17.2 What Can Be Learned from Metabolic Maps? 513 the end product of heterotrophic carbon metabolism, and CO 2 is returned to the at- mosphere for reuse by the photoautotrophs. In effect, solar energy is converted to the chemical energy of organic molecules by photoautotrophs, and heterotrophs recover this energy by metabolizing the organic substances. The flow of energy in the biosphere is thus conveyed within the carbon cycle, and the impetus driving the cycle is light energy. 17.2 What Can Be Learned from Metabolic Maps? Metabolic maps (Figure 17.2) portray the principal reactions of the intermediary metabolism of carbohydrates, lipids, amino acids, nucleotides, and their deriva- tives. These maps are very complex at first glance and seem to be virtually impos- sible to learn easily. Despite their appearance, these maps become easy to follow once the major metabolic routes are known and their functions are understood. The underlying order of metabolism and the important interrelationships between the various pathways then appear as simple patterns against the seemingly compli- cated background. The Metabolic Map Can Be Viewed as a Set of Dots and Lines One interesting transformation of the intermediary metabolism map is to represent each intermediate as a black dot and each enzyme as a line (Figure 17.3). Then, the more than 1000 different enzymes and substrates are represented by just two symbols. This chart has about 520 dots (intermediates). Table 17.2 lists the numbers of dots that have one or two or more lines (enzymes) associated with them. Thus, this table classi- fies intermediates by the number of enzymes that act upon them. A dot connected to just a single line must be either a nutrient, a storage form, an end product, or an ex- cretory product of metabolism. Also, because many pathways tend to proceed in only one direction (that is, they are essentially irreversible under physiological conditions), a dot connected to just two lines is probably an intermediate in only one pathway and has only one fate in metabolism. If three lines are connected to a dot, that intermedi- ate has at least two possible metabolic fates; four lines, three fates; and so on. Note that about 80% of the intermediates connect to only one or two lines and thus have only a single role in the cell. However, intermediates at branch points are subject to a variety of fates. In such instances, the pathway followed is an important regulatory choice. In- deed, whether any substrate is routed down a particular metabolic pathway is the con- sequence of a regulatory decision made in response to the cell’s (or organism’s) mo- mentary requirements for energy or nutrition. The regulation of metabolism is an interesting and important subject to which we will return often. Alternative Models Can Provide New Insights into Pathways Alternative mappings of metabolic reactions have been postulated for several rea- sons. First and most obviously, the sheer complexity of pathways has prompted biochemists to seek simpler portrayals of an organism’s chemistry. Second, tradi- tional metabolite-focused maps (Figure 17.4a) do not provide insight into the spa- tial and temporal organization of the metabolites and the enzymes that intercon- vert them. Even more significantly, the rise of genomics (the study of the whole genomes of organisms), transcriptomics (the study of global messenger RNA ex- pression), and proteomics (the study of the totality of proteins) has provoked fresh conceptions of biological order and function. For example, Juliet Gerrard has proposed that metabolic maps be recast in protein-centric presentations (Figure 17.4b). In such maps, the metabolites and the enzymes that interconvert them are transposed, revealing a new emphasis—the metabolites are “signals” in a cellular network of proteins. Protein-centric maps may be condensed and simplified by realizing that some pathway enzymes are clustered in multienzyme complexes and that metabolites are Lines Dots 1 or 2 410 371 420 511 6 or more 8 TABLE 17.2 Number of Dots (Intermediates) in the Metabolic Map of Figure 17.2, and the Number of Lines Associated with Them 514 ANIMATED FIGURE 17.2 A metabolic map, indicating the reactions of intermediary metabo- lism and the enzymes that catalyze them. More than 500 different chemical intermediates, or metabolites, and a greater number of enzymes are represented here. (Source: From Donald Nicholson, Map #22, © International Union of Biochemistry and Molecular Biology.) See this figure animated at www.cengage.com/login. 17.2 What Can Be Learned from Metabolic Maps? 515 FIGURE 17.3 The metabolic map as a set of dots and lines.The heavy dots and lines trace the central energy- releasing pathways known as glycolysis and the citric acid cycle. (Adapted from Alberts, B., et al.,1989. Molecular Biol- ogy of the Cell, 2nd ed. New York: Garland Publishing Co.) 516 Chapter 17 Metabolism: An Overview literally passed from enzyme to enzyme within such clusters (Figure 17.4c). The re- sult is a simplified representation of metabolic networks, containing only the es- sential signaling information. Metabolic maps are representations of large amounts of information. Conceptualizing them in different formats enables biochemists to analyze vast amounts of information in new and insightful ways. Multienzyme Systems May Take Different Forms The individual metabolic pathways of anabolism and catabolism consist of se- quential enzymatic steps (Figure 17.5). Several types of organization are possible. The enzymes of some multienzyme systems may exist as physically separate, soluble entities, with diffusing intermediates (Figure 17.5a). In other instances, the en- zymes of a pathway are collected to form a discrete multienzyme complex, and the substrate is sequentially modified as it is passed along from enzyme to enzyme (Fig- ure 17.5b). This type of organization has the advantage that intermediates are not lost or diluted by diffusion. In a third pattern of organization, the enzymes common to a pathway reside together as a membrane-bound system (Figure 17.5c). In this case, the enzyme participants (and perhaps the substrates as well) must diffuse in just the two dimensions of the membrane to interact with their neighbors. As research reveals the ultrastructural organization of the cell in ever greater de- tail, more and more of the so-called soluble enzyme systems are found to be physi- cally united into functional complexes. Thus, in many (perhaps all) metabolic path- A (a) E 1 E 2 E 3 E 6 E 4 E 5 BC G E 7 H E 8 I E 9 J E 10 E 14 K E 11 L E 15 P E 12 M E 13 N E 16 DEF O P OP L (b) A E 1 E 6 B C E 7 G E 8 H E 9 I J E 10 D E 11 K E 14 D E 16 E 15 E 12 L E 13 M N E 2 C E 3 D E 4 EF E 5 D C N CF P P E 3 (c) A E 16 DD LL Enzymes 1 and 2 Enzymes 4 and 5 Enzymes 6 to 9 Enzymes 10 and 11 Enzymes 14 and 15 Enzymes 12 and 13 FIGURE 17.4 (a) The traditional view of a metabolic pathway is metabolite-centric. (b) Gerrard has proposed that a protein-centric view is more informative for some purposes. (c) A simplified version of the protein-centric view where proteins in the pathway form multifunctional complexes. 17.3 How Do Anabolic and Catabolic Processes Form the Core of Metabolic Pathways? 517 ways, the consecutively acting enzymes are associated into stable multienzyme com- plexes that are sometimes referred to as metabolons, a word meaning “units of metabolism.” 17.3 How Do Anabolic and Catabolic Processes Form the Core of Metabolic Pathways? Metabolism serves two fundamentally different purposes: the generation of energy to drive vital functions and the synthesis of biological molecules. To achieve these ends, metabolism consists largely of two contrasting processes: catabolism and an- abolism. Catabolic pathways are characteristically energy yielding, whereas anabolic path- ways are energy requiring. Catabolism involves the oxidative degradation of complex nutrient molecules (carbohydrates, lipids, and proteins) obtained either from the environment or from cellular reserves. The breakdown of these molecules by ca- tabolism leads to the formation of simpler molecules such as lactic acid, ethanol, carbon dioxide, urea, or ammonia. Catabolic reactions are usually exergonic, and often the chemical energy released is captured in the form of ATP (see Chapter 3). Because catabolism is oxidative for the most part, part of the chemical energy may be conserved as energy-rich electrons transferred to the coenzymes NAD ϩ and NADP ϩ . These two reduced coenzymes have very different metabolic roles: NAD ϩ reduction is part of catabolism; NADPH oxidation is an important aspect of anabolism. The energy released upon oxidation of NADH is coupled to the phos- phorylation of ADP in aerobic cells, and so NADH oxidation back to NAD ϩ serves to generate more ATP; in contrast, NADPH is the source of the reducing power needed to drive reductive biosynthetic reactions. Thermodynamic considerations demand that the energy necessary for biosyn- thesis of any substance exceed the energy available from its catabolism. Otherwise, (a) (b) (c) FIGURE 17.5 Schematic representation of types of multi- enzyme systems carrying out a metabolic pathway: (a) Physically separate, soluble enzymes with diffusing intermediates. (b) A multienzyme complex.Substrate enters the complex and becomes bound and then se- quentially modified by enzymes E 1 to E 5 before product is released. No intermediates are free to diffuse away. (c) A membrane-bound multienzyme system. 518 Chapter 17 Metabolism: An Overview organisms could achieve the status of perpetual motion machines: A few molecules of substrate whose catabolism yielded more ATP than required for its resynthesis would allow the cell to cycle this substance and harvest an endless supply of energy. Anabolism Is Biosynthesis Anabolism is a synthetic process in which the varied and complex biomolecules (pro- teins, nucleic acids, polysaccharides, and lipids) are assembled from simpler precur- sors. Such biosynthesis involves the formation of new covalent bonds, and an input of chemical energy is necessary to drive such endergonic processes. The ATP gener- ated by catabolism provides this energy. Furthermore, NADPH is an excellent donor of high-energy electrons for the reductive reactions of anabolism. Despite their di- vergent roles, anabolism and catabolism are interrelated in that the products of one provide the substrates of the other (Figure 17.6). Many metabolic intermediates are shared between the two processes, and the precursors needed by anabolic pathways are found among the products of catabolism. Anabolism and Catabolism Are Not Mutually Exclusive Interestingly, anabolism and catabolism occur simultaneously in the cell. The con- flicting demands of concomitant catabolism and anabolism are managed by cells in two ways. First, the cell maintains tight and separate regulation of both catabo- lism and anabolism, so metabolic needs are served in an immediate and orderly fashion. Second, competing metabolic pathways are often localized within differ- ent cellular compartments. Isolating opposing activities within distinct compart- ments, such as separate organelles, avoids interference between them. For exam- ple, the enzymes responsible for catabolism of fatty acids, the fatty acid oxidation pathway, are localized within mitochondria. In contrast, fatty acid biosynthesis takes place in the cytosol. In subsequent chapters, we shall see that the particular mole- cular interactions responsible for the regulation of metabolism become important for an understanding and appreciation of metabolic biochemistry. The Pathways of Catabolism Converge to a Few End Products If we survey the catabolism of the principal energy-yielding nutrients (carbohy- drates, lipids, and proteins) in a typical heterotrophic cell, we see that the degra- dation of these substances involves a succession of enzymatic reactions. In the pres- ence of oxygen (aerobic catabolism), these molecules are degraded ultimately to carbon dioxide, water, and ammonia. Aerobic catabolism consists of three distinct Energy-yielding nutrients Chemical energy H 2 O CO 2 NH 3 Carbohydrates Fats Proteins Cell macromolecules Proteins Polysaccharides Lipids Nucleic acids Energy-poor end products Precursor molecules Amino acids Sugars Fatty acids Nitrogenous bases Catabolism (oxidative, exergonic) Anabolism (reductive, endergonic) ATP ATP ATP ATP ATP NADPH NADPH NADPH NADPH NADPH FIGURE 17.6 Energy relationships between the path- ways of catabolism and anabolism. Oxidative, exergonic pathways of catabolism release free energy and reduc- ing power that are captured in the form of ATP and NADPH, respectively. Anabolic processes are endergonic, consuming chemical energy in the form of ATP and using NADPH as a source of high-energy electrons for reductive purposes. 17.3 How Do Anabolic and Catabolic Processes Form the Core of Metabolic Pathways? 519 stages. In stage 1, the nutrient macromolecules are broken down into their respec- tive building blocks. Despite the great diversity of macromolecules, these building blocks represent a rather limited number of products. Proteins yield up their 20 component amino acids, polysaccharides give rise to carbohydrate units that are convertible to glucose, and lipids are broken down into glycerol and fatty acids (Figure 17.7). In stage 2, the collection of product building blocks generated in stage 1 is further degraded to yield an even more limited set of simpler metabolic intermediates. The Stage 2: Stage 3: Stage 1: Large biomolecules NH 3 Proteins Polysaccharides Lipids The various kinds of proteins, polysaccharides, and fats are broken down into their component building blocks, which are relatively few in number. Amino acids Glycerol, fatty acids Pentoses, hexoses Glucose Glyceraldehyde-3-phosphate Pyruvate Acetyl-CoA Building block molecules The various building blocks are degraded into a common product, the acetyl groups of acetyl-CoA. Glycolysis Oxidative phosphorylation H 2 O Common degradation product Catabolism converges via the citric acid cycle to three principal end products: water, carbon dioxide, and ammonia. Simple, small end products of catabolism End products Citric acid cycle CO 2 FIGURE 17.7 The three stages of catabolism. Stage 1: Proteins, polysaccharides, and lipids are broken down into their component building blocks, which are relatively few in number.Stage 2: The various building blocks are degraded into the common product, the acetyl groups of acetyl-CoA. Stage 3: Catabolism con- verges to three principal end products: water, carbon dioxide, and ammonia. 520 Chapter 17 Metabolism: An Overview deamination of amino acids leaves ␣-keto acid carbon skeletons. Several of these ␣-keto acids are citric acid cycle intermediates and are fed directly into stage 3 catab- olism via this cycle. Others are converted either to the three-carbon ␣-keto acid pyru- vate or to the acetyl groups of acetyl-coenzyme A (acetyl-CoA). Glucose and the glycerol from lipids also generate pyruvate, whereas the fatty acids are broken into two-carbon units that appear as acetyl-CoA. Because pyruvate also gives rise to acetyl-CoA, we see that the degradation of macromolecular nutrients converges to a common end prod- uct, acetyl-CoA (Figure 17.7). The combustion of the acetyl groups of acetyl-CoA by the citric acid cycle and ox- idative phosphorylation to produce CO 2 and H 2 O represents stage 3 of catabolism. The end products of the citric acid cycle, CO 2 and H 2 O, are the ultimate waste products of aerobic catabolism. As we shall see in Chapter 19, the oxidation of acetyl-CoA dur- ing stage 3 metabolism generates most of the energy produced by the cell. Anabolic Pathways Diverge, Synthesizing an Astounding Variety of Biomolecules from a Limited Set of Building Blocks A rather limited collection of simple precursor molecules is sufficient to provide for the biosynthesis of virtually any cellular constituent, be it protein, nucleic acid, lipid, or polysaccharide. All of these substances are constructed from appropriate building blocks via the pathways of anabolism. In turn, the building blocks (amino acids, nucleotides, sugars, and fatty acids) can be generated from metabolites in the cell. For example, amino acids can be formed by amination of the corresponding ␣-keto acid carbon skeletons, and pyruvate can be converted to hexoses for poly- saccharide biosynthesis. Amphibolic Intermediates Play Dual Roles Certain of the central pathways of intermediary metabolism, such as the citric acid cycle, and many metabolites of other pathways have dual purposes—they serve in both catabolism and anabolism. This dual nature is reflected in the designation of such pathways as amphibolic rather than solely catabolic or anabolic. In any event, in con- trast to catabolism—which converges to the common intermediate, acetyl-CoA—the pathways of anabolism diverge from a small group of simple metabolic intermediates to yield a spectacular variety of cellular constituents. Corresponding Pathways of Catabolism and Anabolism Differ in Important Ways The anabolic pathway for synthesis of a given end product usually does not precisely match the pathway used for catabolism of the same substance. Some of the interme- diates may be common to steps in both pathways, but different enzymatic reactions and unique metabolites characterize other steps. A good example of these differences is found in a comparison of the catabolism of glucose to pyruvic acid by the pathway of glycolysis and the biosynthesis of glucose from pyruvate by the pathway called glu- coneogenesis. The glycolytic pathway from glucose to pyruvate consists of 10 enzymes. Although it may seem efficient for glucose synthesis from pyruvate to proceed by a re- versal of all 10 steps, gluconeogenesis uses only seven of the glycolytic enzymes in re- verse, replacing those remaining with four enzymes specific to glucose biosynthesis. In similar fashion, the pathway responsible for degrading proteins to amino acids dif- fers from the protein synthesis system, and the oxidative degradation of fatty acids to two-carbon acetyl-CoA groups does not follow the same reaction path as the biosyn- thesis of fatty acids from acetyl-CoA. Metabolic Regulation Requires Different Pathways for Oppositely Directed Metabolic Sequences A second reason for different pathways serving in opposite metabolic directions is that such pathways must be independently regulated. If ca- tabolism and anabolism passed along the same set of metabolic tracks, equilibrium considerations would dictate that slowing the traffic in one direction by inhibiting Amphi is from the Greek for “on both sides.” 17.3 How Do Anabolic and Catabolic Processes Form the Core of Metabolic Pathways? 521 a particular enzymatic reaction would necessarily slow traffic in the opposite direc- tion. Independent regulation of anabolism and catabolism can be accomplished only if these two contrasting processes move along different routes or, in the case of shared pathways, the rate-limiting steps serving as the points of regulation are cat- alyzed by enzymes that are unique to each opposing sequence (Figure 17.8). ATP Serves in a Cellular Energy Cycle We saw in Chapter 3 that ATP is the energy currency of cells. In phototrophs, ATP is one of the two energy-rich primary products resulting from the transformation of light energy into chemical energy. (The other is NADPH; see the following discus- sion.) In heterotrophs, the pathways of catabolism have as their major purpose the release of free energy that can be captured in the form of energy-rich phosphoric anhydride bonds in ATP. In turn, ATP provides the energy that drives the manifold activities of all living cells—the synthesis of complex biomolecules, the osmotic work involved in transporting substances into cells, the work of cell motility, and the work of muscle contraction. These diverse activities are all powered by energy re- leased in the hydrolysis of ATP to ADP and P i . Thus, there is an energy cycle in cells where ATP serves as the vessel carrying energy from photosynthesis or catabolism to the energy-requiring processes unique to living cells (Figure 17.9). A E 1 B C D E P E 3 E 4 E 5 A P M L K J E 6 E 7 E 8 E 9 E 10 A E 3 E 6 P A E 3 E 6 P E 1 E 2 E 4 E 5 M L K J E 9 E 8 E 7 B C D E E 6 E 2 E 3 E 4 E 5 E 1 E 1 E 2 E 4 E 5 + + ++ E 10 E 2 (a) Regulated step Regulated step Activation of one mode is accompanied by reci p rocal inhibition of the other mode. Catabolic mode Anabolic mode (b) Catabolic mode Anabolic mode FIGURE 17.8 Parallel pathways of catabolism and an- abolism must differ in at least one metabolic step in order that they can be regulated independently. Shown here are two possible arrangements of opposing cata- bolic and anabolic sequences between A and P.(a) The parallel sequences proceed via independent routes. (b) Only one reaction has two different enzymes, a cata- bolic one (E 3 ) and its anabolic counterpart (E 6 ).These provide sites for regulation. ATP O 2 H 2 O a. b. c. Fuels Photosynthesis Light energy The ATP Cycle P i + CO 2 ADP Biosynthesis Osmotic work Cell motility/muscle contraction ATP hydrolysis Catabolism FIGURE 17.9 The ATP cycle in cells. ATP is formed via photosynthesis in phototrophic cells or catabolism in heterotrophic cells. Energy-requiring cellular activities are powered by ATP hydrolysis, liberating ADP and P i . 522 Chapter 17 Metabolism: An Overview NAD ؉ Collects Electrons Released in Catabolism The substrates of catabolism—proteins, carbohydrates, and lipids—are good sources of chemical energy because the carbon atoms in these molecules are in a relatively reduced state (Figure 17.10). In the oxidative reactions of catabolism, reducing equivalents are released from these substrates, often in the form of hydride ions (a proton coupled with two electrons, HϺ Ϫ ). These hydride ions are transferred in en- zymatic dehydrogenase reactions from the substrates to NAD ϩ molecules, reducing them to NADH. A second proton accompanies these reactions, appearing in the overall equation as H ϩ (Figure 17.11). In turn, NADH is oxidized back to NAD ϩ when it transfers its reducing equivalents to electron acceptor systems that are part of the metabolic apparatus of the mitochondria. The ultimate oxidizing agent (e Ϫ ac- ceptor) is O 2 , becoming reduced to H 2 O. Oxidation reactions are exergonic, and the energy released is coupled with the formation of ATP in a process called oxidative phosphorylation. The NAD ϩ –NADH system can be viewed as a shuttle that carries the electrons released from catabolic substrates to the mitochondria, where they are transferred to O 2 , the ultimate elec- tron acceptor in catabolism. In the process, the free energy released is trapped in ATP. The NADH cycle is an important player in the transformation of the chemical energy of carbon compounds into the chemical energy of phosphoric anhydride bonds. Such transformations of energy from one form to another are referred to as energy transduction. Oxidative phosphorylation is one cellular mechanism for en- ergy transduction. Chapter 20 is devoted to electron transport reactions and oxida- tive phosphorylation. CH 2 H > C OH > O C > C O OH > O C O More reduced state Less reduced state FIGURE 17.10 Comparison of the state of reduction of carbon atoms in biomolecules: OCH 2 O (fats) Ͼ OCHOHO (carbohydrates) H E CPO (carbonyls) Ͼ OCOOH (carboxyls) Ͼ CO 2 (carbon dioxide, the final product of catabolism). P + H CH 2 – O O O O P – O O O O N + C NH 2 O CH 2 O OH OH OH OH H NH 2 N N N N CH 3 CH 2 OH Ethyl alcohol Oxidation Reduction N C NH 2 O HH P CH 2 – O O O O P – O O O O CH 2 O OH OH OH OH NH 2 N N N N + CH 3 CH O + Acetaldehyde – NAD + NADH H + FIGURE 17.11 Hydrogen and electrons released in the course of oxidative catabolism are transferred as hydride ions to the pyridine nucleotide, NAD ϩ , to form NADH ϩ H ϩ in dehydrogenase reactions of the type AH 2 ϩ NAD ϩ ⎯⎯→ A ϩ NADH ϩ H ϩ The reaction shown is catalyzed by alcohol dehydrogenase.