosynthesis. Annual Review of Biochemistry 64:315–343.

Kim, K-H., et al., 1989. Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis. The FASEB Journal 3:2250– 2256.

Lardy, H., and Shrago, E., 1990. Biochemical aspects of obesity. Annual Review of Biochemistry 59:689–710.

Liscum, L., and Underwood, K. W., 1995. Intracellular cholesterol transport and compartmentation. Journal of Biological Chemistry 270:15443– 15446.

Lopaschuk, G. D., and Gamble, J., 1994. The 1993 Merck Frosst Award. Acetyl-CoA carboxylase: an important regulator of fatty acid oxidation in the heart. Canadian Journal of Physiology and Pharmacology 72:1101–1109.

McCarthy, A. D., and Hardie, D. G., 1984. Fatty acid synthase—An example of protein evolution by gene fusion. Trends in Biochemical Sciences 9:60–63.

Needleman, P., et al., 1986. Arachidonic acid metabolism. Annual Review of Biochemistry 55:69–102.

Roberts, L. M., Ray, M. J., Shih, T. W., et al., 1997. Structural analysis of apolipoprotein A-I: Limited proteolysis of methionine-reduced and -oxi- dized lipid-free and lipid-bound human Apo A-I. Biochemistry 36:7615.