Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol, the characteristic steroid alcohol of animal tissues, performs a number of essential functions in the body. For example, cholesterol is a structural component of all cell membranes, modulating their fluidity, and, in specialized tissues, cholesterol is a precursor of bile acids, steroid hormones, and vitamin D. Therefore, it is critically important that the cells of the body be assured an appropriate supply of cholesterol. To meet this need, a complex series of transport, biosynthetic, and regulatory mechanisms has evolved. The liver plays a central role in the regulation of the body’s cholesterol homeostasis. For example, cholesterol enters the hepatic cholesterol pool from a number of sources including dietary cholesterol as well as that synthesized de novo by extrahepatic tissues and by the liver itself. Cholesterol is eliminated from the liver as unmodified cholesterol in the bile, or it can be converted to bile salts that are secreted into the intestinal lumen. It


Biochemistry_Lippinco

Biochemistry_Lippinco. tissues and by the liver itself. Cholesterol is eliminated from the liver as unmodified cholesterol in the bile, or it can be converted to bile salts that are secreted into the intestinal lumen. It can also serve as a component of plasma lipoproteins that carry lipids to the peripheral tissues. In humans, the balance between cholesterol influx and efflux is not precise, resulting in a gradual deposition of cholesterol in the tissues, particularly in the endothelial linings of blood vessels. This is a potentially life-threatening occurrence when the lipid deposition leads to plaque formation, causing the narrowing of blood vessels (atherosclerosis) and increased risk of cardio-, cerebro-, and peripheral vascular disease. Figure 18.1 summarizes the major sources of liver cholesterol and the routes by which cholesterol leaves the liver.


Biochemistry_Lippinco

Biochemistry_Lippinco. II. CHOLESTEROL STRUCTURE Cholesterol is a very hydrophobic compound. It consists of four fused hydrocarbon rings (A–D) called the steroid nucleus, and it has an eight-carbon, branched hydrocarbon chain attached to carbon 17 of the D ring. Ring A has a hydroxyl group at carbon 3, and ring B has a double bond between carbon 5 and carbon 6 (Fig. 18.2). A. Sterols


Biochemistry_Lippinco

Biochemistry_Lippinco. Steroids with 8 to 10 carbon atoms in the side chain at carbon 17 and a hydroxyl group at carbon 3 are classified as sterols. Cholesterol is the major sterol in animal tissues. It arises from de novo synthesis and absorption of dietary cholesterol. Intestinal uptake of cholesterol is mediated by the Niemann-Pick C1-like 1 protein, the target of the drug ezetimibe that reduces absorption of dietary cholesterol (see p. 176). [Note: Plant sterols (phytosterols), such as β-sitosterol, are poorly absorbed by humans (5% absorbed as compared to 40% for cholesterol). After entering the enterocytes, they are actively transported back into the intestinal lumen. Defects in the efflux transporter (ABCG5/8) result in the rare condition of sitosterolemia. Because some cholesterol is transported back as well, plant sterols reduce the absorption of dietary cholesterol. Daily ingestion of plant sterol esters supplied, for example, in spreads, is one of a number of dietary strategies to reduce plasma


Biochemistry_Lippinco

Biochemistry_Lippinco. well, plant sterols reduce the absorption of dietary cholesterol. Daily ingestion of plant sterol esters supplied, for example, in spreads, is one of a number of dietary strategies to reduce plasma cholesterol levels (see p. 363).]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Cholesteryl esters Most plasma cholesterol is in an esterified form (with a fatty acid [FA] attached at carbon 3, as shown in Fig. 18.2), which makes the structure even more hydrophobic than free (nonesterified) cholesterol. Cholesteryl esters are not found in membranes and are normally present only in low levels in most cells. Because of their hydrophobicity, cholesterol and its esters must be transported in association with protein as a component of a lipoprotein particle (see p. 227) or be solubilized by phospholipids and bile salts in the bile (see p. 226). III. CHOLESTEROL SYNTHESIS


Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol is synthesized by virtually all tissues in humans, although liver, intestine, adrenal cortex, and reproductive tissues, including ovaries, testes, and placenta, make the largest contributions to the cholesterol pool. As with FA, all the carbon atoms in cholesterol are provided by acetyl coenzyme A (CoA), and nicotinamide adenine dinucleotide phosphate (NADPH) provides the reducing equivalents. The pathway is endergonic, being driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phosphate bond of ATP. Synthesis requires enzymes in the cytosol, the membrane of the smooth endoplasmic reticulum (SER), and the peroxisome. The pathway is responsive to changes in cholesterol concentration, and regulatory mechanisms exist to balance the rate of cholesterol synthesis against the rate of cholesterol excretion. An imbalance in this regulation can lead to an elevation in circulating levels of plasma cholesterol, with the potential for vascular disease.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. 3-Hydroxy-3-methylglutaryl coenzyme A synthesis The first two reactions in the cholesterol biosynthetic pathway are similar to those in the pathway that produces ketone bodies (see Fig. 16.22, p. 196). They result in the production of 3-hydroxy-3-methylglutaryl CoA ([HMG CoA], Fig. 18.3). First, two acetyl CoA molecules condense to form acetoacetyl CoA. Next, a third molecule of acetyl CoA is added by HMG CoA synthase, producing HMG CoA, a six-carbon compound. [Note: Liver parenchymal cells contain two isoenzymes of the synthase. The cytosolic enzyme participates in cholesterol synthesis, whereas the mitochondrial enzyme functions in the pathway for ketone body synthesis.] B. Mevalonate synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Mevalonate synthesis HMG CoA is reduced to mevalonate by HMG CoA reductase. This is the rate-limiting and key regulated step in cholesterol synthesis. It occurs in the cytosol, uses two molecules of NADPH as the reducing agent, and releases CoA, making the reaction irreversible (Fig. 18.4). [Note: HMG CoA reductase is an integral membrane protein of the SER, with its catalytic domain projecting into the cytosol. Regulation of reductase activity is discussed in D. below.] C. Cholesterol synthesis from mevalonate The reactions and enzymes involved in the synthesis of cholesterol from mevalonate are illustrated in Figure 18.5. [Note: The numbers shown in brackets below correspond to numbered reactions shown in this figure.] [1] Mevalonate is converted to 5-pyrophosphomevalonate in two steps, each of which transfers a phosphate group from ATP.


Biochemistry_Lippinco

Biochemistry_Lippinco. [2] A five-carbon isoprene unit, isopentenyl pyrophosphate (IPP), is formed by the decarboxylation of 5-pyrophosphomevalonate. The reaction requires ATP. [Note: IPP is the precursor of a family of molecules with diverse functions, the isoprenoids. Cholesterol is a sterol isoprenoid. Nonsterol isoprenoids include dolichol (see p. 167) and ubiquinone (or, coenzyme Q; see p. 75).] [3] IPP is isomerized to 3,3-dimethylallyl pyrophosphate (DPP). [4] IPP and DPP condense to form 10-carbon geranyl pyrophosphate (GPP).


Biochemistry_Lippinco

Biochemistry_Lippinco. [4] IPP and DPP condense to form 10-carbon geranyl pyrophosphate (GPP). [5] A second molecule of IPP then condenses with GPP to form 15-carbon farnesyl pyrophosphate (FPP). [Note: Covalent attachment of farnesyl to proteins, a process known as prenylation, is one mechanism for anchoring proteins (for example, ras) to the inner face of plasma membranes.] [6] Two molecules of FPP combine, releasing pyrophosphate, and are reduced, forming the 30-carbon compound squalene. [Note: Squalene is formed from six isoprenoid units. Because 3 ATP are hydrolyzed per mevalonate residue converted to IPP, a total of 18 ATP are required to make the polyisoprenoid squalene.] [7] Squalene is converted to the sterol lanosterol by a sequence of two reactions catalyzed by SER-associated enzymes that use molecular oxygen (O2) and NADPH. The hydroxylation of linear squalene triggers the cyclization of the structure to lanosterol.


Biochemistry_Lippinco

Biochemistry_Lippinco. The conversion of lanosterol to cholesterol is a multistep process involving shortening of the side chain, oxidative removal of methyl groups, reduction of double bonds, and migration of a double bond. Smith-Lemli-Opitz syndrome (SLOS), an autosomal-recessive disorder of cholesterol biosynthesis, is caused by a partial deficiency in 7-dehydrocholesterol-7reductase, the enzyme that reduces the double bond in 7dehydrocholesterol (7-DHC), thereby converting it to cholesterol. SLOS is one of several multisystem, embryonic malformation syndromes associated with impaired cholesterol synthesis. [Note: 7-DHC is converted to vitamin D3 in the skin (see p. 390).] D. HMG CoA reductase is the major control point for cholesterol biosynthesis and is subject to different kinds of metabolic control. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Sterol-dependent regulation of gene expression: Expression of the gene for HMG CoA reductase is controlled by the trans-acting factor, sterol regulatory element–binding protein-2 (SREBP-2), which binds DNA at the cis-acting sterol regulatory element (SRE) upstream of the reductase gene. Inactive SREBP-2 is an integral protein of the SER membrane and associates with a second SER membrane protein, SREBP cleavage– activating protein (SCAP). When sterol levels in the SER are low, the SREBP-2–SCAP complex moves from the ER to the Golgi. In the Golgi membrane, SREBP-2 is sequentially acted upon by two proteases, which generate a soluble fragment that enters the nucleus, binds the SRE, and functions as a transcription factor. This results in increased synthesis of HMG CoA reductase and, therefore, increased cholesterol synthesis (Fig. 18.6). However, if sterols are abundant, they bind SCAP at its sterolsensing domain and induce the binding of SCAP to yet other ER membrane proteins, the


Biochemistry_Lippinco

Biochemistry_Lippinco. increased cholesterol synthesis (Fig. 18.6). However, if sterols are abundant, they bind SCAP at its sterolsensing domain and induce the binding of SCAP to yet other ER membrane proteins, the insulin-induced gene proteins (INSIG). This results in the retention of the SCAP–SREBP complex in the SER, thereby preventing the activation of SREBP-2 and leading to downregulation of cholesterol synthesis. [Note: SREBP-1c upregulates expression of enzymes involved in FA synthesis in response to insulin (see p. 184).] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Sterol-accelerated enzyme degradation: The reductase itself is a sterolsensing integral protein of the SER membrane. When sterol levels in the SER are high, the enzyme binds to INSIG proteins. Binding leads to cytosolic transfer, ubiquitination, and proteasomal degradation of the reductase (see p. 247). 3. Sterol-independent phosphorylation/dephosphorylation: HMG CoA reductase activity is controlled covalently through the actions of adenosine monophosphate (AMP)–activated protein kinase ([AMPK] see p. 183) and a phosphoprotein phosphatase (see Fig. 18.6). The phosphorylated form of the enzyme is inactive, whereas the dephosphorylated form is active. [Note: Because AMPK is activated by AMP, cholesterol synthesis, like FA synthesis, is decreased when ATP availability is decreased.] 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Hormonal regulation: The activity of HMG CoA reductase is controlled hormonally. An increase in insulin favors dephosphorylation (activation) of the reductase, whereas an increase in glucagon and epinephrine has the opposite effect. 5. Drug inhibition: The statin drugs (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin) are structural analogs of HMG CoA and are (or are metabolized to) reversible, competitive inhibitors of HMG CoA reductase (Fig. 18.7). They are used to decrease plasma cholesterol levels in patients with hypercholesterolemia. IV. CHOLESTEROL DEGRADATION


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. CHOLESTEROL DEGRADATION Humans cannot metabolize the cholesterol ring structure to carbon dioxide and water. Rather, the intact steroid nucleus is eliminated from the body by conversion to bile acids and bile salts, a small percentage of which is excreted in the feces, and by secretion of cholesterol into the bile, which transports it to the intestine for elimination. [Note: Some of the cholesterol in the intestine is modified by bacteria before excretion. The primary compounds made are the isomers coprostanol and cholestanol, which are reduced derivatives of cholesterol. Together with cholesterol, these compounds make up the bulk of neutral fecal sterols.] V. BILE ACIDS AND BILE SALTS


Biochemistry_Lippinco

Biochemistry_Lippinco. V. BILE ACIDS AND BILE SALTS Bile consists of a watery mixture of organic and inorganic compounds. Phosphatidylcholine (PC), or lecithin (see p. 202), and conjugated bile salts are quantitatively the most important organic components of bile. Bile can either pass directly from the liver, where it is synthesized, into the duodenum through the common bile duct, or be stored in the gallbladder when not immediately needed for digestion. A. Structure


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Structure The bile acids contain 24 carbons, with two or three hydroxyl groups and a side chain that terminates in a carboxyl group. The carboxyl group has a pKa (see p. 6) of ~6. In the duodenum (pH ~6), this group will be protonated in half of the molecules (the bile acids) and deprotonated in the rest (the bile salts). The terms bile acid and bile salt are frequently used interchangeably, however. Both forms have hydroxyl groups that are α in orientation (they lie below the plane of the rings) and methyl groups that are β (they lie above the plane of the rings). Therefore, the molecules have both a polar and a nonpolar surface and can act as emulsifying agents in the intestine, helping prepare dietary fat (triacylglycerol [TAG]) and other complex lipids for degradation by pancreatic digestive enzymes. B. Synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Synthesis Bile acids are synthesized in the liver by a multistep, multiorganelle pathway in which hydroxyl groups are inserted at specific positions on the steroid structure; the double bond of the cholesterol B ring is reduced; and the hydrocarbon chain is shortened by three carbons, introducing a carboxyl group at the end of the chain. The most common resulting compounds, cholic acid (a triol) and chenodeoxycholic acid (a diol), as shown in Figure 18.8, are called primary bile acids. [Note: The rate-limiting step in bile acid synthesis is the introduction of a hydroxyl group at carbon 7 of the steroid nucleus by 7-α-hydroxylase, a SER-associated cytochrome P450 monooxygenase found only in liver. Expression of the enzyme is downregulated by bile acids (Fig. 18.9)]. C. Conjugation


Biochemistry_Lippinco

Biochemistry_Lippinco. Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine (an end product of cysteine metabolism) by an amide bond between the carboxyl group of the bile acid and the amino group of the added compound. These new structures include glycocholic and glycochenodeoxycholic acids and taurocholic and taurochenodeoxycholic acids (Fig. 18.10). The ratio of glycine to taurine forms in the bile is ~3/1. Addition of glycine or taurine results in the presence of a carboxyl group with a lower pKa (from glycine) or a sulfonate group (from taurine), both of which are fully ionized (negatively charged) at the alkaline pH of bile. The conjugated, ionized bile salts are more effective detergents than the unconjugated ones because of their enhanced amphipathic nature. Therefore, only the conjugated forms are found in the bile. Individuals with genetic deficiencies in the conversion of cholesterol to bile acids are treated with exogenously supplied


Biochemistry_Lippinco

Biochemistry_Lippinco. nature. Therefore, only the conjugated forms are found in the bile. Individuals with genetic deficiencies in the conversion of cholesterol to bile acids are treated with exogenously supplied chenodeoxycholic acid.


Biochemistry_Lippinco

Biochemistry_Lippinco. Bile salts provide the only significant mechanism for cholesterol excretion, both as a metabolic product of cholesterol and as a solubilizer of cholesterol in bile. D. Bacterial action on bile salts Bacteria of the intestinal microbiota (see p. 372) can deconjugate (remove glycine and taurine) bile salts. They can also dehydroxylate carbon 7, producing secondary bile salts such as deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. E. Enterohepatic circulation


Biochemistry_Lippinco

Biochemistry_Lippinco. Bile salts secreted into the intestine are efficiently reabsorbed (>95%) and reused. The liver actively secretes bile salts via the bile salt export pump. In the intestine, they are reabsorbed in the terminal ileum via the apical sodium (Na+)–bile salt cotransporter and returned to the blood via a separate transport system. [Note: Lithocholic acid is only poorly absorbed.] They are efficiently taken up from blood by the hepatocytes via an isoform of the cotransporter and reused. [Note: Albumin binds bile salts and transports them through the blood as was seen with FA (see p. 181).] The continuous process of secretion of bile salts into the bile, their passage through the duodenum where some are deconjugated then dehydroxylated to secondary bile salts, their uptake in the ileum, and their subsequent return to the liver as a mixture of primary and secondary forms is termed the enterohepatic circulation (Fig. 18.11). Between 15 and 30 g of bile salts are secreted from the liver into the


Biochemistry_Lippinco

Biochemistry_Lippinco. subsequent return to the liver as a mixture of primary and secondary forms is termed the enterohepatic circulation (Fig. 18.11). Between 15 and 30 g of bile salts are secreted from the liver into the duodenum each day, yet only ~0.5 g (<3%) is lost daily in the feces. Approximately 0.5 g/day is synthesized from cholesterol in the liver to replace the amount lost. Bile acid sequestrants, such as cholestyramine, bind bile salts in the gut and prevent their reabsorption, thereby promoting their excretion. They are used in the treatment of hypercholesterolemia because the removal of bile salts relieves the inhibition on bile acid synthesis in the liver, thereby diverting additional cholesterol into that pathway. [Note: Dietary fiber also binds bile salts and increases their excretion (see p. 365).]


Biochemistry_Lippinco

F. Bile salt deficiency: Cholelithiasis

Biochemistry_Lippinco. F. Bile salt deficiency: Cholelithiasis


Biochemistry_Lippinco

Biochemistry_Lippinco. The movement of cholesterol from the liver into the bile must be accompanied by the simultaneous secretion of phospholipid and bile salts. If this dual process is disrupted and more cholesterol is present than can be solubilized by the bile salts and PC present, the cholesterol may precipitate in the gallbladder, leading to cholesterol gallstone disease or cholelithiasis (Fig. 18.12). This disorder is typically caused by a decrease of bile acids in the bile. Cholelithiasis also may result from increased secretion of cholesterol into bile, as seen with the use of fibrates (for example, gemfibrozil) to reduce cholesterol (and TAG) in the blood. Laparoscopic cholecystectomy (surgical removal of the gallbladder through a small incision) is currently the treatment of choice. However, for patients who are unable to undergo surgery, oral administration of chenodeoxycholic acid to supplement the body’s supply of bile acids results in a gradual (months to years) dissolution of the gallstones.


Biochemistry_Lippinco

Biochemistry_Lippinco. who are unable to undergo surgery, oral administration of chenodeoxycholic acid to supplement the body’s supply of bile acids results in a gradual (months to years) dissolution of the gallstones. [Note: Cholesterol stones account for >85% of cases of cholelithiasis, with bilirubin and mixed stones accounting for the rest].


Biochemistry_Lippinco

Biochemistry_Lippinco. VI. PLASMA LIPOPROTEINS The plasma lipoproteins are spherical macromolecular complexes of lipids and proteins (apolipoproteins). The lipoprotein particles include chylomicrons, verylow-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They differ in lipid and protein composition, size, density (Fig. 18.13), and site of origin. [Note: Because lipoprotein particles constantly interchange lipids and apolipoproteins, the actual apolipoprotein and lipid content of each class of particles is somewhat variable.] Lipoproteins function both to keep their component lipids soluble as they transport them in the plasma and to provide an efficient mechanism for transporting their lipid contents to (and from) the tissues. In humans, the transport system is less perfect than in other animals and, as a result, humans experience a gradual deposition of lipid (especially cholesterol) in tissues. A. Composition


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Composition Lipoproteins are composed of a neutral lipid core (containing TAG and cholesteryl esters) surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified (free) cholesterol (Fig. 18.14). These amphipathic compounds are oriented such that their polar portions are exposed on the surface of the lipoprotein, thereby rendering the particle soluble in aqueous solution. The TAG and cholesterol carried by the lipoproteins are obtained either from the diet (exogenous source) or from de novo synthesis (endogenous source). [Note: The cholesterol (C) content of plasma lipoproteins is now routinely measured in fasting blood. Total C = LDL-C + HDL-C + VLDL-C, where VLDL-C is calculated by dividing TAG by 5 because the TAG/cholesterol ratio is 5/1 in VLDL. The goal value for total cholesterol is <200 mg/dl.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Size and density: Chylomicrons are the lipoprotein particles lowest in density and largest in size and that contain the highest percentage of lipid (as TAG) and the lowest percentage of protein. VLDL and LDL are successively denser, having higher ratios of protein to lipid. HDL particles are the smallest and densest. Plasma lipoproteins can be separated on the basis of their electrophoretic mobility, as shown in Figure 18.15, or on the basis of their density by ultracentrifugation. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Apolipoproteins: The apolipoproteins associated with lipoprotein particles have a number of diverse functions, such as providing recognition sites for cell-surface receptors and serving as activators or coenzymes for enzymes involved in lipoprotein metabolism. Some of the apolipoproteins are required as essential structural components of the particles and cannot be removed (in fact, the particles cannot be produced without them), whereas others are transferred freely between lipoproteins. Apolipoproteins are divided by structure and function into several major classes, denoted by letters, with each class having subclasses (for example, apolipoprotein [apo] C-I, apo C-II, and apo CIII). [Note: The functions of all the apolipoproteins are not yet known.] B. Chylomicron metabolism


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Chylomicron metabolism Chylomicrons are assembled in intestinal mucosal cells and carry dietary (exogenous) TAG, cholesterol, fat-soluble vitamins, and cholesteryl esters to the peripheral tissues (Fig. 18.16). [Note: TAG account for close to 90% of the lipids in a chylomicron.] 1. Apolipoprotein synthesis: Apo B-48 is unique to chylomicrons. Its synthesis begins on the rough ER (RER), and it is glycosylated as it moves through the RER and Golgi. [Note: Apo B-48 is so named because it constitutes the N-terminal 48% of the protein encoded by the gene for apo B. Apo B-100, which is synthesized by the liver and found in VLDL and LDL, represents the entire protein encoded by this gene. Posttranscriptional editing (see p. 474) of a cytosine to a uracil in intestinal apo B-100 messenger RNA (mRNA) creates a nonsense (stop) codon (see p. 449), allowing translation of only 48% of the mRNA.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Chylomicron assembly: Many enzymes involved in TAG, cholesterol, and phospholipid synthesis are located in the SER. Assembly of the apolipoprotein and lipid into chylomicrons requires microsomal triglyceride transfer protein ([MTP] see p. 177), which loads apo B-48 with lipid. This occurs before transition from the ER to the Golgi, where the particles are packaged in secretory vesicles. These fuse with the plasma membrane releasing the lipoproteins, which then enter the lymphatic system and, ultimately, the blood. [Note: Chylomicrons leave the lymphatic system via the thoracic duct that empties into the left subclavian vein.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Nascent chylomicron modification: The particle released by the intestinal mucosal cell is called a nascent chylomicron because it is functionally incomplete. When it reaches the plasma, the particle is rapidly modified, receiving apo E (which is recognized by hepatic receptors) and apo C. The latter includes apo C-II, which is necessary for the activation of lipoprotein lipase (LPL), the enzyme that degrades the TAG contained in the chylomicron. The source of these apolipoproteins is circulating HDL (see Fig. 18.16). [Note: Apo C-III on TAG-rich lipoproteins inhibits LPL.] 4. Triacylglycerol degradation by lipoprotein lipase: LPL is an extracellular enzyme that is anchored to the capillary walls of most tissues but predominantly those of adipose tissue and cardiac and skeletal muscle.


Biochemistry_Lippinco

Biochemistry_Lippinco. The adult liver does not express this enzyme. [Note: A hepatic lipase is found on the surface of endothelial cells of the liver. It plays a role in TAG degradation in chylomicrons and VLDL and is important in HDL metabolism (see p. 234).] LPL, activated by apo C-II on circulating chylomicrons, hydrolyzes the TAG in these particles to FA and glycerol. The FA are stored (in adipose) or used for energy (in muscle). The glycerol is taken up by the liver, converted to dihydroxyacetone phosphate (an intermediate of glycolysis), and used in lipid synthesis or gluconeogenesis. [Note: Patients with a deficiency of LPL or apo C-II (type I hyperlipoproteinemia or familial chylomicronemia) show a dramatic accumulation (≥1,000 mg/dl) of chylomicron-TAG in the plasma (hypertriacylglycerolemia) even in the fasted state. They are at increased risk for acute pancreatitis. Treatment is the reduction of dietary fat.] 5.


Biochemistry_Lippinco

Biochemistry_Lippinco. Lipoprotein lipase expression: LPL is synthesized by adipose tissue and by cardiac and skeletal muscle. Expression of the tissue-specific isozymes is regulated by nutritional state and hormonal level. For example, in the fed state (elevated insulin levels), LPL synthesis is increased in adipose but decreased in muscle tissue. Fasting (decreased insulin) favors LPL synthesis in muscle. [Note: The highest concentration of LPL is in cardiac muscle, reflecting the use of FA to provide much of the energy needed for cardiac function.] 6.


Biochemistry_Lippinco

Biochemistry_Lippinco. Chylomicron remnant formation: As the chylomicron circulates, and >90% of the TAG in its core is degraded by LPL, the particle decreases in size and increases in density. In addition, the C apolipoproteins (but not apo B or E) are returned to HDL. The remaining particle, called a remnant, is rapidly removed from the circulation by the liver, whose cell membranes contain lipoprotein receptors that recognize apo E (see Fig. 18.16). Chylomicron remnants bind to these receptors and are taken into the hepatocytes by endocytosis. The endocytosed vesicle then fuses with a lysosome, and the apolipoproteins, cholesteryl esters, and other components of the remnant are hydrolytically degraded, releasing amino acids, free cholesterol, and FA. The receptor is recycled. [Note: The mechanism of receptor-mediated endocytosis is illustrated for LDL in Fig. 18.20.] C. Very-low-density lipoprotein metabolism


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Very-low-density lipoprotein metabolism VLDL are produced in the liver (Fig. 18.17). They are composed predominantly of endogenous TAG (~60%), and their function is to carry this lipid from the liver (site of synthesis) to the peripheral tissues. There, the TAG is degraded by LPL, as discussed for chylomicrons (see p. 228). [Note: Nonalcoholic fatty liver (hepatic steatosis) occurs in conditions in which there is an imbalance between hepatic TAG synthesis and the secretion of VLDL. Such conditions include obesity and type 2 diabetes mellitus (see p. 343).] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Release from the liver: VLDL are secreted directly into the blood by the liver as nascent particles containing apo B-100. They must obtain apo CII and apo E from circulating HDL (see Fig. 18.17). As with chylomicrons, apo C-II is required for activation of LPL. [Note: Abetalipoproteinemia is a rare hypolipoproteinemia caused by a defect in MTP, leading to an inability to load apo B with lipid. Consequently, few VLDL or chylomicrons are formed, and TAG accumulates in the liver and intestine. Absorption of fat-soluble vitamins is decreased. LDL are low.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Modification in the circulation: As VLDL pass through the circulation, TAG is degraded by LPL, causing the VLDL to decrease in size and become denser. Surface components, including the C and E apolipoproteins, are returned to HDL, but the particles retain apo B-100. Additionally, some TAG are transferred from VLDL to HDL in an exchange reaction that concomitantly transfers cholesteryl esters from HDL to VLDL. This exchange is accomplished by cholesteryl ester transfer protein (CETP), as shown in Figure 18.18.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Conversion to low-density lipoproteins: With these modifications, the VLDL is converted in the plasma to LDL. Intermediate-density lipoproteins (IDL) of varying sizes are formed during this transition. IDL can also be taken up by liver cells through receptor-mediated endocytosis that uses apo E as the ligand. Apo E is normally present in three isoforms, E-2 (the least common), E-3 (the most common), and E-4. Apo E-2 binds poorly to receptors, and patients who are homozygotic for apo E-2 are deficient in the clearance of IDL and chylomicron remnants. These individuals have familial type III hyperlipoproteinemia (familial dysbetalipoproteinemia or broad beta disease), with hypercholesterolemia and premature atherosclerosis. [Note: The apo E-4 isoform confers increased susceptibility to an earlier age of onset of the late-onset form of Alzheimer disease. The effect is dose dependent, with homozygotes being at greatest risk. Estimates of the risk vary.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Low-density lipoprotein metabolism LDL particles contain much less TAG than their VLDL predecessors and have a high concentration of cholesterol and cholesteryl esters (Fig. 18.19). About 70% of plasma cholesterol is in LDL. 1. Receptor-mediated endocytosis: The primary function of LDL particles is to provide cholesterol to the peripheral tissues (or return it to the liver). They do so by binding to plasma membrane LDL receptors that recognize apo B-100 (but not apo B-48). Because these LDL receptors can also bind apo E, they are known as apo B-100/apo E receptors. A summary of the uptake and degradation of LDL particles is presented in Figure 18.20. [Note: The numbers in brackets below refer to corresponding numbers on that figure.] A similar mechanism of receptor-mediated endocytosis is used for the uptake and degradation of chylomicron remnants and IDL by the liver.


Biochemistry_Lippinco

Biochemistry_Lippinco. [1] LDL receptors are negatively charged glycoproteins that are clustered in pits on cell membranes. The cytosolic side of the pit is coated with the protein clathrin, which stabilizes the pit.


Biochemistry_Lippinco

Biochemistry_Lippinco. [2] After binding, the LDL–receptor complex is endocytosed. [Note: Defects in the synthesis of functional LDL receptors causes a significant elevation in plasma LDL-C. Patients with such deficiencies have type IIa hyperlipidemia (familial hypercholesterolemia [FH]) and premature atherosclerosis. Autosomal dominant hypercholesterolemia can also be caused by defects in apo B-100 that reduce its binding to the receptor and by increased activity of a protease, proprotein convertase subtilisin/kexin type 9 (PCSK9), which promotes internalization and lysosomal degradation of the receptor. PCSK9 inhibitors are now available for the treatment of hypercholesterolemia.] [3] The vesicle containing LDL loses its clathrin coat and fuses with other similar vesicles, forming larger vesicles called endosomes.


Biochemistry_Lippinco

Biochemistry_Lippinco. [4] The pH of the endosome falls (due to the proton-pumping activity of endosomal ATPase), which allows separation of the LDL from its receptor. The receptors then migrate to one side of the endosome, whereas the LDL stay free within the lumen of the vesicle.


Biochemistry_Lippinco

Biochemistry_Lippinco. [5] The receptors can be recycled, whereas the lipoprotein remnants in the vesicle are transferred to lysosomes and degraded by lysosomal acid hydrolases, releasing free cholesterol, amino acids, FA, and phospholipids. These compounds can be reutilized by the cell. [Note: Lysosomal storage diseases result from rare autosomal-recessive deficiencies in the ability to hydrolyze lysosomal cholesteryl esters (Wolman disease) or to transport free cholesterol out of the lysosome (Niemann-Pick disease, type C).] 2. Endocytosed cholesterol and cholesterol homeostasis: The chylomicron remnant–, IDL-, and LDL-derived cholesterol affects cellular cholesterol content in several ways (see Fig. 18.20). First, expression of the gene for HMG CoA reductase is inhibited by high cholesterol, and de novo cholesterol synthesis decreases as a result. Additionally, degradation of the reductase is accelerated. Second, synthesis of new LDL receptor protein is reduced by decreasing the expression of the LDL


Biochemistry_Lippinco

Biochemistry_Lippinco. synthesis decreases as a result. Additionally, degradation of the reductase is accelerated. Second, synthesis of new LDL receptor protein is reduced by decreasing the expression of the LDL receptor gene, thus limiting further entry of LDL-C into cells. [Note: As was seen with the reductase gene (see p. 222), transcriptional regulation of the LDL receptor gene involves an SRE and SREBP-2. This allows coordinate regulation of the expression of these proteins.] Third, if the cholesterol is not required immediately for some structural or synthetic purpose, it is esterified by acyl CoA:cholesterol acyltransferase (ACAT). ACAT transfers a FA from a fatty acyl CoA to cholesterol, producing a cholesteryl ester that can be stored in the cell (Fig. 18.21). The activity of ACAT is enhanced in the presence of increased intracellular cholesterol.


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Biochemistry_Lippinco. 3. Uptake by macrophage scavenger receptors: In addition to the highly specific and regulated receptor-mediated pathway for LDL uptake described above, macrophages possess high levels of scavenger receptor activity. These receptors, known as scavenger receptor class A (SR-A), can bind a broad range of ligands and mediate the endocytosis of chemically modified LDL in which the lipid or apo B component has been oxidized. Unlike the LDL receptor, the scavenger receptor is not downregulated in response to increased intracellular cholesterol. Cholesteryl esters accumulate in macrophages and cause their transformation into “foam” cells, which participate in the formation of atherosclerotic plaque (Fig. 18.22). LDL-C is the primary cause of atherosclerosis. E. High-density lipoprotein metabolism


Biochemistry_Lippinco

Biochemistry_Lippinco. E. High-density lipoprotein metabolism HDL comprise a heterogeneous family of lipoproteins with a complex metabolism that is not yet completely understood. HDL particles are formed in the blood by the addition of lipid to apo A-1, an apolipoprotein made and secreted by the liver and intestine. Apo A-1 accounts for ~70% of the apolipoproteins in HDL. HDL perform a number of important functions, including the following. 1. Apolipoprotein supply: HDL particles serve as a circulating reservoir of apo C-II (the apolipoprotein that is transferred to VLDL and chylomicrons and is an activator of LPL) and apo E (the apolipoprotein required for the receptor-mediated endocytosis of IDL and chylomicron remnants). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Nonesterified cholesterol uptake: Nascent HDL are disc-shaped particles containing primarily phospholipid (largely PC) and apo A, C, and E. They take up cholesterol from nonhepatic (peripheral) tissues and return it to the liver as cholesteryl esters (Fig. 18.23). [Note: HDL particles are excellent acceptors of nonesterified cholesterol as a result of their high concentration of phospholipids, which are important solubilizers of cholesterol.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol esterification: The cholesterol taken up by HDL is immediately esterified by the plasma enzyme lecithin:cholesterol acyltransferase (LCAT, also known as PCAT, in which P stands for phosphatidylcholine, the source of the FA). This enzyme is synthesized and secreted by the liver. LCAT binds to nascent HDL and is activated by apo A-I. LCAT transfers the FA from carbon 2 of PC to cholesterol. This produces a hydrophobic cholesteryl ester, which is sequestered in the core of the HDL, and lysophosphatidylcholine, which binds to albumin. [Note: Esterification maintains the cholesterol concentration gradient, allowing continued efflux of cholesterol to HDL.] As the discoidal nascent HDL accumulates cholesteryl esters, it first becomes a spherical, relatively cholesteryl ester–poor HDL3 and, eventually, a cholesteryl ester–rich HDL2 particle that carries these esters to the liver. Hepatic lipase, which degrades TAG and phospholipids, participates in the conversion of HDL2 to HDL3


Biochemistry_Lippinco

Biochemistry_Lippinco. and, eventually, a cholesteryl ester–rich HDL2 particle that carries these esters to the liver. Hepatic lipase, which degrades TAG and phospholipids, participates in the conversion of HDL2 to HDL3 (see Fig. 18.23). CETP (see p. 231) transfers some of the cholesteryl esters from HDL to VLDL in exchange for TAG, relieving product inhibition of LCAT. Because VLDL are catabolized to LDL, the cholesteryl esters transferred by CETP are ultimately taken up by the liver (see p. 231).


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Biochemistry_Lippinco. 4.


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Biochemistry_Lippinco. Reverse cholesterol transport: The selective transfer of cholesterol from peripheral cells to HDL and from HDL to the liver for bile acid synthesis or disposal via the bile is a key component of cholesterol homeostasis. This process of reverse cholesterol transport (RCT) is, in part, the basis for the inverse relationship seen between plasma HDL concentration and atherosclerosis and for the designation of HDL as the “good” cholesterol carrier. [Note: Exercise and estrogen raise HDL levels.] RCT involves efflux of cholesterol from peripheral cells to HDL, esterification of the cholesterol by LCAT, binding of the cholesteryl ester–rich HDL (HDL2) to liver (and, perhaps, steroidogenic cells), selective transfer of the cholesteryl esters into these cells, and release of lipid-depleted HDL (HDL3). The efflux of cholesterol from peripheral cells is mediated primarily by the transport protein ABCA1. [Note: Tangier disease is a very rare deficiency of ABCA1 and is characterized by the virtual


Biochemistry_Lippinco

Biochemistry_Lippinco. The efflux of cholesterol from peripheral cells is mediated primarily by the transport protein ABCA1. [Note: Tangier disease is a very rare deficiency of ABCA1 and is characterized by the virtual absence of HDL particles due to degradation of lipid-poor apo A-1.] Cholesteryl ester uptake by the liver is mediated by the cell-surface receptor SR-B1 (scavenger receptor class B type 1) that binds HDL (see p. 232 for SR-A receptors). The HDL particle itself is not taken up. Instead, there is selective uptake of the cholesteryl ester from the HDL particle. [Note: Low HDL-C is a risk factor for atherosclerosis.]


Biochemistry_Lippinco

Biochemistry_Lippinco. ABCA1 is an ATP-binding cassette (ABC) protein. ABC proteins use energy from ATP hydrolysis to transport materials, including lipids, in and out of cells and across intracellular compartments. In addition to Tangier disease, defects in specific ABC proteins result in sitosterolemia, cystic fibrosis, X-linked adrenoleukodystrophy, respiratory distress syndrome due to decreased surfactant secretion, and liver disease due to decreased bile salt secretion. F. Lipoprotein (a) and heart disease


Biochemistry_Lippinco

Biochemistry_Lippinco. Lipoprotein (a), or Lp(a), is nearly identical in structure to an LDL particle. Its distinguishing feature is the presence of an additional apolipoprotein molecule, apo(a), which is covalently linked at a single site to apo B-100. Circulating levels of Lp(a) are determined primarily by genetics. However, factors such as diet may play some role, as trans FA have been reported to increase it. The physiologic function of Lp(a) is unknown. When present in large quantities in the plasma, Lp(a) is associated with an increased risk of coronary heart disease. [Note: Apo(a) is structurally homologous to plasminogen, the precursor of a blood protease whose target is fibrin, the main protein component of blood clots (see Chapter 35 online). It is hypothesized that elevated Lp(a) slows the breakdown of blood clots that trigger heart attacks because it competes with plasminogen for binding to fibrin.] Niacin reduces Lp(a), as well as LDL-C and TAG, and raises HDLC. VII. STEROID HORMONES


Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol is the precursor of all classes of steroid hormones: glucocorticoids (for example, cortisol), mineralocorticoids (for example, aldosterone), and the sex hormones (that is, androgens, estrogens, and progestins), as shown in Figure 18.24. [Note: Glucocorticoids and mineralocorticoids are collectively called corticosteroids.] Synthesis and secretion occur in the adrenal cortex (cortisol, aldosterone, and androgens), ovaries and placenta (estrogens and progestins), and testes (testosterone). Steroid hormones are transported by the blood from their sites of synthesis to their target organs. Because of their hydrophobicity, they must be complexed with a plasma protein. Albumin can act as a nonspecific carrier and does carry aldosterone. However, specific steroid-carrier plasma proteins bind the steroid hormones more tightly than does albumin (for example, corticosteroid-binding globulin, or transcortin, is responsible for transporting cortisol). A number of genetic diseases are


Biochemistry_Lippinco

Biochemistry_Lippinco. bind the steroid hormones more tightly than does albumin (for example, corticosteroid-binding globulin, or transcortin, is responsible for transporting cortisol). A number of genetic diseases are caused by deficiencies in specific steps in the biosynthesis of steroid hormones. Some representative diseases are described in Figure 18.25.


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A. Synthesis

Biochemistry_Lippinco. A. Synthesis


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Biochemistry_Lippinco. Synthesis involves shortening the hydrocarbon chain of cholesterol and hydroxylating the steroid nucleus. The initial and rate-limiting reaction converts cholesterol to the 21-carbon pregnenolone. It is catalyzed by the cholesterol side-chain cleavage enzyme, a cytochrome P450 (CYP) mixed function oxidase of the inner mitochondrial membrane (see p. 149) that is also known as P450scc and desmolase. NADPH and O2 are required for the reaction. The cholesterol substrate can be newly synthesized, taken up from lipoproteins, or released by an esterase from cholesteryl esters stored in the cytosol of steroidogenic tissues. The cholesterol moves to the outer mitochondrial membrane. An important control point is the subsequent movement from the outer to the inner mitochondrial membrane. This process is mediated by StAR (steroidogenic acute regulatory) protein. Pregnenolone is the parent compound for all steroid hormones (see Fig. 18.25). It is oxidized and then isomerized to progesterone,


Biochemistry_Lippinco

Biochemistry_Lippinco. is mediated by StAR (steroidogenic acute regulatory) protein. Pregnenolone is the parent compound for all steroid hormones (see Fig. 18.25). It is oxidized and then isomerized to progesterone, which is further modified to the other steroid hormones by CYP protein–catalyzed hydroxylation reactions in the SER and mitochondria. A defect in the activity or amount of an enzyme in this pathway can lead to a deficiency in the synthesis of hormones beyond the affected step and to an excess in the hormones or metabolites before that step. Because all members of the pathway have potent biologic activity, serious metabolic imbalances occur with enzyme deficiencies (see Fig. 18.25). Collectively, these disorders are known as the congenital adrenal hyperplasias (CAH), because they result in enlarged adrenals. [Note: Addison disease, due to autoimmune destruction of the adrenal cortex, is characterized by adrenocortical insufficiency.]


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B. Adrenal cortical steroid hormones

Biochemistry_Lippinco. B. Adrenal cortical steroid hormones


Biochemistry_Lippinco

Biochemistry_Lippinco. Steroid hormones are synthesized and secreted in response to hormonal signals. The corticosteroids and androgens are made in different regions of the adrenal cortex and are secreted into blood in response to different signals. [Note: The adrenal medulla makes catecholamines (see p. 285).] 1. Cortisol: Its production in the middle layer (zona fasciculata) of the adrenal cortex is controlled by the hypothalamus, to which the pituitary gland is attached (Fig. 18.26). In response to severe stress (for example, infection), corticotropin-releasing hormone (CRH), produced by the hypothalamus, travels through capillaries to the anterior lobe of the pituitary, where it induces the production and secretion of adrenocorticotropic hormone (ACTH), a peptide. ACTH stimulates the adrenal cortex to synthesize and secrete the glucocorticoid cortisol, the stress hormone. [Note: ACTH binds to a membrane G protein–coupled receptor, resulting in cyclic AMP (cAMP) production and activation of protein


Biochemistry_Lippinco

Biochemistry_Lippinco. synthesize and secrete the glucocorticoid cortisol, the stress hormone. [Note: ACTH binds to a membrane G protein–coupled receptor, resulting in cyclic AMP (cAMP) production and activation of protein kinase A ([PKA] see p. 94). PKA phosphorylates and activates both the esterase that converts cholesteryl ester to free cholesterol and StAR protein.] Cortisol allows the body to respond to stress through its effects on intermediary metabolism (for example, increased gluconeogenesis) and the inflammatory and immune responses (which are decreased). As cortisol levels rise, the release of CRH and ACTH is inhibited. [Note: The reduction of cortisol in CAH results in a rise in ACTH that causes adrenal hyperplasia.] 2. Aldosterone: Its production in the outer layer (zona glomerulosa) of the adrenal cortex is induced by a decrease in the plasma Na+/potassium (K+) ratio and by the hormone angiotensin II (Ang-II). Ang-II (an octapeptide) is produced from angiotensin I ([Ang-I] a decapeptide) by


Biochemistry_Lippinco

Biochemistry_Lippinco. cortex is induced by a decrease in the plasma Na+/potassium (K+) ratio and by the hormone angiotensin II (Ang-II). Ang-II (an octapeptide) is produced from angiotensin I ([Ang-I] a decapeptide) by angiotensinconverting enzyme (ACE), an enzyme found predominantly in the lungs but also distributed widely in the body. [Note: Ang-I is produced in the blood by cleavage of an inactive precursor, angiotensinogen, secreted by the liver. Cleavage is catalyzed by renin, made and secreted by the kidneys.] Ang-II binds to cell surface receptors. However, in contrast to ACTH, its effects are mediated through the phosphatidylinositol 4,5bisphosphate pathway (see p. 205) and not by cAMP. Aldosterone’s primary effect is on the kidney tubules, where it stimulates Na+ and water uptake and K+ excretion (Fig. 18.27). [Note: An effect of aldosterone is an increase in blood pressure. Competitive inhibitors of ACE are used to treat renin-dependent hypertension.] 3. Androgens: Both the inner (zona


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Biochemistry_Lippinco. (Fig. 18.27). [Note: An effect of aldosterone is an increase in blood pressure. Competitive inhibitors of ACE are used to treat renin-dependent hypertension.] 3. Androgens: Both the inner (zona reticularis) and middle layers of the adrenal cortex produce androgens, primarily dehydroepiandrosterone and androstenedione. Although adrenal androgens themselves are weak, they are converted by aromatase (CYP19) to testosterone, a stronger androgen, in the testes and to estrogens in the ovaries (primarily) of premenopausal women. [Note: Postmenopausal women produce estrogen at extragonadal sites such as the breast. Aromatase inhibitors are used in the treatment of estrogen-responsive breast cancer in these women.]


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Biochemistry_Lippinco. C. Gonadal steroid hormones The testes and ovaries (gonads) synthesize hormones necessary for sexual differentiation and reproduction. A single hypothalamic-releasing factor, gonadotropin-releasing hormone, stimulates the anterior pituitary to release the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Like ACTH, LH and FSH bind to surface receptors and cause an increase in cAMP. LH stimulates the testes to produce testosterone and the ovaries to produce estrogens and progesterone (see Fig. 18.27). FSH regulates the growth of ovarian follicles and stimulates testicular spermatogenesis. D. Mechanism


Biochemistry_Lippinco

Biochemistry_Lippinco. Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific cytosolic or nuclear receptor. These receptor–ligand complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements [HRE]) in association with coactivator proteins, thereby causing increased transcription of targeted genes (Fig. 18.28). An HRE is found in the promoter or an enhancer element (see p. 440) for genes that respond to a specific steroid hormone, thus insuring coordinated regulation of these genes. Hormone–receptor complexes can also inhibit transcription in association with corepressors. [Note: The binding of a hormone to its receptor causes a conformational change in the receptor that uncovers its DNA-binding domain, allowing the complex to interact through a zinc finger motif with the appropriate DNA sequence. Receptors for the steroid hormones, plus those for thyroid hormone, retinoic acid (see p. 386), and


Biochemistry_Lippinco

Biochemistry_Lippinco. allowing the complex to interact through a zinc finger motif with the appropriate DNA sequence. Receptors for the steroid hormones, plus those for thyroid hormone, retinoic acid (see p. 386), and 1,25-dihydroxycholecalciferol (vitamin D; see p. 390), are members of a superfamily of structurally related gene regulators that function in a similar way.]


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Biochemistry_Lippinco. E. Further metabolism Steroid hormones are generally converted into inactive metabolic excretion products in the liver. Reactions include reduction of unsaturated bonds and the introduction of additional hydroxyl groups. The resulting structures are made more soluble by conjugation with glucuronic acid or sulfate (from 3′phosphoadenosyl-5′-phosphosulfate; see p. 162). These conjugated metabolites are fairly water soluble and do not need protein carriers. They are eliminated in feces and urine. VIII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol is a hydrophobic compound, with a single hydroxyl group located at carbon 3 of the A ring, to which a fatty acid (FA) can be attached, producing an even more hydrophobic cholesteryl ester. Cholesterol is synthesized by virtually all human tissues, although primarily by the liver, intestine, adrenal cortex, and reproductive tissues (Fig. 18.29). All the carbon atoms are provided by acetyl coenzyme A (CoA), and nicotinamide adenine dinucleotide phosphate provides the reducing equivalents. The pathway is driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phosphate bond of ATP. Synthesis requires enzymes of the cytosol, smooth endoplasmic reticulum (SER), and peroxisomes. The rate-limiting and regulated step in cholesterol synthesis is catalyzed by the SER-membrane protein hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, which produces mevalonate from HMG CoA. The enzyme is regulated by a number of mechanisms: 1) increased expression of


Biochemistry_Lippinco

Biochemistry_Lippinco. by the SER-membrane protein hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, which produces mevalonate from HMG CoA. The enzyme is regulated by a number of mechanisms: 1) increased expression of the reductase gene when cholesterol levels are low, via the transcription factor, sterol regulatory element–binding protein-2 (SREBP-2), bound to a sterol regulatory element (SRE), resulting in increased enzyme and, therefore, cholesterol, synthesis; 2) accelerated degradation of the reductase protein when cholesterol levels are high; 3) phosphorylation (causing inactivation of reductase activity) by adenosine monophosphate–activated protein kinase [AMPK] and dephosphorylation (activation) by a phosphoprotein phosphatase; and 4) hormonal regulation by insulin and glucagon. Statins are competitive inhibitors of HMG CoA reductase. These drugs are used to decrease plasma cholesterol in patients with hypercholesterolemia. The ring structure of cholesterol cannot be degraded in humans.


Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol is eliminated from the body either by conversion to bile salts or by secretion into the bile. Bile salts and phosphatidylcholine (PC) are quantitatively the most important organic components of bile. The rate-limiting step in bile acid synthesis is catalyzed by cholesterol-7-αhydroxylase, which is inhibited by bile acids. Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine, producing the conjugated bile salts glycocholic or taurocholic acid and glycochenodeoxycholic or taurochenodeoxycholic acid. Bile salts (deprotonated) are more amphipathic than bile acids (protonated) and, therefore, are more effective emulsifiers of dietary fat. Intestinal bacteria can remove the glycine and taurine as well as a hydroxyl group from the steroid nucleus, producing the secondary bile salts, deoxycholic and lithocholic acids. Bile salts are efficiently reabsorbed (>95%) in the intestinal ileum by a sodium–bile salt cotransporter, returned


Biochemistry_Lippinco

Biochemistry_Lippinco. nucleus, producing the secondary bile salts, deoxycholic and lithocholic acids. Bile salts are efficiently reabsorbed (>95%) in the intestinal ileum by a sodium–bile salt cotransporter, returned to the blood, and carried by albumin back to the liver where they are taken up by the hepatic isoform of the cotransporter and reused (enterohepatic circulation, which bile acid sequestrants reduce). If more cholesterol enters the bile than can be solubilized by the available bile salts and PC, cholesterol gallstone disease (cholelithiasis) can occur.


Biochemistry_Lippinco

Biochemistry_Lippinco. The plasma lipoproteins (see Fig. 18.29) include chylomicrons, verylow-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They function to keep lipids (primarily triacylglycerol [TAG] and cholesteryl esters) soluble as they transport them between tissues. Lipoproteins are composed of a neutral lipid (TAG, cholesteryl esters, or both) core surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified cholesterol. Chylomicrons are assembled in intestinal mucosal cells from dietary lipids (primarily TAG). Each nascent chylomicron particle has one molecule of apolipoprotein (apo) B-48. They are released from the cells into the lymphatic system and travel to the blood, where they receive apo C-II and apo E from HDL. Apo C-II activates endothelial lipoprotein lipase (LPL), which degrades the TAG in chylomicrons to FA and glycerol. The FA that are released are stored (in adipose


Biochemistry_Lippinco

Biochemistry_Lippinco. apo C-II and apo E from HDL. Apo C-II activates endothelial lipoprotein lipase (LPL), which degrades the TAG in chylomicrons to FA and glycerol. The FA that are released are stored (in adipose tissue) or used for energy (in muscle). The glycerol is metabolized by the liver. Patients with a deficiency of LPL or apo C-II show a dramatic accumulation of chylomicrons in the plasma (type I hyperlipoproteinemia or familial chylomicronemia) even if fasted. After most of the TAG is removed, apo C-II is returned to HDL, and the chylomicron remnant, carrying most of the dietary cholesterol, binds to a liver receptor that recognizes apo E. The particle is endocytosed, and its contents degraded by lysosomal enzymes. Defective uptake of these remnants (and IDL) causes type III hyperlipoproteinemia or dysbetalipoproteinemia. Nascent VLDL are produced in the liver and are composed predominantly of TAG. They contain a single molecule of apo B-100. Like chylomicrons, VLDL receive apo C-II and apo E


Biochemistry_Lippinco

Biochemistry_Lippinco. dysbetalipoproteinemia. Nascent VLDL are produced in the liver and are composed predominantly of TAG. They contain a single molecule of apo B-100. Like chylomicrons, VLDL receive apo C-II and apo E from HDL in the plasma. VLDL carry hepatic TAG to the peripheral tissues where LPL degrades the lipid. Additionally, the VLDL particle receives cholesteryl esters from HDL in exchange for TAG. This process is accomplished by cholesteryl ester transfer protein (CETP). VLDL in the plasma is first converted to IDL and then to LDL, a much smaller, denser particle. Apo C-II and apo E are returned to HDL, but the LDL retains apo B-100, which is recognized by receptors on peripheral tissues and the liver. LDL undergo receptor-mediated endocytosis, and their contents are degraded in the lysosomes. The protease proprotein convertase subtilisin/kexin type 9 (PCSK9) prevents receptor recycling. Defects in the synthesis of functional LDL receptors causes type IIa hyperlipoproteinemia (familial


Biochemistry_Lippinco

Biochemistry_Lippinco. The protease proprotein convertase subtilisin/kexin type 9 (PCSK9) prevents receptor recycling. Defects in the synthesis of functional LDL receptors causes type IIa hyperlipoproteinemia (familial hypercholesterolemia [FH]). The endocytosed cholesterol decreases expression of HMG CoA reductase (and LDL receptors) through prevention of SREBP-2 binding to the SRE. Some of it can be esterified by acyl CoA:cholesterol acyltransferase (ACAT) and stored. HDL are created by lipidation of apo A-1 synthesized in the liver and intestine. They have a number of functions, including 1) serving as a circulating reservoir of apo C-II and apo E for chylomicrons and VLDL; 2) removing cholesterol from peripheral tissues via ABCA1 and esterifying it using lecithin:cholesterol acyl transferase (LCAT), a liver-synthesized plasma enzyme that is activated by apo A-1; and 3) delivering these cholesteryl esters to the liver (reverse cholesterol transport) for uptake via scavenger receptor-B1 (SR-B1).


Biochemistry_Lippinco

Biochemistry_Lippinco. Cholesterol is the precursor of all classes of steroid hormones, which include glucocorticoids, mineralocorticoids, and the sex hormones (androgens, estrogens, and progestins). Synthesis, using primarily cytochrome P450 mixed function oxidases, occurs in the adrenal cortex (cortisol in the zona fasciculata, aldosterone in the zona glomerulosa, and androgens in the zona reticularis), ovaries and placenta (estrogens and progestins), and testes (testosterone). The initial and rate-limiting step is the conversion of cholesterol to pregnenolone by the side-chain cleavage enzyme P450scc. Deficiencies in synthesis lead to congenital adrenal hyperplasia (CAH). Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific intracellular receptor. These receptor–hormone complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements) in association with coactivator proteins, thereby causing increased


Biochemistry_Lippinco

Biochemistry_Lippinco. complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements) in association with coactivator proteins, thereby causing increased transcription of targeted genes. In association with corepressors, transcription is decreased.


Biochemistry_Lippinco

Biochemistry_Lippinco. lipoproteins; TAG = triacylglycerol; NADPH = nicotinamide adenine dinucleotide phosphate; C = carbon. Choose the ONE best answer. 8.1. Mice were genetically engineered to contain hydroxymethylglutaryl coenzyme A reductase in which serine 871, a phosphorylation site, was replaced by alanine. Which of the following statements concerning the modified form of the enzyme is most likely to be correct? A. The enzyme is nonresponsive to ATP depletion. B. The enzyme is nonresponsive to statin drugs. C. The enzyme is nonresponsive to the sterol response element–sterol response element–binding protein system. D. The enzyme is unable to be degraded by the ubiquitin–proteasome system.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. The enzyme is nonresponsive to the sterol response element–sterol response element–binding protein system. D. The enzyme is unable to be degraded by the ubiquitin–proteasome system. Correct answer = A. The reductase is regulated by covalent phosphorylation and dephosphorylation. Depletion of ATP results in a rise in adenosine monophosphate (AMP), which activates AMP kinase (AMPK), thereby phosphorylating and inactivating the reductase. In the absence of the serine, a common phosphorylation site, the enzyme cannot be phosphorylated by AMPK. The enzyme is also regulated physiologically through changes in transcription and degradation and pharmacologically by statin drugs (competitive inhibitors), but none of these depends on serine phosphorylation.


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Biochemistry_Lippinco. 8.2. Calculate the amount of cholesterol in the low-density lipoproteins in an individual whose fasting blood gave the following lipid-panel test results: total cholesterol = 300 mg/dl, high-density lipoprotein cholesterol = 25 mg/dl, triglycerides = 150 mg/dl. A. 55 mg/dl B. 95 mg/dl C. 125 mg/dl D. 245 mg/dl Correct answer = D. The total cholesterol in the blood of a fasted individual is equal to the sum of the cholesterol in low-density lipoproteins plus the cholesterol in high-density lipoproteins plus the cholesterol in very-lowdensity lipoproteins (VLDL). This last term is calculated by dividing the triacylglycerol value by 5 because cholesterol accounts for about 1/5 of the volume of VLDL in fasted blood. For Questions 18.3 and 18.4, use the following scenario.


Biochemistry_Lippinco

Biochemistry_Lippinco. For Questions 18.3 and 18.4, use the following scenario. A young girl with a history of severe abdominal pain was taken to her local hospital at 5 a.m. in severe distress. Blood was drawn, and the plasma appeared milky, with the triacylglycerol level >2,000 mg/dl (normal = 4–150 mg/dl). The patient was placed on a diet extremely limited in fat but supplemented with medium-chain triglycerides. 8.3. Which of the following lipoprotein particles are most likely responsible for the appearance of the patient’s plasma? A. Chylomicrons B. High-density lipoproteins C. Intermediate-density lipoproteins D. Low-density lipoproteins E. Very-low-density lipoproteins


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Chylomicrons B. High-density lipoproteins C. Intermediate-density lipoproteins D. Low-density lipoproteins E. Very-low-density lipoproteins Correct answer = A. The milky appearance of her plasma was a result of triacylglycerol-rich chylomicrons. Because 5 a.m. is presumably several hours after her evening meal, the patient must have difficulty degrading these lipoprotein particles. Intermediate-, low-, and high-density lipoproteins contain primarily cholesteryl esters, and, if one or more of these particles was elevated, it would cause hypercholesterolemia. Very-low-density lipoproteins do not cause the described milky appearance of plasma. 8.4. Which one of the following proteins is most likely to be deficient in this patient? A. Apolipoprotein A-I B. Apolipoprotein B-48 C. Apolipoprotein C-II D. Cholesteryl ester transfer protein E. Microsomal triglyceride transfer protein


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Apolipoprotein A-I B. Apolipoprotein B-48 C. Apolipoprotein C-II D. Cholesteryl ester transfer protein E. Microsomal triglyceride transfer protein Correct answer = C. The triacylglycerol (TAG) in chylomicrons is degraded by endothelial lipoprotein lipase (LPL), which requires apolipoprotein (apo) C-II as a coenzyme. Deficiency of LPL or apo C-II results in decreased ability to degrade chylomicrons to their remnants, which get cleared (via apo E) by liver receptors. Apo A-I is the coenzyme for lecithin:cholesterol acyltransferase; apo B-48 is the characteristic structural protein of chylomicrons; cholesteryl ester transfer protein catalyzes the cholesteryl ester–TAG exchange between high-density and very-low-density lipoproteins (VLDL); and microsomal triglyceride transfer protein is involved in the formation, not degradation, of chylomicrons (and VLDL). 8.5. Complete the table below for an individual with classic 21-α-hydroxylase deficiency relative to a normal individual.


Biochemistry_Lippinco

Biochemistry_Lippinco. 8.5. Complete the table below for an individual with classic 21-α-hydroxylase deficiency relative to a normal individual. How might the results be changed if this individual were deficient in 17-αhydroxylase, rather than 21-α-hydroxylase? Classic 21-α-hydroxylase deficiency causes mineralocorticoids (aldosterone) and glucocorticoids (cortisol) to be virtually absent. Because aldosterone increases blood pressure, and cortisol increases blood glucose, their deficiencies result in a decrease in blood pressure and blood glucose, respectively. Cortisol normally feeds back to inhibit adrenocorticotropic hormone (ACTH) release by the pituitary, and, so, its absence results in an elevation in ACTH. The loss of 21-α-hydroxylase pushes progesterone and pregnenolonetoandrogensynthesisand,therefore,causesandrostenedionelevelstorise.With17-α-hydroxylasedeficiency,sexhormonesynthesiswouldbedecreased.Mineralocorticoidproductionwouldbeincreased,leadingtohypertension.


Biochemistry_Lippinco

Biochemistry_Lippinco. UNIT IV Nitrogen Metabolism Amino Acids: Nitrogen Disposal 19 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Unlike fats and carbohydrates, amino acids are not stored by the body. That is, no protein exists whose sole function is to maintain a supply of amino acids for future use. Therefore, amino acids must be obtained from the diet, synthesized de novo, or produced from the degradation of body protein. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded. The first phase of catabolism involves the removal of the α-amino groups (usually by transamination and subsequent oxidative deamination), forming ammonia and the corresponding α-keto acids, the carbon skeletons of amino acids. A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea (Fig. 19.1), which is quantitatively the most important route for disposing of nitrogen from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the α-keto acids are converted to common intermediates of energy-producing metabolic


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Biochemistry_Lippinco. from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the α-keto acids are converted to common intermediates of energy-producing metabolic pathways. These compounds can be metabolized to carbon dioxide (CO2) and water (H2O), glucose, fatty acids, or ketone bodies by the central pathways of metabolism described in Chapters 8–13 and 16.


Biochemistry_Lippinco

Biochemistry_Lippinco. II. OVERALL NITROGEN METABOLISM Amino acid catabolism is part of the larger process of the metabolism of nitrogen-containing molecules. Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (such as creatinine, see p. 287). The role of body proteins in these transformations involves two important concepts: the amino acid pool and protein turnover. A. Amino acid pool


Biochemistry_Lippinco

Biochemistry_Lippinco. Free amino acids are present throughout the body, such as in cells, blood, and the extracellular fluids. For the purpose of this discussion, envision all of these amino acids as if they belonged to a single entity, called the amino acid pool. This pool is supplied by three sources: 1) amino acids provided by the degradation of endogenous (body) proteins, most of which are reutilized; 2) amino acids derived from exogenous (dietary) protein; and 3) nonessential amino acids synthesized from simple intermediates of metabolism (Fig. 19.2). Conversely, the amino acid pool is depleted by three routes: 1) synthesis of body protein, 2) consumption of amino acids as precursors of essential nitrogen-containing small molecules, and 3) conversion of amino acids to glucose, glycogen, fatty acids, and ketone bodies or oxidation to CO2 + H2O (see Fig. 19.2). Although the amino acid pool is small (comprising ~90–100 g of amino acids) in comparison with the amount of protein in the body (~12 kg in a


Biochemistry_Lippinco

Biochemistry_Lippinco. ketone bodies or oxidation to CO2 + H2O (see Fig. 19.2). Although the amino acid pool is small (comprising ~90–100 g of amino acids) in comparison with the amount of protein in the body (~12 kg in a 70-kg man), it is conceptually at the center of whole-body nitrogen metabolism.


Biochemistry_Lippinco

Biochemistry_Lippinco. In healthy, well-fed individuals, the input to the amino acid pool is balanced by the output. That is, the amount of amino acids contained in the pool is constant. The amino acid pool is said to be in a steady state, and the individual is said to be in nitrogen balance (see p. 367). B. Protein turnover Most proteins in the body are constantly being synthesized and then degraded (turned over), permitting the removal of abnormal or unneeded proteins. For many proteins, regulation of synthesis determines the concentration of protein in the cell, with protein degradation assuming a minor role. For other proteins, the rate of synthesis is constitutive (that is, essentially constant), and cellular levels of the protein are controlled by selective degradation. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Rate: In healthy adults, the total amount of protein in the body remains constant because the rate of protein synthesis is just sufficient to replace the protein that is degraded. This process, called protein turnover, leads to the hydrolysis and resynthesis of 300–400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins (for example, many regulatory proteins and misfolded proteins) are rapidly degraded, having half-lives measured in minutes or hours. Long-lived proteins, with half-lives of days to weeks, constitute the majority of proteins in the cell. Structural proteins, such as collagen, are metabolically stable and have half-lives measured in months or years. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Protein degradation: There are two major enzyme systems responsible for degrading proteins: the ATP-dependent ubiquitin (Ub)–proteasome system of the cytosol and the ATP-independent degradative enzyme system of the lysosomes. Proteasomes selectively degrade damaged or short-lived proteins. Lysosomes use acid hydrolases (see p. 162) to nonselectively degrade intracellular proteins (autophagy) and extracellular proteins (heterophagy), such as plasma proteins, that are taken into the cell by endocytosis.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Ubiquitin–proteasome system: Proteins selected for degradation by the cytosolic ubiquitin–proteasome system are first modified by the covalent attachment of Ub, a small, globular, nonenzymic protein that is highly conserved across eukaryotic species. Ubiquitination of the target substrate occurs through isopeptide linkage of the α-carboxyl group of the C-terminal glycine of Ub to the ε-amino group of a lysine in the protein substrate by a three-step, enzyme-catalyzed, ATP-dependent process. [Note: Enzyme 1 (E1, an activating enzyme) activates Ub, which is then transferred to E2 (a conjugating enzyme). E3 (a ligase) identifies the protein to be degraded and interacts with E2-Ub. There are many more E3 proteins than there are E1 or E2.] The consecutive addition of four or more Ub molecules to the target protein generates a polyubiquitin chain. Proteins tagged with Ub chains are recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome (Fig. 19.3).


Biochemistry_Lippinco

Biochemistry_Lippinco. to the target protein generates a polyubiquitin chain. Proteins tagged with Ub chains are recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome (Fig. 19.3). The proteasome unfolds, deubiquitinates, and cuts the target protein into fragments that are then further degraded by cytosolic proteases to amino acids, which enter the amino acid pool. The Ub is recycled. It is noteworthy that the selective degradation of proteins by the ubiquitin–proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires ATP hydrolysis.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. Degradation signals: Because proteins have different half-lives, it is clear that protein degradation cannot be random but, rather, is influenced by some structural aspect of the protein that serves as a degradation signal, which is recognized and bound by an E3. The half-life of a protein is also influenced by the amino (N)-terminal residue, the so-called N-end rule, and ranges from minutes to hours. Destabilizing N-terminal amino acids include arginine and posttranslationally modified amino acids such as acetylated alanine. In contrast, serine is a stabilizing amino acid. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly ubiquitinated and degraded and, therefore, have short half-lives. III. DIETARY PROTEIN DIGESTION


Biochemistry_Lippinco

Biochemistry_Lippinco. III. DIETARY PROTEIN DIGESTION Most of the nitrogen in the diet is consumed in the form of protein, typically amounting to 70–100 g/day in the American diet (see Fig. 19.2). Proteins are generally too large to be absorbed by the intestine. [Note: An example of an exception to this rule is that newborns can take up maternal antibodies in breast milk.] Therefore, proteins must be hydrolyzed to yield di-and tripeptides as well as individual amino acids, which can be absorbed. Proteolytic enzymes responsible for degrading proteins are produced by three different organs: the stomach, the pancreas, and the small intestine (Fig. 19.4). A. Digestion by gastric secretion The digestion of proteins begins in the stomach, which secretes gastric juice, a unique solution containing hydrochloric acid (HCl) and the proenzyme pepsinogen. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. The digestion of proteins begins in the stomach, which secretes gastric juice, a unique solution containing hydrochloric acid (HCl) and the proenzyme pepsinogen. 1. Hydrochloric acid: Stomach HCl is too dilute (pH 2–3) to hydrolyze proteins. The acid, secreted by the parietal cells of the stomach, functions instead to kill some bacteria and to denature proteins, thereby making them more susceptible to subsequent hydrolysis by proteases. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Pepsin: This acid-stable endopeptidase is secreted by the chief cells of the stomach as an inactive zymogen (or proenzyme), pepsinogen. [Note: In general, zymogens contain extra amino acids in their sequences that prevent them from being catalytically active. Removal of these amino acids permits the proper folding required for an active enzyme.] In the presence of HCl, pepsinogen undergoes a conformational change that allows it to cleave itself (autocatalysis) to the active form, pepsin, which releases polypeptides and a few free amino acids from dietary proteins. B. Digestion by pancreatic enzymes


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Digestion by pancreatic enzymes On entering the small intestine, the polypeptides produced in the stomach by the action of pepsin are further cleaved to oligopeptides and amino acids by a group of pancreatic proteases that include both endopeptidases (that cleave within) and exopeptidases (that cut at an end). [Note: Bicarbonate (HCO3−), secreted by the pancreas in response to the intestinal hormone secretin, raises the intestinal pH.] 1. Specificity: Each of these enzymes has a different specificity for the amino acid R-groups adjacent to the susceptible peptide bond (Fig. 19.5). For example, trypsin cleaves only when the carbonyl group of the peptide bond is contributed by arginine or lysine. These enzymes, like pepsin described above, are synthesized and secreted as inactive zymogens. 2. Zymogen release: The release and activation of the pancreatic zymogens are mediated by the secretion of cholecystokinin, a polypeptide hormone of the small intestine (see p. 176). 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Zymogen release: The release and activation of the pancreatic zymogens are mediated by the secretion of cholecystokinin, a polypeptide hormone of the small intestine (see p. 176). 3. Zymogen activation: Enteropeptidase (also called enterokinase), a serine protease synthesized by and present on the luminal (apical) surface of intestinal mucosal cells (enterocytes) of the brush border, converts the pancreatic zymogen trypsinogen to trypsin by removal of a hexapeptide from the N-terminus of trypsinogen. Trypsin subsequently converts other trypsinogen molecules to trypsin by cleaving a limited number of specific peptide bonds in the zymogen. Thus, enteropeptidase unleashes a cascade of proteolytic activity because trypsin is the common activator of all the pancreatic zymogens (see Fig. 19.5). 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Digestion abnormalities: In individuals with a deficiency in pancreatic secretion (for example, because of chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas), the digestion and absorption of fat and protein are incomplete. This results in the abnormal appearance of lipids in the feces (a condition called steatorrhea; see p. 177) as well as undigested protein. Celiac disease (celiac sprue) is a disease of malabsorption resulting from immune-mediated damage to the small intestine in response to ingestion of gluten (or gliadin produced from gluten), a protein found in wheat, barley, and rye. C. Digestion of oligopeptides by small intestine enzymes The luminal surface of the enterocytes contains aminopeptidase, an exopeptidase that repeatedly cleaves the N-terminal residue from oligopeptides to produce even smaller peptides and free amino acids. D. Amino acid and small peptide intestinal absorption


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Amino acid and small peptide intestinal absorption Most free amino acids are taken into enterocytes via sodium-dependent secondary active transport by solute carrier (SLC) proteins of the apical membrane. At least seven different transport systems with overlapping amino acid specificities are known. Di-and tripeptides, however, are taken up by a proton-linked peptide transporter (PepT1). The peptides are then hydrolyzed to free amino acids. Regardless of their source, free amino acids are released from enterocytes into the portal system by sodium-independent transporters of the basolateral membrane. Therefore, only free amino acids are found in the portal vein after a meal containing protein. These amino acids are either metabolized by the liver or released into the general circulation. [Note: Branched-chain amino acids (BCAA) are not metabolized by the liver but, instead, are sent from the liver to muscle via the blood.] E. Absorption abnormalities


Biochemistry_Lippinco

Biochemistry_Lippinco. The small intestine and the proximal tubules of the kidneys have common transport systems for amino acid uptake. Consequently, a defect in any one of these systems results in an inability to absorb particular amino acids into the intestine and into the kidney tubules. For example, one system is responsible for the uptake of cystine and the dibasic amino acids ornithine, arginine, and lysine (represented as COAL). In the inherited disorder cystinuria, this carrier system is defective, and all four amino acids appear in the urine (Fig. 19.6). Cystinuria occurs at a frequency of 1 in 7,000 individuals, making it one of the most common inherited diseases and the most common genetic error of amino acid transport. The disease expresses itself clinically by the precipitation of cystine to form kidney stones (calculi), which can block the urinary tract. Oral hydration is an important part of treatment for this disorder. [Note: Defects in the uptake of tryptophan by a neutral amino acid


Biochemistry_Lippinco

Biochemistry_Lippinco. form kidney stones (calculi), which can block the urinary tract. Oral hydration is an important part of treatment for this disorder. [Note: Defects in the uptake of tryptophan by a neutral amino acid transporter can result in Hartnup disorder and pellagra-like (see p. 384) dermatologic and neurologic symptoms.]


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. NITROGEN REMOVAL FROM AMINO ACIDS The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the α-amino group is essential for producing energy from any amino acid and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted as urea, with the carbon skeletons being metabolized. This section describes transamination and oxidative deamination, reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen (see p. 253). A. Transamination: Funneling amino groups to glutamate


Biochemistry_Lippinco

Biochemistry_Lippinco. The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate (Fig. 19.7), producing an α-keto acid (derived from the original amino acid) and glutamate. α-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thereby becoming glutamate. Glutamate produced by transamination can be oxidatively deaminated (see B. below) or used as an amino group donor in the synthesis of nonessential amino acids. This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (also called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body. All amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino groups by deamination (see pp. 265–266).] 1. Substrate specificity: Each


Biochemistry_Lippinco

Biochemistry_Lippinco. threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino groups by deamination (see pp. 265–266).] 1. Substrate specificity: Each aminotransferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always α-ketoglutarate. Two important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as shown in Figure 19.8.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Alanine aminotransferase: ALT is present in many tissues. The enzyme catalyzes the transfer of the amino group of alanine to α-ketoglutarate, resulting in the formation of pyruvate and glutamate. The reaction is readily reversible. However, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. [Note: In effect, glutamate acts as a collector of nitrogen from most amino acids.] b. Aspartate aminotransferase: AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST primarily transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle (see p. 255). Like other transaminations, the AST reaction is reversible.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Mechanism: All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B6; see p. 382), which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme. Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an α-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme. Figure 19.9 shows these two component reactions for the transamination catalyzed by AST. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Equilibrium: For most transamination reactions, the equilibrium constant is near 1. This allows the reaction to function in both amino acid degradation through removal of α-amino groups (for example, after consumption of a protein-rich meal) and biosynthesis of nonessential amino acids through addition of amino groups to the carbon skeletons of α-keto acids (for example, when the supply of amino acids from the diet is not adequate to meet the synthetic needs of cells). 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Diagnostic value: Aminotransferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contents during normal cell turnover. Elevated plasma levels of aminotransferases indicate damage to cells rich in these enzymes. For example, physical trauma or a disease process can cause cell lysis, resulting in release of intracellular enzymes into the blood. Two aminotransferases, AST and ALT, are of particular diagnostic value when they are found in the plasma. a.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Hepatic disease: Plasma AST and ALT are elevated in nearly all hepatic diseases but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. ALT is more specific than AST for liver disease, but the latter is more sensitive because the liver contains larger amounts of AST. Serial measurements of AST and ALT (liver function tests) are often useful in determining the course of liver damage. Figure 19.10 shows the early release of ALT into the blood, following ingestion of a liver toxin. [Note: The elevation in bilirubin results from hepatocellular damage that decreases the hepatic conjugation and excretion of bilirubin (see p. 282).] b. Nonhepatic disease: Aminotransferases may be elevated in nonhepatic diseases such as those that cause damage to cardiac or skeletal muscle. However, these disorders can usually be distinguished clinically from liver disease.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Oxidative deamination: Amino group removal


Biochemistry_Lippinco

Biochemistry_Lippinco. In contrast to transamination reactions that transfer amino groups, oxidative deamination reactions result in the liberation of the amino group as free ammonia (Fig. 19.11). These reactions occur primarily in the liver and kidney. They provide α-keto acids that can enter the central pathways of energy metabolism and ammonia, which is a source of nitrogen in hepatic urea synthesis. [Note: Ammonia exists primarily as ammonium (NH4+) in aqueous solution, but it is the unionized form (NH3) that crosses membranes.] 1. Glutamate dehydrogenase: As described above, the amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with α-ketoglutarate. Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination, a reaction catalyzed by glutamate dehydrogenase ([GDH], see Fig. 19.11). Therefore, the sequential action of transamination (resulting in the transfer of amino groups from most amino acids to α-ketoglutarate to


Biochemistry_Lippinco

Biochemistry_Lippinco. catalyzed by glutamate dehydrogenase ([GDH], see Fig. 19.11). Therefore, the sequential action of transamination (resulting in the transfer of amino groups from most amino acids to α-ketoglutarate to produce glutamate) and the oxidative deamination of that glutamate (regenerating α-ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Coenzymes: GDH, a mitochondrial enzyme, is unusual in that it can use either nicotinamide adenine dinucleotide (NAD+) or its phosphorylated reduced form (NADPH) as a coenzyme (see Fig. 19.11). NAD+ is used primarily in oxidative deamination (the simultaneous loss of ammonia coupled with the oxidation of the carbon skeleton, as shown in Fig. 19.12A), whereas NADPH is used in reductive amination (the simultaneous gain of ammonia coupled with the reduction of the carbon skeleton, as shown in Fig. 19.12B). NADP(H) = nicotinamide adenine dinucleotide phosphate. b.


Biochemistry_Lippinco

Biochemistry_Lippinco. NADP(H) = nicotinamide adenine dinucleotide phosphate. b. Reaction direction: The direction of the reaction depends on the relative concentrations of glutamate, α-ketoglutarate, and ammonia and the ratio of oxidized to reduced coenzymes. For example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia (see Fig. 19.12A). High ammonia levels are required to drive the reaction to glutamate synthesis. c. Allosteric regulators: Guanosine triphosphate is an allosteric inhibitor of GDH, whereas adenosine diphosphate is an activator. Therefore, when energy levels are low in the cell, amino acid degradation by GDH is high, facilitating energy production from the carbon skeletons derived from amino acids.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. d-Amino acid oxidase: D-Amino acids (see p. 5) are supplied by the diet but are not used in the synthesis of mammalian proteins. They are, however, efficiently metabolized to α-keto acids, ammonia, and hydrogen peroxide in the peroxisomes of liver and kidney cells by flavin adenine dinucleotide–dependent D-amino acid oxidase (DAO). The αketo acids can enter the general pathways of amino acid metabolism and be reaminated to L-isomers or catabolized for energy. [Note: DAO degrades D-serine, the isomeric form of serine that modulates N-methylD-aspartate (NMDA)-type glutamate receptors. Increased DAO activity has been linked to increased susceptibility to schizophrenia. DAO also converts glycine to glyoxylate (see p. 263).] L-Amino acid oxidases are found in snake venom. C. Ammonia transport to the liver


Biochemistry_Lippinco

Biochemistry_Lippinco. Two mechanisms are available in humans for the transport of ammonia from peripheral tissues to the liver for conversion to urea. Both are important in, but not exclusive to, skeletal muscle. The first uses glutamine synthetase to combine ammonia with glutamate to form glutamine, a nontoxic transport form of ammonia (Fig. 19.13). The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to glutamate and ammonia (see p. 256). The glutamate is oxidatively deaminated to ammonia and α-ketoglutarate by GDH. The ammonia is converted to urea. The second transport mechanism involves the formation of alanine by the transamination of pyruvate produced from both aerobic glycolysis and metabolism of the succinyl coenzyme A (CoA) generated by the catabolism of the BCAA isoleucine and valine. Alanine is transported in the blood to the liver, where it is transaminated by ALT to pyruvate. The pyruvate is used to synthesize glucose, which can enter the blood and be used


Biochemistry_Lippinco

Biochemistry_Lippinco. and valine. Alanine is transported in the blood to the liver, where it is transaminated by ALT to pyruvate. The pyruvate is used to synthesize glucose, which can enter the blood and be used by muscle, a pathway called the glucose–alanine cycle. The glutamate product of ALT can be deaminated by GDH, generating ammonia. Thus, both alanine and glutamine carry ammonia to the liver.


Biochemistry_Lippinco

Biochemistry_Lippinco. V. UREA CYCLE is the major disposal form of amino groups derived from amino acids and accounts for ~90% of the nitrogen-containing components of urine. One nitrogen of the urea molecule is supplied by free ammonia and the other nitrogen by aspartate. [Note: Glutamate is the immediate precursor of both ammonia (through oxidative deamination by GDH) and aspartate nitrogen (through transamination of oxaloacetate by AST).] The carbon and oxygen of urea are derived from CO2 (as HCO3−). Urea is produced by the liver and then is transported in the blood to the kidneys for excretion in the urine. A. Reactions The first two reactions leading to the synthesis of urea occur in the mitochondrial matrix, whereas the remaining cycle enzymes are located in the cytosol (Fig. 19.14). [Note: Gluconeogenesis (see p. 117) and heme synthesis (see p. 278) also involve both the mitochondrial matrix and the cytosol.]


Biochemistry_Lippinco

Biochemistry_Lippinco. Pi = inorganic phosphate; NAD(H) = nicotinamide adenine dinucleotide; MD = malate dehydrogenase. 1. Carbamoyl phosphate formation: Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I (CPS I) is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial GDH (see Fig. 19.11). Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. CPS I requires N-acetylglutamate (NAG) as a positive allosteric activator (see Fig. 19.14). [Note: Carbamoyl phosphate synthetase II participates in the biosynthesis of pyrimidines (see p. 302). It does not require NAG, uses glutamine as the nitrogen source, and occurs in the cytosol.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Citrulline formation: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamylase (OTC) as the phosphate is released as inorganic phosphate. The reaction product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline move across the inner mitochondrial membrane via an antiporter. These basic amino acids are not incorporated into cellular proteins because there are no codons for them (see p. 447).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the tricarboxylic acid (TCA) cycle (see p. 109). 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Argininosuccinate formation: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea. 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Argininosuccinate cleavage: Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The arginine serves as the immediate precursor of urea. The fumarate is hydrated to malate, providing a link with several metabolic pathways. Malate can be oxidized by malate dehydrogenase to oxaloacetate, which can be transaminated to aspartate (see Fig. 19.8) and enter the urea cycle (see Fig. 19.14). Alternatively, malate can be transported into mitochondria via the malate–aspartate shuttle (see p. 80), reenter the TCA cycle, and get oxidized to oxaloacetate, which can be used for gluconeogenesis (see p. 120). [Note: Malate oxidation generates NADH for oxidative phosphorylation (see p. 77), thereby reducing the energy cost of the urea cycle.] 5.