The concepts of conformation and dynamics developed in Sections 1 through

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1 SECTIN 6 Basic Concepts and Design of Metabolism Learning bjectives ow are dietary proteins, carbohydrates and lipids digested? ow is the release of pancreatic enzymes coordinated with digestion in the stomach? Why is ATP an energy-rich molecule? ow can ATP power reactions that would otherwise not take place? What is the relation between the oxidation state of a carbon molecule and its usefulness as a fuel? The concepts of conformation and dynamics developed in Sections 1 through 5 especially those dealing with the specificity and catalytic power of enzymes, the regulation of their catalytic activity, and the transport of molecules and ions across membranes enable us to now ask questions fundamental to biochemistry: 1. ow does a cell extract energy and reducing power from its environment? 2. ow does a cell synthesize the building blocks of its macromolecules and then the macromolecules themselves? These processes are carried out by a highly integrated network of chemical reactions that are collectively known as metabolism or intermediary metabolism.

2 Metabolism can be subdivided into two categories: catabolism and anabolism. Catabolism is the set of reactions that extract biologically useful energy from environmental sources, such as meals. Anabolism is the set of reactions that use biological useful energy to synthesize new biomolecules, supramolecular complexes, and cells themselves. More than a thousand chemical reactions take place in even as simple an organism as Escherichia coli. The array of reactions may seem overwhelming at first glance. owever, closer scrutiny reveals that metabolism has a coherent design containing many common motifs. These motifs include the use of an energy currency and the repeated appearance of a limited number of activated intermediates. In fact, a group of about 100 molecules play central roles in and are processed using similar pathways in all forms of life. Moreover, these metabolic pathways are also regulated in common ways. Before a cell can begin the metabolism of a molecule for anabolic or catabolic purposes, the molecule must be made accessible to the cell. In higher organisms such as human beings, converting meals into accessible biomolecules begins in the digestive track with the biochemical process of digestion. We begin this section with an investigation of how meals dietary forms of the biochemicals required for survival are converted into biochemicals that enter metabolic pathways. Next, we will examine some general principles and motifs of metabolism to provide a foundation for the more detailed studies to follow. Chapter 13: Digestion Chapter 14: Metabolism

3 CAPTER 13 Digestion: Turning a Meal into Cellular Biochemicals 13.1 Digestion Prepares Large Biomolecules for Use in Metabolism 13.2 Proteases Digest Proteins Into Amino Acids and Peptides 13.3 Dietary Carbohydrates Are Digested by -Amylase 13.4 The Digestion of Lipids Is Complicated by Their ydrophobicity 13.5 Metabolism in Context: Cell Signaling Facilitates Digestion Growing requires vast amounts of energy and biochemical building blocks. These needs do not disappear as we age but are required to maintain our bodies against the wear and tear of living. The energy and building blocks come in the form of food, which must be converted into biochemicals in the process of digestion. [Stuart Pearce/Agefotostock.] 194 FATS Fatty acids and glycerol xidative phosphorylation PLYSACCARIDES Glucose and other sugars Acetyl CoA Citric acid cycle 8 e ATP 2 CoA 2 PRTEINS Amino acids 2 C 2 Stage 1 Stage 2 Stage 3 Figure 13.1 The stages of catabolism. The extraction of energy from fuels comprises three stages. The generation of energy from the oxidation of foodstuffs takes place in three stages (Figure 13.1). In the first stage, large molecules in food are broken down into smaller units. This process is digestion. Proteins are hydrolyzed to the 20 varieties of amino acids, polysaccharides are hydrolyzed to simple sugars such as glucose, and fats are hydrolyzed to fatty acids. This stage is strictly a preparation stage; no useful energy is captured at this point. In the second stage, these numerous small molecules are degraded to a few simple units that play a central role in metabolism. In fact, most of them sugars, fatty acids, glycerol, and several amino acids are converted into acetyl CoA, the activated twocarbon unit that is the fuel for the final stages of aerobic metabolism. Some ATP is generated in this stage, but the amount is small compared with that obtained in the third stage. In the third stage, ATP is produced from the complete oxidation of acetyl CoA. The third stage consists of the citric acid cycle and oxidative phosphorylation, which are the final common pathways in the oxidation of fuel molecules. Acetyl CoA brings the breakdown products of proteins, sugars, and fats into the citric acid cycle (also called the tricarboxylic acid cycle or Krebs cycle), where they are completely oxidized to C 2. In this chapter, we will focus on stage 1 digestion and leave stages 2 and 3 for later chapters.

4 Digestion of Proteins Figure 13.2 Pizza. Foods provide a pleasurable means of obtaining energy and building blocks for biological systems. [Food Collection/Superstock.] 13.1 Digestion Prepares Large Biomolecules for Use in Metabolism Let us begin our study of digestion by examining what happens after we take a bite of pizza, a delicious concoction of lipids, carbohydrates, and proteins (Figure 13.2). Digestion begins in the mouth, where teeth, tongue, and saliva are employed to homogenize a bite of pizza, converting it into an aqueous slurry that is more readily attacked by digestive enzymes than a piece of poorly chewed food would be. Subsequent to homogenization, the food passes into the stomach, where two principle activities take place. First, the proteins are denatured by the acidic environment of the stomach, where the p is maintained at values ranging from 1 to 2 by an ATP-dependent proton pump similar to the Na K ATPase (p. 164). This denaturation renders protein a better substrate for the degradation that will take place later. Second, the process of protein degradation begins in the stomach with action of the proteolytic enzyme pepsin. The action of pepsin yields protein fragments that will be further degraded by the proteases of the intestine. Moreover, protein digestion in the stomach stimulates the pancreas to release a host of digestive enzymes into intestine, where the digestion of proteins, lipids, and carbohydrates begins in earnest. The pancreas also releases large amounts of sodium bicarbonate (NaC 3 ) that serves to neutralize the p of the food as it leaves the acidic environment of the stomach. The gall bladder contributes to digestion by secreting bile salts that are required for lipid digestion. In the next three sections, we will study the digestion of proteins, carbohydrates, and lipids in detail Proteases Digest Proteins into Amino Acids and Peptides uman beings ingest about 70 to 100 g of proteins daily. In regard to our pizza, the meat and cheese provide most of the protein, which must be degraded so that the individual amino acids will be available for use in metabolic pathways. In addition to proteins in foods, from 50 to 100 g of protein per day is sloughed off the cells of the intestine in the wear and tear of digestion; this protein is degraded, and the amino acids are salvaged. As mentioned earlier, the process of protein degradation begins in the stomach with action of the pepsin, a protease. Proteases, or proteolytic enzymes, are a class of enzymes that break the peptide bonds between amino acids, thus digesting proteins. The remarkable enzyme pepsin shows optimal activity in the p range of 1 to 2, conditions so acidic that other proteins are denatured.

5 Digestion: Turning a Meal into Cellular Biochemicals NaC 3 Gall bladder Digestive enzymes Pancreas Secretin CCK Food Small intestine The partly digested proteins as well as carbohydrates and lipids then move from the acidic environment of the stomach to the beginning of the small intestine. The low p of the food stimulates the cells of the small intestine to release the hormone secretin (Figure 13.3). Secretin, in turn, stimulates the release of sodium bicarbonate (NaC 3 ), which neutralizes the p of the food. The polypeptide products of pepsin digestion also stimulate the release of the hormone cholecystokinin (CCK) by the intestinal cells. The pancreas responds to CCK (p. 200) by releasing a host of digestive enzymes into intestine, where the digestion of proteins, lipids, and carbohydrates begins. The digestive enzymes of the pancreas are secreted as inactive precursors called zymogens or proenzymes (Table 13.1). Proteins + Cl + Pepsin ligopeptides Stomach Initial digestion products Figure 13.3 The hormonal control of digestion. Cholecystokinin (CCK) is secreted by specialized intestinal cells and causes the secretion of bile salts from the gall bladder and digestive enzymes from the pancreas. Secretin stimulates sodium bicarbonate (NaC 3 ) secretion, which neutralizes the stomach acid. [After D. Randall, W. Burggren, and K. French, Eckert Animal Physiology, 5th ed. (W.. Freeman and Company, 2002), p. 658.] Table 13.1 Gastric and pancreatic zymogens Site of synthesis Zymogen Active enzyme Stomach Pepsinogen Pepsin Pancreas Chymotrypsinogen Chymotrypsin Pancreas Trypsinogen Trypsin Pancreas Procarboxypeptidase Carboxypeptidase Pancreas Proelastase Elastase Before their secretion, zymogens exist in granules near the cell membrane. In response to CCK, the granules fuse with the cell membrane, expelling their contents into the lumen of the intestine. The zymogens are activated when a part of the inactive precursor is proteolytically cleaved. Indeed, the stomach-enzyme pepsin is itself secreted as a zymogen called pepsinogen. Pepsinogen has a small amount of enzyme activity and can activate itself to some degree in an acidic environment. The active pepsin activates the remaining pepsinogen. The enzyme enteropeptidase, secreted by the epithelial cells of the small intestine, activates the pancreatic zymogen trypsinogen to form trypsin, which in turn activates the remaining pancreatic zymogens (Figure 13.4). Enteropeptidase Trypsinogen Trypsin Figure 13.4 Zymogen activation by proteolytic cleavage. Enteropeptidase initiates the activation of the pancreatic zymogens by activating trypsin, which then activates more trypsinogen as well as the other zymogens. Active enzymes are shown in yellow; zymogens are shown in orange. Chymotrypsinogen Proelastase Chymotrypsin Elastase Procarboxypeptidase Carboxypeptidase Prolipase Lipase The pancreatic proteases hydrolyze the proteins into small fragments called oligopeptides, but digestion is completed by enzymes called peptidases that are attached to the external surfaces of the intestinal cells. These enzymes cleave the oligopeptides into amino acids and di- and tripeptides that can be transported into an intestinal cell by transporters. At least seven different transporters exist, each specific to a different group of amino acids. The amino acids are subsequently released into the blood for use by other tissues (Figure 13.5).

6 LUMEN INTESTINAL CELL BLD Proteins Proteolytic enzymes Amino acids Amino acids Tripeptides Dipeptides Peptidases ligopeptides Peptidase Figure 13.5 The digestion and absorption of proteins. Protein digestion is primarily a result of the activity of enzymes secreted by the pancreas. Peptidases associated with the intestinal epithelium further digest proteins. The amino acids and di- and tripeptides are absorbed into the intestinal cells by specific transporters. Free amino acids are then released into the blood for use by other tissues Dietary Carbohydrates Are Digested by -Amylase ow are the crust and vegetables that topped our pizza converted into biochemicals? These ingredients are sources of carbohydrates, both complex, such as starch and glycogen (p. 130), and simple, such as sucrose (p. 129). Like proteins, dietary carbohydrates are digested into molecules that can be readily absorbed by the intestine. The most common end products are the monosaccharides glucose, fructose, and galactose. ur primary sources of carbohydrates are the complex carbohydrates such as starch and glycogen (present in the meat of our pizza). These branched homopolymers of glucose are digested primarily by the pancreatic enzyme a-amylase, which cleaves the -1,4 bonds of starch and glycogen but not the a-1,6 bonds (Figure 13.6). The products are the Baking pizza in an oven converts the dough into crust and solves a biochemical problem as well: starch is difficult to digest without first being hydrated, and heat allows the starch to absorb water. Starch -Amylase Maltotriose -Limit dextrin Maltose Glucose Figure 13.6 The digestion of starch by -amylase. Amylase hydrolyzes starch into simple sugars. The -1,4 bonds are shown in green. The -1,6 bonds are red. The sites of -amylase digestion are indicated by the small green arrows. 197

7 Digestion: Turning a Meal into Cellular Biochemicals di- and trisaccharides maltose and maltotriose as well as the material not digestible by the -amylase, because of the -1,6 bonds, called the limit dextrin. Maltase converts maltose into glucose, and a-glucosidase digests maltotriose and any other oligosaccharides that may have escaped digestion by the amylase. - Dextrinase further digests limit dextrin into simple sugars. Maltase and -glucosidase are on the surfaces of the intestinal cells. The digestion of disaccharides is simpler than the digestion of complex carbohydrates. Sucrose, a disaccharide consisting of glucose and fructose contributed by the vegetables, is digested by sucrase. Lactase degrades the milk-sugar lactose into glucose and galactose. Sucrase and lactase also reside on the surfaces of intestinal cells. The monosaccharides are then transported into the cell and, subsequently, into the bloodstream, where they can travel to other tissues to be used as fuel. Recall that the transport of glucose across the membranes of intestinal epithelial cells is a secondary active-transport process carried out by the sodium glucose cotransporter (p. 167). C 3 3 C C 3 Glycocholate Figure 13.7 Glycocholate. Bile salts, such as glycocholate, facilitate lipid digestion in the intestine The Digestion of Lipids Is Complicated by Their ydrophobicity As we consume our pizza, slice by slice, we may notice that the box has greasy stains. These stains are caused by the lipids in our meal. The main sources of lipids in the pizza are the meat and the cheese, which, as already noted, are sources of protein as well. Most lipids are ingested in the form of triacylglycerides and must be degraded to fatty acids for absorption across the intestinal epithelium. Lipid digestion presents a problem, because unlike carbohydrates and proteins, these molecules are not soluble in water. ow can the lipids be degraded to fatty acids if the lipids are not soluble in the same medium as the degradative enzymes are? Moreover, the lipid digestion products fatty acids also are not water soluble; so, when digestion has taken place, how does fatty acid transport happen? Lipids are prepared for digestion in the stomach. The grinding and mixing that takes place in the stomach converts lipids into an emulsion, a mixture of lipid droplets and water. N C Common emulsions include mayonnaise and shaken oil-andvinegar salad dressing. After the lipids leave the stomach, emulsification is enhanced with the aid of bile salts, amphipathic molecules synthesized from cholesterol in the liver and secreted from the gall bladder (Figure 13.7). These molecules insert into the lipid droplets, making the triacylglycerides more readily digested. Triacylglycerides are degraded to free fatty acids and monoacylglycerol by enzymes secreted by the pancreas called lipases (Figure 13.8), which attach to the surface of a lipid droplet. Pancreatic lipases are also released into the intestine as proenzymes that are subsequently activated. The final digestion R 2 2 C C 2 C Triacylglycerol R 1 R 3 2 Lipase R 3 R 2 2 C C C 2 Diacylglycerol R 1 2 Lipase R 1 C 2 R 2 C C 2 Monoacylglycerol Figure 13.8 The action of pancreatic lipases. Lipases secreted by the pancreas convert triacylglycerols into fatty acids and monoacylglycerol for absorption into the intestine.

8 products, free fatty acids and monoacylglycerol, are carried in micelles to the intestinal epithelium where they are absorbed across the plasma membrane. Micelles are globular structures formed by small lipids in aqueous solutions (Figure 13.9). In a micelle, the polar head groups of the fatty acids and monoacylglycerol are in contact with the aqueous solution and the hydrocarbon chains are sequestered in the interior of the micelle. Micelle formation is also facilitated by bile salts. If the production of bile salts is inadequate due to liver disease, large amounts of fats (as much as 30 g per day) are excreted in the feces. This condition is referred to as steatorrhea, after stearic acid, a common fatty acid. In the intestine, the triacylglycerides are resynthesized from fatty acids and the monoacylglycerol and then packaged into lipoprotein transport particles called chylomicrons, stable particles approximately 2000 Å (200 nm) in diameter. These particles are composed mainly of triacylglycerides, with some proteins on the surface. Chylomicrons also function in the transport of fat-soluble vitamins and cholesterol. The chylomicrons are released into the lymph system and then into the blood (Figure 13.10). After a meal rich in lipids, the blood appears milky because of the high content of chylomicrons. These particles bind to membrane-bound lipoprotein lipases, primarily at adipose tissue and muscle, where the triacylglycerides are once again degraded into free fatty acids and monoacylglycerol for transport into the tissue. The triacylglycerides are then resynthesized and stored. In the muscle, they can be oxidized to provide energy, as will be discussed in Chapter 26. Figure 13.9 A diagram of a section of a micelle. Ionized fatty acids generated by the action of lipases readily form micelles. QUICK QUIZ Explain why a person who has a trypsinogen deficiency will suffer from more digestion difficulties than will a person lacking most other zymogens. LUMEN MUCSAL CELL Triacylglycerides 2 0 ther lipids and proteins Lipases Fatty acids + Monoacylglycerols Triacylglycerides Chylomicrons To lymph system Figure Chylomicron formation. Free fatty acids and monoacylglycerols are absorbed by intestinal epithelial cells. Triacylglycerols are resynthesized and packaged with other lipids and proteins to form chylomicrons, which are then released into the lymph system. Biological Insight Snake Venoms Digest from the Inside ut Most animals ingest food and, in response to this ingestion, produce enzymes that digest the food. Many venomous snakes, on the other hand, do the opposite. They inject digestive enzymes into their prospective meals to begin the digestion process from the inside out, before they even consume the meals. Snake venom consists of 50 to 60 different protein and peptide components that differ among species of snake and possibly even among individual snakes of the same species. Consider rattlesnakes (Figure 13.11). Rattlesnake venom contains a host of enzymes that digest the tissues of the victim. Phospholipases digest cell membranes at the site of the snakebite, causing a loss of cellular components. The phospholipases also disrupt the membranes of red blood cells, destroying them (a process called hemolysis). Collagenase digests the protein collagen, a major component of connective tissue, Figure A rattlesnake poised to strike. Rattlesnakes inject digestive enzymes into their prospective meals. [Steve amblin/alamy.] 199

9 Digestion: Turning a Meal into Cellular Biochemicals whereas hyaluronidase digests hyaluronidate, a glycosaminoglycan (p. 133) component of connective tissue. The combined action of both collagenase and hyaluronidase is to destroy tissue at the site of the bite, enabling the venom to spread more readily throughout the victim. Various proteolytic enzymes in the venom degrade basement membranes and components of the extracellular matrix, leading to severe tissue damage. Some venoms contain proteolytic enzymes that stimulate the formation of blood clots as well as enzymes that digest blood clots. The net effect of these enzymes acting in concert may be to deplete all clotting factors from the blood, and so clots do not form. Venoms also contain various peptides that have neurotoxic activities. The neurotoxins immobilize the prey while the digestive enzymes reduce the size of the prey to make swallowing easier Metabolism in Context: Cell Signaling Facilitates Digestion In Chapter 12, we examined the basics of signal transduction. Signaling pathways coordinate all biological processes, and digestion is no exception. Among the many signal molecules that help to regulate digestion and appetite, we will briefly examine one, cholecystokinin. As stated earlier, CCK is a peptide hormone secreted by the endocrine cells of the upper intestine (see Figure 13.3). The secretion of CCK, in turn, results in the secretion of a battery of digestive enzymes by the pancreas and bile salts from the gall bladder. CCK exerts its effects by binding to a seventransmembrane receptor in the membranes of the target tissues. Like all 7TM receptors, the CCK receptor is associated with a trimeric G protein. The binding of CCK to the receptor activates G q, which in turn initiates the phosphatidylinositol cascade (Figure and p. 180). Calcium is released into the cytoplasm from the endoplasmic reticulum through the IP 3 -activated calcium channel, and protein CCK + CCK receptor Binding Activated receptor Activated G q protein GTP exchange for GDP on G q Dissociation of trimeric G protein Activated phospholipase C Conversion of phosphatidylinositol 4,5-bisphosphate into IP 3 and diacylglycerol Figure The binding of cholecystokinin to its receptor activates the phosphatidylinositol pathway. Cholecystokinin directs the release of calcium and the activation of protein kinase C, resulting in the secretion of bile salts and digestive enzymes. IP 3 opens calcium channels Zymogen granule fuses with the cell membrane Zymogens released Dyacylglycerol activates protein kinase C

10 kinase C is activated by diacylglycerol and calcium. These events result in the fusion of secretory granules with the plasma membrane. Pancreatic zymogen-containing granules discharge their contents into the intestine, where the zymogens are activated as described in Figure Thus, digestion proceeds in response to CCK, which signals that substrates are available for the digestive enzymes. 201 Key Terms SUMMARY 13.1 Digestion Prepares Large Biomolecules for Use in Metabolism Digestion begins in the mouth, where food is homogenized into an aqueous slurry susceptible to enzyme digestion. The homogenized food then passes into the stomach, an acid environment. The low p of the stomach denatures proteins, thus preparing them for degradation Proteases Digest Proteins into Amino Acids and Peptides Protein digestion begins in the stomach with the action of the proteolytic enzyme pepsin. The digestion products of pepsin stimulate the release of the hormone cholecystokinin from specialized cells in the upper intestine. Cholecystokinin stimulates the release of bile salts from the gall bladder and digestion enzymes from the pancreas in the form of zymogens or proenzymes. Enteropeptidase converts trypsinogen into trypsin, which, in turn, activates the other zymogens Dietary Carbohydrates Are Digested by -Amylase Complex carbohydrates such as starch and glycogen are degraded by -amylase, which cleaves the -1,4 bonds of starch and glycogen. The products of -amylase digestion are the disaccharide maltose and limit dextrin, a carbohydrate rich in -1,6 bonds. Limit dextrin is digested by -dextrinase. Sucrase and lactase digest the disaccharides sucrose and lactose, respectively The Digestion of Lipids Is Complicated by Their ydrophobicity Because dietary lipids, mostly triacylglycerides, are not water soluble, they must be converted into an emulsion, a mixture of lipid droplets and water, to be digested by lipases secreted by the pancreas. Bile salts, provided by the gall bladder, facilitate the formation of the emulsion. The products of lipase digestion free fatty acids and monoacylglycerol form micelles for absorption by the intestine. In the cells of the intestine, triacylglycerides are re-formed and packaged into lipoprotein particles called chylomicrons for transport in the lymph system and the blood Metabolism in Context: Cell Signaling Facilitates Digestion Cholecystokinin, a hormone released by cells of the upper intestine, acts on the pancreas to stimulate zymogen secretion. CCK binds to a seventransmembrane receptor, activating a G-protein signal that in turn activates the phosphatidylinositol cascade. The end result is that zymogencontaining granules in the pancreas fuse with the cell membrane, discharging their contents into the intestine so that digestion takes place. Key Terms digestion (p. 195) pepsin (p. 195) proteolytic enzymes (proteases) (p. 195) secretin (p. 196) cholecystokinin (p. 196) zymogens (proenzymes) (p. 196) enteropeptidase (p. 196) trypsinogen (p. 196) trypsin (p. 196) -amylase (p. 197) emulsion (p. 198) bile salts (p. 198) lipase (p. 198) micelle (p. 199) chylomicron (p. 199)

11 Digestion: Turning a Meal into Cellular Biochemicals Answer to QUICK QUIZ Trypsin, which is formed from trypsinogen, activates most of the other zymogens. ence, a deficiency in trypsinogen would lead to a loss of activity of virtually all of the enzymes required for digestion. n the other hand, loss of a zymogen for lipase, for instance, would impair only lipid digestion, without affecting the digestion of other molecules. Problems 1. Necessary but not sufficient. Why is digestion required for fuel metabolism even though no useful energy is harnessed in the process? 2. Mother knows best. When your mother told you to chew your food well, she had your best biochemical interests at heart. Explain. 3. Zymogen activation. When very low concentrations of pepsinogen are added to an acidic medium, how does the half-time of activation depend on zymogen concentration? 4. Safeguard. Trypsin inhibitor is a pancreatic polypeptide that binds trypsin with very high affinity, preventing it from digesting proteins. Why might a lack of trypsin inhibitor cause pancreatitus (inflammation of the pancreas)? 5. Not too al dente. The digestion of macaroni is more efficient after the pasta has been heated in water. Why is this the case? 6. Accessibility matters. Why is emulsification required for efficient lipid digestion? 7. umors are necessary. ow would a lack of bile salts affect digestion? 8. Precautions. Why are most digestive enzymes produced as zymogens? Selected readings for this chapter can be found online at