The word metabolism derives from the Greek word for “change.” Metabolism represents the sum of the chemical changes that convert nutrients, the “raw materials” necessary to nourish living organisms, into energy and the chemically complex finished products of cells. Metabolism consists of literally hundreds of enzymatic reactions organized into discrete pathways. These pathways proceed in a stepwise fashion, transforming substrates into end products through many specific chemical intermediates. Metabolism is sometimes referred to as intermediary metabolism to reflect this aspect of the process. Metabolic maps (Figure 18.1) portray virtually all of the principal reactions of the intermediary metabolism of carbohydrates, lipids, amino acids, nucleotides, and their derivatives. These maps are very complex at first glance and seem to be virtually impossible 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 complicated background.

Figure 18.1• A metabolic map, indicating the reactions of intermediary metabolism and the enzymes that catalyze them. Over 500 different chemical intermediates, or metabolites, and a greater number of enzymes are represented here. ((c) 1997 20th edition, designed by and courtesy of D. E. Nicholson, University of Leeds , U. K., and the Sigma Chemical Co. )

The Metabolic Map 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 18.2). Then, the more than 1000 different enzymes and substrates are represented by just two symbols. This chart has about 520 dots (intermediates). Table 18.1 lists the numbers of dots that have one or two or more lines (enzymes) associated with them.

Table 18.1

Thus, this table classifies 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 excretory 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 intermediate 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 limited purpose in the cell. However, many intermediates are subject to a variety of fates. In such instances, the pathway followed is an important regulatory choice. Indeed, whether any substrate is routed down a particular metabolic pathway is the consequence of a regulatory decision made in response to the cell’s (or organism’s) momentary requirements for energy or nutrition. The regulation of metabolism is an interesting and important subject to which we will return often.

Figure 18.2 • 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 Biology of the Cell, 2nd ed . New York : Garland Publishing Co. )

18.1 ^• Virtually All Organisms Have the Same Basic Set of Metabolic Pathways

One of the great unifying principles of modern biology is that organisms show marked similarity in their major pathways of metabolism. Given the almost unlimited possibilities within organic chemistry, this generality would appear most unlikely. Yet it’s true, and it provides strong evidence that all life has descended from a common ancestral form. All forms of nutrition and almost all metabolic pathways evolved in early prokaryotes prior to the appearance of eukaryotes 1 billion years ago. For example, glycolysis , the metabolic pathway by which energy is released from glucose and captured in the form of ATP under anaerobic conditions, is common to almost every cell. It is believed to be the most ancient of metabolic pathways, having arisen prior to the appearance of oxygen in abundance in the atmosphere. All organisms, even those that can synthesize their own glucose, are capable of glucose degradation and ATP synthesis via glycolysis. Other prominent pathways are also virtually ubiquitous among organisms.

Metabolic Diversity

Although most cells have the same basic set of central metabolic pathways, different cells (and, by extension, different organisms) are characterized by the alternative pathways they might express. These pathways offer a wide diversity of metabolic possibilities. For instance, organisms are often classified according to the major metabolic pathways they exploit to obtain carbon or energy. Classification based on carbon requirements defines two major groups, autotrophs and heterotrophs. Autotrophs are organisms that can use just carbon dioxide as their sole source of carbon. Heterotrophs require an organic form of carbon, such as glucose, in order to synthesize other essential carbon compounds.
            Classification based on energy sources also gives two groups: phototrophs and chemotrophs . Phototrophs are photosynthetic organisms, which use light as a source of energy. Chemotrophs use organic compounds such as glucose or, in some instances, oxidizable inorganic substances such as Fe²⁺, NO₂^-, NH₄⁺, or elemental sulfur as sole sources of energy. Typically, the energy is extracted through oxidation-reduction reactions. Based on these characteristics, every organism falls into one of four categories (Table 18.2).

Metabolic Diversity Among the Five Kingdoms

Prokaryotes (the kingdom Monera —bacteria) show a greater metabolic diversity than all the four eukaryotic kingdoms ( Protoctista [previously called Protozoa], Fungi, Plants, and Animals) put together. Prokaryotes are variously chemoheterotrophic, photoautotrophic, photoheterotrophic, or chemoautotrophic. No protoctista are chemoautotrophs; fungi and animals are exclusively chemoheterotrophs; plants are characteristically photoautotrophs, although some are heterotrophic in their mode of carbon acquisition.

The Role of O₂ in Metabolism

A further metabolic distinction among organisms is whether or not they can use oxygen as an electron acceptor in energy-producing pathways. Those that can are called aerobes or aerobic organisms; others, termed anaerobes, can subsist without O₂. Organisms for which O₂ is obligatory for life are called obligate aerobes; humans are an example. Some species, the so-called facultative anaerobes, can adapt to anaerobic conditions by substituting other electron acceptors for O₂ in their energy-producing pathways; Escherichia coli is an example. Yet others cannot use oxygen at all and are even poisoned by it; these are the obligate anaerobes. Clostridium botulinum, the bacterium that produces botulin toxin, is representative.

The Flow of Energy in the Biosphere and the Carbon and Oxygen Cycles Are Intimately Related

The primary source of energy for life is the sun. Photoautotrophs utilize light energy to drive the synthesis of organic molecules, such as carbohydrates, from atmospheric CO₂ and water (Figure 18.3). Heterotrophic cells then use these organic products of photosynthetic cells both as fuels and as building blocks, or precursors, for the biosynthesis of their own unique complement of biomolecules. Ultimately, CO₂ is the end product of heterotrophic carbon metabolism, and CO₂ is returned to the atmosphere 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.

Figure 18.3 • The flow of energy in the biosphere is coupled primarily to the carbon and oxygen cycles.

18.2 ^• Metabolism Consists of Catabolism (Degradative Pathways) and Anabolism (Biosynthetic 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 anabolism. Catabolic pathways are characteristically energy-yielding, whereas anabolic pathways 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 catabolism 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 (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⁺ and NADP⁺. These two reduced coenzymes have very different metabolic roles: NAD⁺ reduction is part of catabolism; NADPH oxidation is an important aspect of anabolism. The energy released upon oxidation of NADH is coupled to the phosphorylation of ADP in aerobic cells, and so NADH oxidation back to NAD⁺ 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 biosynthesis of any substance exceed the energy available from its catabolism. Otherwise, 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 (proteins, nucleic acids, polysaccharides, and lipids) are assembled from simpler precursors. Such biosynthesis involves the formation of new covalent bonds, and an input of chemical energy is necessary to drive such endergonic processes. The ATP generated by catabolism provides this energy. Furthermore, NADPH is an excellent donor of high-energy electrons for the reductive reactions of anabolism. Despite their divergent roles, anabolism and catabolism are interrelated in that the products of one provide the substrates of the other (Figure 18.4). Many metabolic intermediates are shared between the two processes, and the precursors needed by anabolic pathways are found among the products of catabolism.

Figure 18.4 ^• Energy relationships between the pathways of catabolism and anabolism. Oxidative, exergonic pathways of catabolism release free energy and reducing 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.

Anabolism and Catabolism Are Not Mutually Exclusive

Interestingly, anabolism and catabolism occur simultaneously in the cell. The conflicting demands of concomitant catabolism and anabolism are managed by cells in two ways. First, the cell maintains tight and separate regulation of both catabolism and anabolism, so that metabolic needs are served in an immediate and orderly fashion. Second, competing metabolic pathways are often localized within different cellular compartments. Isolating opposing activities within distinct compartments, such as separate organelles, avoids interference between them. For example, 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 molecular interactions responsible for the regulation of metabolism become important to an understanding and appreciation of metabolic biochemistry.

Modes of Enzyme Organization in Metabolic Pathways

The individual metabolic pathways of anabolism and catabolism consist of sequential enzymatic steps (Figure 18.5). Several types of organization are possible. The enzymes of some multienzyme systems may exist as physically separate, soluble entities, with diffusing intermediates (Figure 18.5a). In other instances, the enzymes 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 (Figure 18.5b). 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 18.5c). 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.

Figure 18.5 • ^{Schematic representation} of types of multienzyme systems carrying out a metabolic pathway: (a) Physically separate, soluble enzymes with diffusing intermediates. (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes E₁ to E₅ before product is released. No intermediates are free to diffuse away. (c) A membrane-bound multienzyme system.

amphi · from the Greek for “on both sides”

            As research reveals the ultrastructural organization of the cell in ever greater detail, more and more of the so-called soluble enzyme systems are found to be physically united into functional complexes. Thus, in many (perhaps all) metabolic pathways, the consecutively acting enzymes are associated into stable multienzyme complexes that are sometimes referred to as metabolons, a word meaning “units of metabolism.”

The Pathways of Catabolism Converge to a Few End Products

If we survey the catabolism of the principal energy-yielding nutrients (carbohydrates, lipids, and proteins) in a typical heterotrophic cell, we see that the degradation of these substances involves a succession of enzymatic reactions. In the presence of oxygen (aerobic catabolism), these molecules are degraded ultimately to carbon dioxide, water, and ammonia. Aerobic catabolism consists of three distinct stages. In stage 1, the nutrient macromolecules are broken down into their respective building blocks. Given 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 18.6).

Figure 18.6 ^• The three stages of catabolism. Stage I: Proteins, polysaccharides, and lipids are broken down into their component building blocks, which are relatively few in number. Stage II: The various building blocks are degraded into the common product, the acetyl groups of acetyl-CoA. Stage III: Catabolism converges to three principal end products: water, carbon dioxide, and ammonia.

            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 deamination of amino acids leaves a- keto acid carbon skeletons. Several of these a- keto acids are citric acid cycle intermediates and are fed directly into stage 3 catabolism via this cycle. Others are converted either to the three-carbon a-keto acid pyruvate 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 product, acetyl-CoA (Figure 18.6).

            The combustion of the acetyl groups of acetyl-CoA by the citric acid cycle and oxidative phosphorylation to produce CO₂ and H₂O represents stage 3 of catabolism. The end products of the citric acid cycle, CO₂ and H₂O, are the ultimate waste products of aerobic catabolism. As we shall see in Chapter 20, the oxidation of acetyl-CoA during 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 a-keto acid carbon skeletons, and pyruvate can be converted to hexoses for polysaccharide biosynthesis.

Amphibolic Intermediates

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 contrast 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 intermediates 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 gluconeogenesis. 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 reversal of all 10 steps, gluconeogenesis uses only seven of the glycolytic enzymes in reverse, replacing the remaining three with four enzymes specific to glucose biosynthesis. In similar fashion, the pathway responsible for degrading proteins to amino acids differs 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 biosynthesis of fatty acids from acetyl-CoA .

Metabolic Regulation Favors 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 catabolism and anabolism passed along the same set of metabolic tracks, equilibrium considerations would dictate that slowing the traffic in one direction by inhibiting a particular enzymatic reaction would necessarily slow traffic in the opposite direction. 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 catalyzed by enzymes that are unique to each opposing sequence (Figure 18.7).

Figure 18.7 • Parallel pathways of catabolism and anabolism must differ in at least one metabolic step in order that they can be regulated independently. Shown here are two possible arrangements of opposing catabolic and anabolic sequences between A and P. (a) The parallel sequences proceed via independent routes. (b) Only one reaction has two different enzymes, a catabolic one (E₃) and its anabolic counterpart (E₆). These provide sites for regulation.

The ATP 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 discussion.) 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, the work of muscle contraction. These diverse activities are all powered by energy released 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 18.8).

Figure 18.8 ^• 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.

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 18.9).

Figure 18.9 • Comparison of the state of reduction of carbon atoms in biomolecules: —CH₂O— (fats) > —CHOH — (carbohydrates) > —C=O (carbonyls) > —COOH ( carboxyls ) > CO₂ (carbon dioxide, the final product of catabolism).

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, H^:-). These hydride ions are transferred in enzymatic dehydrogenase reactions from the substrates to NAD⁺ molecules, reducing them to NADH. A second proton accompanies these reactions, appearing in the overall equation as H⁺ (Figure 18.10). In turn, NADH is oxidized back to NAD⁺ 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^-acceptor) is O₂, becoming reduced to H₂O.

        Oxidation reactions are exergonic, and the energy released is coupled with the formation of ATP in a process called oxidative phosphorylation. The NAD⁺-NADH system can be viewed as a shuttle that carries the electrons released from catabolic substrates to the mitochondria, where they are transferred to O₂, the ultimate electron 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 energy transduction. Chapter 21 is devoted to electron transport reactions and oxidative phosphorylation.

Figure 18.10 • Hydrogen and electrons released in the course of oxidative catabolism are transferred as hydride ions to the pyridine nucleotide, NAD⁺, to form NADH + H⁺ in dehydrogenase reactions of the type
            AH₂ + NAD⁺ → A + NADH + H⁺
The reaction shown is catalyzed by alcohol dehydrogenase.

NADPH Provides the Reducing Power for Anabolic Processes

Figure 18.11• ^{Transfer of reducing equivalents from catbolism to anabolism via the NADPH cycle.}

Whereas catabolism is fundamentally an oxidative process, anabolism is, by its contrasting nature, reductive. The biosynthesis of the complex constituents of the cell begins at the level of intermediates derived from the degradative pathways of catabolism; or, less commonly, biosynthesis begins with oxidized substances available in the inanimate environment, such as carbon dioxide. When the hydrocarbon chains of fatty acids are assembled from acetyl-CoA units, activated hydrogens are needed to reduce the carbonyl (C=O) carbon of acetyl-CoA into a-CH₂— at every other position along the chain. When glucose is synthesized from CO₂ during photosynthesis in plants, reducing power is required. These reducing equivalents are provided by NADPH, the usual source of high-energy hydrogens for reductive biosynthesis. NADPH is generated when NADP⁺ is reduced with electrons in the form of hydride ions. In heterotrophic organisms, these electrons are removed from fuel molecules by NADP⁺-specific dehydrogenases. In these organisms, NADPH can be viewed as the carrier of electrons from catabolic reactions to anabolic reactions (Figure 18.11). In photosynthetic organisms, the energy of light is used to pull electrons from water and transfer them to NADP⁺; O₂ is a by-product of this process.

18.3 ^• Experimental Methods to Reveal Metabolic Pathways

Armed with the knowledge that metabolism is organized into pathways of successive reactions, we can appreciate by hindsight the techniques employed by early biochemists to reveal their sequence. A major intellectual advance took place at the end of the 19th century when Eduard Buchner showed that the fermentation of glucose to yield ethanol and carbon dioxide can occur in extracts of broken yeast cells. Until this discovery, many thought that metabolism was a vital property, unique to intact cells; even the eminent microbiologist Louis Pasteur, who contributed so much to our understanding of fermentation, was a vitalist, one of those who believed that the processes of living substance transcend the laws of chemistry and physics. After Buchner’s revelation, biochemists searched for intermediates in the transformation of glucose and soon learned that inorganic phosphate was essential to glucose breakdown. This observation gradually led to the discovery of a variety of phosphorylated organic compounds that serve as intermediates along the fermentative pathway.

        An important tool for elucidating the steps in the pathway was the use of metabolic inhibitors. Adding an enzyme inhibitor to a cell-free extract caused an accumulation of intermediates in the pathway prior to the point of inhibition (Figure 18.12). Each inhibitor was specific for a particular site in the sequence of metabolic events. As the arsenal of inhibitors was expanded, the individual steps in metabolism were revealed.

Figure 18.12 • The use of inhibitors to reveal the sequence of reactions in a metabolic pathway. (a) Control: Under normal conditions, the steady-state concentrations of a series of intermediates will be determined by the relative activities of the enzymes in the pathway. (b) Plus inhibitor: In the presence of an inhibitor (in this case, an inhibitor of enzyme 4), intermediates upstream of the metabolic block (B, C, and D) accumulate, revealing themselves as intermediates in the pathway. The concentration of intermediates lying downstream (E and F) will fall.

Mutations Create Specific Metabolic Blocks

Isotopic Tracers as Metabolic Probes

Another widely used approach to the elucidation of metabolic sequences is to “feed” cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard: radioactive isotopes, such as ¹⁴C, and stable “heavy” isotopes, such as ¹⁸O or ¹⁵N (Table 18.3).

Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable “tags” for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13).

Figure 18.13 ^• One of the earliest experiments using a radioactive isotope as a metabolic tracer. Cells of Chlorella (a green alga) synthesizing carbohydrate from carbon dioxide were exposed briefly (5 sec) to ¹⁴C-labeled CO₂. The products of CO₂ incorporation were then quickly isolated from the cells, separated by two-dimensional paper chromatography, and observed via autoradiographic exposure of the chromatogram. Such experiments identified radioactive 3-phosphoglycerate (PGA) as the primary product of CO₂ fixation. The 3-phosphoglycerate was labeled in the 1-position (in its carboxyl group). Radioactive compounds arising from the conversion of 3-phosphoglycerate to other metabolic intermediates included phosphoenolpyruvate (PEP), malic acid, triose phosphate, alanine, and sugar phosphates and diphosphates.
(Photograph courtesy of Professor Melvin Calvin, Lawmann Berkeley Laboratory, University of California, Berkeley.)

Heavy Isotopes

            CO₂+H₂O ® (CH₂O ) +O₂

            C¹⁶O₂+2 H₂¹⁸O ® (CH₂¹⁶O ) +18O₂+H₂¹⁶O

NMR as a Metabolic Probe

A technology analogous to isotopic tracers is provided by nuclear magnetic resonance (NMR) spectroscopy. The atomic nuclei of certain isotopes, such as the naturally occurring isotope of phosphorus, ³¹P, have magnetic moments. The resonance frequency of a magnetic moment is influenced by the local chemical environment. That is, the NMR signal of the nucleus is influenced in an identifiable way by the chemical nature of its neighboring atoms in the compound. In many ways, these nuclei are ideal tracers because their signals contain a great deal of structural information about the environment around the atom, and thus the nature of the compound containing the atom. Transformations of substrates and metabolic intermediates labeled with magnetic nuclei can be traced by following changes in NMR spectra. Furthermore, NMR spectroscopy is a noninvasive procedure. Whole-body NMR spectrometers are being used today in hospitals to directly observe the metabolism (and clinical condition) of living subjects (Figure 18.14). NMR promises to be a revolutionary tool for clinical diagnosis and for the investigation of metabolism in situ (literally “in site,” meaning, in this case, “where and as it happens”).

Figure 18.14 ^• With NMR spectroscopy one can observe the metabolism of a living subject in real time. These NMR spectra show the changes in ATP, creatine-P (phosphocreatine), and P_i levels in the forearm muscle of a human subjected to 19 minutes of exercise. Note that the three P atoms of ATP ( a ,b, and g- ) have different chemical shifts, reflecting their different chemical environments.

Metabolic Pathways Are Compartmentalized Within Cells

Although the interior of a prokaryotic cell is not subdivided into compartments by internal membranes, the cell still shows some segregation of metabolism. For example, certain metabolic pathways, such as phospholipid synthesis and oxidative phosphorylation, are localized in the plasma membrane. Also, protein biosynthesis is carried out on ribosomes.

        In contrast, eukaryotic cells are extensively compartmentalized by an endomembrane system. Each of these cells has a true nucleus bounded by a double membrane called the nuclear envelope. The nuclear envelope is continuous with the endomembrane system, which is composed of differentiated regions: the endoplasmic reticulum; the Golgi complex; various membrane-bounded vesicles such as lysosomes, vacuoles, and microbodies; and, ultimately, the plasma membrane itself. Eukaryotic cells also possess mitochondria and, if they are photosynthetic, chloroplasts. Disruption of the cell membrane and fractionation of the cell contents into the component organelles have allowed an analysis of their respective functions (Figure 18.15).

Each compartment is dedicated to specialized metabolic functions, and the enzymes appropriate to these specialized functions are confined together within the organelle. In many instances, the enzymes of a metabolic sequence occur together within the organellar membrane. Thus, the flow of metabolic intermediates in the cell is spatially as well as chemically segregated. For example, the 10 enzymes of glycolysis are found in the cytosol, but pyruvate, the product of glycolysis, is fed into the mitochondria. These organelles contain the citric acid cycle enzymes, which oxidize pyruvate to CO₂. The great amount of energy released in the process is captured by the oxidative phosphorylation system of mitochondrial membranes and used to drive the formation of ATP (Figure 18.16).

Figure 18.16 ^• Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation.

18.4 ^• Nutrition

Protein

Carbohydrate

The principal purpose of carbohydrate in the diet is production of metabolic energy. Simple sugars are metabolized in the glycolytic pathway (see Chapter 19). Complex carbohydrates are degraded into simple sugars, which then can enter the glycolytic pathway. Carbohydrates are also essential components of nucleotides, nucleic acids, glycoproteins, and glycolipids. Human metabolism can adapt to a wide range of dietary carbohydrate levels, but the brain requires glucose for fuel. When dietary carbohydrate consumption exceeds the energy needs of the organism, excess carbohydrate is converted to triacylglycerols and glycogen for long-term energy storage. On the other hand, when dietary carbohydrate intake is low, ketone bodies are formed from acetate units to provide metabolic fuel for the brain.

Lipid

Fiber

SPECIAL FOCUS:
Vitamins

Vitamins are essential nutrients that are required in the diet, usually in trace amounts, because they cannot be synthesized by the organism itself. The requirement for any given vitamin depends on the organism. Not all “vitamins” are required by all organisms. Vitamins required in the human diet are listed in Table 18.4. These important substances are traditionally distinguished as being either water-soluble or fat-soluble. Except for vitamin C (ascorbic acid), the water-soluble vitamins are all components or precursors of important biological substances known as coenzymes. These are low-molecular-weight molecules that bring unique chemical functionality to certain enzyme reactions. Coenzymes may also act as carriers of specific functional groups, such as methyl groups and acyl groups. The side chains of the common amino acids provide only a limited range of chemical reactivities and carrier properties. Coenzymes, acting in concert with appropriate enzymes, provide a broader range of catalytic properties for the reactions of metabolism. Coenzymes are typically modi-fied by these reactions and are then converted back to their original forms by other enzymes, so that small amounts of these substances can be used repeatedly. The coenzymes derived from the water-soluble vitamins are listed in Table 18.4. Each of these will be discussed in this chapter. The fat-soluble vitamins are not directly related to coenzymes, but they play essential roles in a variety of critical biological processes, including vision, maintenance of bone structure, and blood coagulation. The mechanisms of action of fat-soluble vitamins are not as well understood as their water-soluble counterparts, but modern research efforts are gradually closing this gap.

Vitamin B₁: Thiamine and Thiamine Pyrophosphate

Figure 18.17 ^•  Thiamine pyrophosphate (TPP), the active form of vitamin B₁, is formed by the action of TPP-synthetase.

As shown in Figure 18.17, thiamine is composed of a substituted thiazole ring joined to a substituted pyrimidine by a methylene bridge. It is the precursor of thiamine pyrophosphate (TPP), a coenzyme involved in reactions of carbohydrate metabolism in which bonds to carbonyl carbons (aldehydes or ketones) are synthesized or cleaved. In particular, the decarboxylations of a-keto acids and the formation and cleavage of a-hydroxyketones depend on thiamine pyrophosphate. The first of these is illustrated in Figure 18.18 by (a) the decarboxylation of pyruvate by yeast pyruvate decarboxylase to yield carbon dioxide and acetaldehyde. An example of the formation and cleavage of a-hydroxyketones is presented in Figure 18.18 (b) the condensation of two molecules of pyruvate in the acetolactate synthase reaction. Another example is provided by a reaction from the pentose phosphate pathway (Chapters 22 and 23) called the transketolase reaction. This latter reaction is referred to as an a-ketol transfer for obvious reasons.

Figure 18.18 ^• Thiamine pyrophosphate participates in (a) the decarboxylation of a-keto acids and (b) the formation and cleavage of a-hydroxyketones.

Vitamins Containing Adenine Nucleotides

Several classes of vitamins are related to, or are precursors of, coenzymes that contain adenine nucleotides as part of their structure. These coenzymes include the flavin dinucleotides, the pyridine dinucleotides, and coenzyme A. The adenine nucleotide portion of these coenzymes does not participate actively in the reactions of these coenzymes; rather, it enables the proper enzymes to recognize the coenzyme. Specifically, the adenine nucleotide greatly increases both the affinity and the specificity of the coenzyme for its site on the enzyme, owing to its numerous sites for hydrogen bonding, and also the hydrophobic and ionic bonding possibilities it brings to the coenzyme structure.

Nicotinic Acid and the Nicotinamide Coenzymes

Figure 18.19 • The structures and redox states of the nicotinamide coenzymes. Hydride ion (H:^-, a proton with two electrons) transfers to NAD⁺ to produce NADH.

Nicotinamide is an essential part of two important coenzymes: nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺) (Figure 18.19). The reduced forms of these coenzymes are NADH and NADPH. The nicotinamide coenzymes (also known as pyridine nucleotides) are electron carriers. They play vital roles in a variety of enzyme-catalyzed oxidation-reduction reactions. (NAD⁺ is an electron acceptor in oxidative (catabolic) pathways and NADPH is an electron donor in reductive (biosynthetic) pathways.) These reactions involve direct transfer of hydride anion either to NAD(P)⁺ or from NAD(P)H. The enzymes that facilitate such transfers are thus known as dehydrogenases. The hydride anion contains two electrons, and thus NAD⁺ and NADP⁺ act exclusively as two-electron carriers. The C-4 position of the pyridine ring, which can either accept or donate hydride ion, is the reactive center of both NAD and NADP. The quaternary nitrogen of the nicotinamide ring functions as an electron sink to facilitate hydride transfer to NAD⁺, as shown in Figure 18.20. The adenine portion of the molecule is not directly involved in redox processes.

Figure 18.20 • NAD⁺ and NADP⁺ participate exclusively in two-electron transfer reactions. For example, alcohols can be oxidized to ketones or aldehydes via hydride transfer to NAD(P)⁺.

            Examination of the structures of NADH and NADPH reveals that the 4-position of the nicotinamide ring is pro-chiral , meaning that while this carbon is not chiral, it would be if either of its hydrogens were replaced by something else. As shown in Figure 18.20, the hydrogen “projecting” out of the page toward you is the “pro-R” hydrogen because, if a deuterium is substituted at this position, the molecule would have the R-configuration. Substitution of the other hydrogen would yield an S-configuration. An interesting aspect of the enzymes that require nicotinamide coenzymes is that they are stereospecific and withdraw hydrogen from either the pro-R or the pro-S position selectively. This stereospecificity arises from the fact that enzymes (and the active sites of enzymes) are inherently asymmetric structures. These same enzymes are stereo-specific with respect to the substrates as well.
            The NAD- and NADP-dependent dehydrogenases catalyze at least six different types of reactions: simple hydride transfer, deamination of an amino acid to form an a-keto acid, oxidation of b-hydroxy acids followed by decarboxylation of the b-keto acid intermediate, oxidation of aldehydes, reduction of isolated double bonds, and the oxidation of carbon-nitrogen bonds (as with dihydrofolate reductase ).

Human Biochemistry
Niacin and Pellegra
Pellegra, a disease characterized by dermatitis, diarrhea, and dementia, has been known for centuries. It was once prevalent in the southern part of the United States and is still a common problem in some parts of Spain, Italy, and Romania. Pellegra was once thought to be an infectious disease, but Joseph Goldberger showed early in this century that it could be cured by dietary actions. Soon thereafter, it was found that brewer’s yeast would prevent pellegra in humans. Studies of a similar disease in dogs, called blacktongue, eventually led to the identification of nicotinic acid as the relevant dietary factor. Elvehjem and his colleagues at the University of Wisconsin in 1937 isolated nicotin -amide from liver, and showed that it and nicotinic acid could prevent and cure blacktongue in dogs.	That same year, nicotin-amide and nicotinic acid were both shown to be able to cure pellegra in humans. Interestingly, plants and many animals can synthesize nicotinic acid from tryptophan and other precursors, and nicotinic acid is thus not a true vitamin for these species. However, if dietary intake of tryptophan is low, nicotinic acid is required for optimal health. Nicotinic acid, which is beneficial to humans and animals, is structurally related to nicotine, a highly toxic tobacco alkaloid. In order to avoid confusion of nicotinic acid and nicotinamide with nicotine itself, niacin was adopted as a common name for nicotinic acid. Cowgill, at Yale University, suggested the name from the letters of three words—nicotinic, acid, and vitamin.
	The structures of pyridine, nicotinic acid, nicotinamide, and nicotine.

Riboflavin and the Flavin Coenzymes

Riboflavin, or vitamin B₂, is a constituent and precursor of both riboflavin 5'-phosphate, also known as flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The name riboflavin is a synthesis of the names for the molecule’s component parts, ribitol and flavin. The structures of riboflavin, FMN, and FAD are shown in Figure 18.21.

Figure 18.21 ^•  The structures of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Flavin coenzymes bind tightly to the enzymes that use them, with typical dissociation constants in the range of 10^-8 to 10^-11 M, so that only very low levels of free flavin coenzymes occur in most cells. Even in organisms that rely on the nicotinamide coenzymes (NADH and NADPH) for many of their oxidation-reduction cycles, the flavin coenzymes fill essential roles. Flavins are stronger oxidizing agents than NAD⁺ and NADP⁺. They can be reduced by both one-electron and two-electron pathways and can be reoxidized easily by molecular oxygen. Enzymes that use flavins to carry out their reactions— flavoenzymes —are involved in many kinds of oxidation-reduction reactions.

The isoalloxazine ring is the core structure of the various flavins. Because the ribityl group is not a true pentose sugar (it is a sugar alcohol) and is not joined to riboflavin in a glycosidic bond, the molecule is not truly a “nucleotide,” and the terms flavin mononucleotide and dinucleotide are incorrect. Nonetheless, these designations are so deeply ingrained in common biochemical usage that the erroneous nomenclature persists. The flavins have a characteristic bright yellow color and take their name from the Latin flavus for “yellow.” As shown in Figure 18.22, the oxidized form of the isoalloxazine structure absorbs light around 450 nm (in the visible region) and also at 350 to 380 nm. The color is lost, however, when the ring is reduced or “bleached.” Similarly, the enzymes that bind flavins, known as flavoenzymes, can be yellow, red, or green in their oxidized states. Nevertheless, these enzymes also lose their color on reduction of the bound flavin group.

Figure 18.22 ^• The redox states of FAD and FMN. The boxes correspond to the colors of each of these forms. The atoms primarily involved in electron transfer are indicated by red shading in the oxidized form, white in the semiquinone form, and blue in the reduced form.

            Flavin coenzymes can exist in any of three different redox states. Fully oxidized flavin is converted to a semiquinone by a one-electron transfer, as shown in Figure 18.22. At physiological pH, the semiquinone is a neutral radical, blue in color, with a l_max of 570 nm. The semiquinone possesses a pK_a of about 8.4. When it loses a proton at higher pH values, it becomes a radical anion, displaying a red color with a l_max of 490 nm. The semiquinone radical is particularly stable, owing to extensive delocalization of the unpaired electron across the p-electron system of the isoalloxazine . A second one-electron transfer converts the semiquinone to the completely reduced dihydroflavin as shown in Figure 18.22.
            Access to three different redox states allows flavin coenzymes to participate in one-electron transfer and two-electron transfer reactions. Partly because of this, flavoproteins catalyze many different reactions in biological systems and work together with many different electron acceptors and donors. These include two-electron acceptor/donors, such as NAD⁺ and NADP⁺, one- or two-electron carriers, such as quinones , and a variety of one-electron acceptor/donors, such as cytochrome proteins. Many of the components of the respiratory electron transport chain are one-electron acceptor/donors. The stability of the flavin semiquinone state allows flavoproteins to function as effective electron carriers in respiration processes (Chapter 21).

Pantothenic Acid and Coenzyme A

Pantothenic acid, sometimes called vitamin B₃, is a vitamin that makes up one part of a complex coenzyme called coenzyme A ( CoA) (Figure 18.23). Pantothenic acid is also a constituent of acyl carrier proteins. Coenzyme A consists of 3', 5'-adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage. Phosphopantetheine in turn consists of three parts: b-mercaptoethylamine linked to b-alanine, which makes an amide bond with a branched-chain dihydroxy acid. As was the case for the nicotinamide and flavin coenzymes, the adenine nucleotide moiety of CoA acts as a recognition site, increasing the affinity and specificity of CoA binding to its enzymes.

Figure 18.23 • The structure of coenzyme A. Acyl groups form thioester linkages with the -SH group of the b -mercaptoethylamine moiety.

            The two main functions of coenzyme A are

            (a)        activation of acyl groups for transfer by nucleophilic attack and

            (b)        activation of the a-hydrogen of the acyl group for abstraction as a proton .

Both of these functions are mediated by the reactive sulfhydryl group on CoA , which forms thioester linkages with acyl groups.

            The activation of acyl groups for transfer by CoA can be appreciated by comparing the hydrolysis of the thioester bond of acetyl-CoA with hydrolysis of a simple oxygen ester:

            Ethyl acetate + H₂O ® acetate + ethanol + H⁺         DG°' = 220.0 kJ/mol

            Acetyl- SCoA + H₂O ® acetate + Coa-SH + H⁺   DG°' = 231.5 kJ/mol

Hydrolysis of the thioester is more favorable than that of oxygen esters, presumably because the carbon-sulfur bond has less double bond character than the corresponding carbon-oxygen bond. This means that transfer of the acetyl group from acetyl-CoA to a given nucleophile (Figure 18.24) will be more spontaneous than transfer of an acetyl group from an oxygen ester. For this reason, acetyl-CoA is said to have a high group-transfer potential.

Figure 18.24 ^• Acyl transfer from acyl-CoA to a nucleophile is more favorable than transfer of an acyl group from an oxygen ester.

            The 4-phosphopantetheine group of CoA is also utilized (for essentially the same purposes) in acyl carrier proteins ( ACPs ) involved in fatty acid biosynthesis (see Chapter 25). In acyl carrier proteins, the 4-phosphopantetheine is covalently linked to a serine hydroxyl group. Pantothenic acid is an essential factor for the metabolism of fat, protein, and carbohydrates in the tricarboxylic acid cycle and other pathways. In view of its universal importance in metabolism, it is surprising that pantothenic acid deficiencies are not a more serious problem in humans, but this vitamin is abundant in almost all foods, so that deficiencies are rarely observed.

Figure 18.25 • The tautomeric forms of pyridoxal-5-phosphate (PLP).

Vitamin B₆: Pyridoxine and Pyridoxal Phosphate

The biologically active form of vitamin B₆ is pyridoxal-5-phosphate (PLP), a coenzyme that exists under physiological conditions in two tautomeric forms (Figure 18.25). PLP participates in the catalysis of a wide variety of reactions involving amino acids, including transaminations, a- and b-decarboxylations , b- and g-eliminations, racemizations, and aldol reactions (Figure 18.26). Note that these reactions include cleavage of any of the bonds to the amino acid alpha carbon, as well as several bonds in the side chain. The remarkably versatile chemistry of PLP is due to its ability to

     (a) form stable Schiff base ( aldimine ) adducts with a-amino groups of amino acids, and

     (b)  act as an effective electron sink to stabilize reaction intermediates.

Figure 18.26 • The seven classes of reactions catalyzed by pyridoxal-5-phosphate.

            The Schiff base formed by PLP and its role as an electron sink are illustrated in Figure 18.27. In nearly all PLP-dependent enzymes, PLP in the absence of substrate is bound in a Schiff base linkage with the Î-NH ₂ group of an active site lysine. Rearrangement to a Schiff base with the arriving substrate is a transaldiminization reaction. One key to PLP chemistry is the protonation of the Schiff base, which is stabilized by H bonding to the ring oxygen, increasing the acidity of the C_a proton [as shown in (3) of Figure 18.27]. The carbanion formed by loss of the C_a proton is stabilized by electron delocalization into the pyridinium ring, with the positively charged ring nitrogen acting as an electron sink. Another important intermediate is formed by protonation of the aldehyde carbon of PLP. As shown, this produces a new substrate - PLP Schiff base, which plays a role in transamination reactions and increases the acidity of the proton at C_b, a feature important in gelimination reactions.