Chapter 2:  Small Molecules, Energy, and Biosynthesis

Cell Chemistry Is Based on Carbon Compounds ¹

A living cell is composed of a restricted set of elements, four of which (C, H, N, and O) make up nearly 99% of its weight. This composition differs markedly from that of the earth's crust and is evidence of a distinctive type of chemistry (Figure 2-1). What is this special chemistry, and how did it evolve?

The most abundant substance of the living cell is water. It accounts for about 70% of a cell's weight, and most intracellular reactions occur in an aqueous environment. Life on this planet began in the ocean, and the conditions in that primeval environment put a permanent stamp on the chemistry of living things. All organisms have been designed around the special properties of water, such as its polar character, its ability to form hydrogen bonds, and its high surface tension. Water will completely surround polar molecules, for example, while tending to push nonpolar molecules together into larger assemblies. Some important properties of water are summarized in Panel 2-1 (pp. 48-49).

If we disregard water, nearly all of the molecules in a cell are carbon compounds, which are the subject matter of organic chemistry. Carbon is outstanding among all the elements on earth for its ability to form large molecules; silicon is a poor second. The carbon atom, because of its small size and four outer-shell electrons, can form four strong covalent bonds with other atoms. Most important, it can join to other carbon atoms to form chains and rings and thereby generate large and complex molecules with no obvious upper limit to their size. The other abundant atoms in the cell (H, N, and O) are also small and able to make very strong covalent bonds (Panel 2-2, pp. 50-51).

A typical covalent bond in a biological molecule has an energy of 15 to 170 Kcal/mole, depending on the atoms involved. Since the average thermal energy at body temperature is only 0.6 kcal/mole, even an unusually energetic collision with another molecule will leave a covalent bond intact. Specific catalysts, however, can rapidly break or rearrange covalent bonds. Biology is made possible by the combination of the stability of covalent bonds under physiological conditions and the ability of biological catalysts (called enzymes) to break and rearrange these bonds in a controlled way in selected molecules.

In principle, the simple rules of covalent bonding between carbon and other elements permit an infinitely large number of compounds. Although the number of different carbon compounds in a cell is very large, it is only a tiny subset of what is theoretically possible. In some cases we can point to good reasons why this compound or that performs a given biological function; more often it seems that the actual "choice" was one among many reasonable alternatives and therefore something of an accident (Figure 2-2). Once established in an ancient cell, certain chemical themes and patterns of reaction were preserved, with variations, during billions of years of cellular evolution. Apparently, the development of new classes of compounds was only rarely necessary or useful.

Cells Use Four Basic Types of Small Molecules ²

Certain simple combinations of atoms - such as the methyl (-CH₃), hydroxyl (-OH), carboxyl (-COOH), and amino (-NH₂) groups - recur repeatedly in biological molecules. Each such group has distinct chemical and physical properties that influence the behavior of whatever molecule the group occurs in. The main types of chemical groups and some of their salient properties are summarized in Panel 2-2 (pp. 50-51).

The atomic weights of H, C, N, and O are 1, 12, 14, and 16, respectively. The small organic molecules of the cell have molecular weights in the range 100 to 1000 and contain up to 30 or so carbon atoms. They are usually found free in solution, where some of them form a pool of intermediates from which large polymers, called macromolecules, are made. They are also essential intermediates in the chemical reactions that transform energy derived from food into usable forms (discussed below).

The small molecules amount to about one-tenth of the total organic matter in a cell, and (at a rough estimate) only on the order of a thousand different kinds are present (Table 2-1). All biological molecules are synthesized from and broken down to the same simple compounds. Both synthesis and breakdown occur through sequences of chemical changes that are limited in scope and follow definite rules. As a consequence, the compounds in a cell are chemically related and can be classified into a small number of distinct families. Since the macromolecules in a cell, which form the subject of Chapter 3, are assembled from these small molecules, they belong to corresponding families.

Broadly speaking, cells contain just four major families of small organic molecules: the simple sugars, the fatty acids, the amino acids, and the nucleotides. Each of these families contains many different members with common chemical features. Although some cellular compounds do not fit into these categories, the four families, and especially the macromolecules made from them, account for a surprisingly large fraction of the mass of every cell (Table 2-1).

Sugars Are Food Molecules of the Cell ³

The simplest sugars - the monosaccharides - are compounds with the general formula (CH₂O)_n, where n is an integer from 3 through 7. Glucose,for example, has the formula C₆H₁₂O₆ (Figure 2-3). As shown in Figure 2-3, sugars can exist in either a ring or an open-chain form. In their open-chain form sugars contain a number of hydroxyl groups and either one aldehyde (_H> C=O) or one ketone (> C=O) group. The aldehyde or ketone group plays a special role. First, it can react with a hydroxyl group in the same molecule to convert the molecule into a ring; in the ring form the carbon of the original aldehyde or ketone group can be recognized as the only one that is bonded to two oxygens. Second, once the ring is formed, this carbon can become further linked to one of the carbons bearing a hydroxyl group on another sugar molecule, creating a disaccharide (Panel 2-3, pp. 52-53). The addition of more monosaccharides in the same way results in oligosaccharides of increasing length (trisaccharides, tetrasaccharides, and so on) up to very large polysaccharide molecules with thousands of monosaccharide units. Because each monosaccharide has several free hydroxyl groups that can form a link to another monosaccharide (or to some other compound), the number of possible polysaccharide structures is extremely large. Even a simple disaccharide consisting of two glucose residues can exist in eleven different varieties (Figure 2-4), while three different hexoses (C₆H₁₂O₆) can join together to make several thousand different trisaccharides. It is very difficult to determine the structure of any particular polysaccharide because one needs to determine the sites of linkage between each sugar unit and its neighbors. With present methods, for instance, it takes longer to determine the arrangement of half a dozen linked sugars (those in a glycoprotein, for example) than to determine the nucleotide sequence of a DNA molecule containing many thousands of nucleotides (where each unit is joined to the next in exactly the same way).

Glucose is the principal food compound of many cells. A series of oxidative reactions (see p. 62) leads from this hexose to various smaller sugar derivatives and eventually to CO₂ and H₂O. The net result can be written

In the course of glucose breakdown, energy and "reducing power," both of which are essential in biosynthetic reactions, are salvaged and stored, mainly in the form of ATP in the case of energy and NADH for reducing power. We discuss the structures and functions of these two crucial molecules later in the chapter.

Simple polysaccharides composed only of glucose residues - principally glycogen in animal cells and starch in plant cells - are used to store energy for future use. But sugars have functions in addition to the production and storage of energy. Important extracellular structural materials (such as cellulose) are composed of simple polysaccharides, and smaller but more complex chains of sugar molecules are often covalently linked to proteins in glycoproteins and to lipids in glycolipids.

Fatty Acids Are Components of Cell Membranes ⁴

A fatty acid molecule, such as palmitic acid (Figure 2-5), has two distinct regions: a long hydrocarbon chain, which is hydrophobic (water insoluble) and not very reactive chemically, and a carboxylic acid group, which is ionized in solution (COO^-), extremely hydrophilic (water soluble), and readily reacts with a hydroxyl or an amino group on a second molecule to form esters and amides. In fact, almost all of the fatty acid molecules in a cell are covalently linked to other molecules by their carboxylic acid group. The many different fatty acids found in cells differ in the length of their hydrocarbon chains and the number and position of the carbon-carbon double bonds they contain (Panel 2-4, pp. 54-55).

Fatty acids are a valuable source of food since they can be broken down to produce more than twice as much usable energy, weight for weight, as glucose. They are stored in the cytoplasm of many cells in the form of droplets of triglyceride molecules, which consist of three fatty acid chains, each joined to a glycerol molecule (Panel 2-4, pp. 54-55); these molecules are the animal fats familiar from everyday experience. When required to provide energy, the fatty acid chains can be released from triglycerides and broken down into two-carbon units. These two-carbon units, present as the acetyl group in a water-soluble molecule called acetyl CoA, are then further degraded in various energy-yielding reactions, which we describe below.

But the most important function of fatty acids is in the construction of cell membranes. These thin, impermeable sheets that enclose all cells and surround their internal organelles are composed largely of phospholipids, which are small molecules that resemble triglycerides in that they are constructed mostly from fatty acids and glycerol. In phospholipids, however, the glycerol is joined to two rather than three fatty acid chains. The remaining site on the glycerol is coupled to a negatively charged phosphate group, which is in turn attached to another small hydrophilic compound, such as ethanolamine, choline, or serine.

Each phospholipid molecule, therefore, has a hydrophobic tail - composed of the two fatty acid chains - and a hydrophilic polar head group, where the phosphate is located. A small amount of phospholipid will spread over the surface of water to form a monolayer of phospholipid molecules; in this thin film, the hydrophobic tail regions pack together very closely facing the air and the hydrophilic head groups are in contact with the water (Panel 2-4, pp. 54-55). Two such films can combine tail to tail in water to make a phospholipid sandwich, or lipid bilayer, an extremely important assembly that is the structural basis of all cell membranes (discussed in Chapter 10).

Amino Acids Are the Subunits of Proteins ⁵

The common amino acids are chemically varied, but they all contain a carboxylic acid group and an amino group, both linked to a single carbon atom (called the alpha-carbon; Figure 2-6). They serve as subunits in the synthesis of proteins, which are long linear polymers of amino acids joined head to tail by a peptide bond between the carboxylic acid group of one amino acid and the amino group of the next (Figure 2-7). Although there are many different possible amino acids, only 20 are common in proteins, each with a different side chain attached to the alpha-carbon atom (Panel 2-5, pp. 56-57). The same 20 amino acids occur over and over again in all proteins, including those made by bacteria, plants, and animals. Although the choice of these particular 20 amino acids probably occurred by chance in the course of evolution, the chemical versatility they provide is vitally important. For example, 5 of the 20 amino acids have side chains that can carry a charge (Figure 2-8), whereas the others are uncharged but reactive in specific ways (Panel 2-5, pp. 56-57). As we shall see, the properties of the amino acid side chains, in aggregate, determine the properties of the proteins they constitute and underlie all of the diverse and sophisticated functions of proteins.

Nucleotides Are the Subunits of DNA and RNA ⁶

In nucleotides one of several different nitrogen-containing ring compounds (often referred to as bases because they can combine with H⁺ in acidic solutions) is linked to a five-carbon sugar (either ribose or deoxyribose) that carries a phosphate group. There is a strong family resemblance between the different nitrogen-containing rings found in nucleotides. Cytosine (C), thymine (T), and uracil (U) are called pyrimidine compounds because they are all simple derivatives of a six-membered pyrimidine ring; guanine (G) and adenine (A) are purine compounds, with a second five-membered ring fused to the six-membered ring. Each nucleotide is named by reference to the unique base that it contains (Panel 2-6, pp.58-59).

Nucleotides can act as carriers of chemical energy. The triphosphate ester of adenine, ATP (Figure 2-9), above all others, participates in the transfer of energy in hundreds of individual cellular reactions. Its terminal phosphate is added using energy from the oxidation of foodstuffs, and this phosphate can be split off readily by hydrolysis to release energy that drives energetically unfavorable biosynthetic reactions elsewhere in the cell. As we discuss later, other nucleotide derivatives serve as carriers for the transfer of particular chemical groups, such as hydrogen atoms or sugar residues, from one molecule to another. And a cyclic phosphate-containing adenine derivative, cyclic AMP, serves as a universal signaling molecule within cells.

The special significance of nucleotides is in the storage of biological information. Nucleotides serve as building blocks for the construction of nucleic acids, long polymers in which nucleotide subunits are covalently linked by the formation of a phosphate ester between the 3'-hydroxyl group on the sugar residue of one nucleotide and the 5'-phosphate group on the next nucleotide (Figure 2-10). There are two main types of nucleic acids, differing in the type of sugar that forms their polymeric backbone. Those based on the sugar ribose are known as ribonucleic acids, or RNA, and contain the four bases A, U, G, and C. Those based on deoxyribose (in which the hydroxyl at the 2' position of ribose is replaced by a hydrogen) are known as deoxyribonucleic acids, or DNA, and contain the four bases A, T, G, and C. The sequence of bases in a DNA or RNA polymer represents the genetic information of the living cell. The ability of the bases from different nucleic acid molecules to recognize each other by noncovalent interactions (called base-pairing) - G with C, and A with either T (in DNA) or U (in RNA) - underlies all of heredity and evolution, as explained in Chapter 3.

Summary

Living organisms are autonomous, self-propagating chemical systems. They are made from a distinctive and restricted set of small carbon-based molecules that are essentially the same for every living species. The main categories are sugars, fatty acids, amino acids, and nucleotides. Sugars are a primary source of chemical energy for cells and can be incorporated into polysaccharides for energy storage. Fatty acids are also important for energy storage, but their most significant function is in the formation of cell membranes. Polymers consisting of amino acids constitute the remarkably diverse and versatile macromolecules known as proteins. Nucleotides play a central part in energy transfer and also are the subunits from which the informational macromolecules, RNA and DNA, are made.

Introduction

Cells must obey the laws of physics and chemistry. The rules of mechanics and of the conversion of one form of energy to another apply just as much to a cell as to a steam engine. There are, however, puzzling features of a cell that, at first sight, seem to place it in a special category. It is common experience that things left to themselves eventually become disordered: buildings crumble, dead organisms become oxidized, and so on. This general tendency is expressed in the second law of thermodynamics, which states that the degree of disorder in the universe (or in any isolated system in the universe) can only increase.

The puzzle is that living organisms maintain, at every level, a very high degree of order; and as they feed, develop, and grow, they appear to create this order out of raw materials that lack it. Order is strikingly apparent in large structures such as a butterfly wing or an octopus eye, in subcellular structures such as a mitochondrion or a cilium, and in the shape and arrangement of molecules from which these structures are built. The constituent atoms have been captured, ultimately, from a relatively disorganized state in the environment and locked together into a precise structure. Even a nongrowing cell requires constant ordering processes for survival since all of its organized structures are subject to spontaneous accidents and must be repaired continually. How is this possible thermodynamically? The answer is that the cell draws in fuel from its environment and releases heat as a waste product. The cell is therefore not an isolated system in the thermodynamic sense.

Biological Order Is Made Possible by the Release of Heat Energy from Cells ⁸

As already mentioned, the second law of thermodynamics states that the amount of order in the universe (that is, in a cell plus its environment) must always decrease. Therefore, the continuous increase in order inside a living cell must be accompanied by an even greater increase in disorder in the cell's environment. Heat is energy in its most disordered form - the random commotion of molecules - and heat is released from the cell by the reactions that order the molecules it contains. The increase in random motion, including bond distortions, of the molecules in the rest of the universe creates a disorder that more than compensates for the increased order in the cell, as required by the laws of thermodynamics for spontaneous processes. In this way the release of heat by a cell to its surroundings allows it to become more highly ordered internally at the same time that the universe as a whole becomes more disordered (Figure 2-11).

It is important to note that the cell will achieve nothing by producing heat unless the heat-generating reactions are directly linked with the processes that generate molecular order in the cell. Such linked reactions are said to be coupled, as we explain later. It is the tight coupling of heat production to an increase in order that distinguishes the metabolism of the cell from the wasteful burning of fuel in a fire.

The creation of order inside the cell is at the expense of the degradation of fuel energy. For plants this fuel energy is initially derived from the electromagnetic radiation of the sun; for animals it is derived from the energy stored in the covalent bonds of the organic molecules that animals eat. Since these organic nutrients are themselves produced by photosynthetic organisms such as green plants, however, the sun is in fact the ultimate energy source for animals also.

Photosynthetic Organisms Use Sunlight to Synthesize Organic Compounds ⁹

Solar energy enters the living world (the biosphere) by means of the photosynthesis carried out by photosynthetic organisms - either plants or bacteria. In photosynthesis electromagnetic energy is converted into chemical bond energy. At the same time, however, part of the energy of sunlight is converted into heat energy, and the release of this heat to the environment increases the disorder of the universe and thereby drives the photosynthetic process.

The reactions of photosynthesis are described in detail in Chapter 14. In broad terms, they occur in two distinct stages. In the first (the light-activated reactions) the visible radiation impinging on a pigment molecule drives the transfer of electrons from water to NADPH and at the same time provides the energy needed for the synthesis of ATP. In the second (the dark reactions) the ATP and NADPH are used to drive a series of "carbon-fixation" reactions in which CO₂ from the air is used to form sugar molecules (Figure 2-12).

The net result of photosynthesis, so far as the green plant is concerned, can be summarized by the equation

This simple equation hides the complex nature of the reactions, which involve many linked reaction steps. Furthermore, although the initial fixation of CO₂ results in sugars, subsequent metabolic reactions soon convert these into the many other small and large molecules essential to the plant cell.

Chemical Energy Passes from Plants to Animals

Animals and other nonphotosynthetic organisms cannot capture energy from sunlight directly and so have to survive on "secondhand" energy obtained by eating plants or on "thirdhand" energy obtained by eating other animals. The organic molecules made by plant cells provide both building blocks and fuel to the organisms that feed on them. All types of plant molecules can serve this purpose - sugars, proteins, polysaccharides, lipids, and many others.

The transactions between plants and animals are not all one-way. Plants, animals, and microorganisms have existed together on this planet for so long that many of them have become an essential part of the others' environment. The oxygen released by photosynthesis, for example, is consumed in the combustion of organic molecules by nearly all organisms, and some of the CO₂ molecules that are "fixed" today into larger organic molecules by photosynthesis in a green leaf were yesterday released into the atmosphere by the respiration of an animal. Thus, carbon utilization is a cyclic process that involves the biosphere as a whole and crosses boundaries between individual organisms (Figure 2-13). Similarly, atoms of nitrogen, phosphorus, and sulfur can, in principle, be traced from one biological molecule to another in a series of similar cycles.

Cells Obtain Energy by the Oxidation of Biological Molecules ¹⁰

The carbon and hydrogen atoms in the molecules taken up as food materials by a cell can serve as fuel because they are not in their most stable form. The earth's atmosphere contains a great deal of oxygen, and in the presence of oxygen the most energetically stable form of carbon is as CO₂ and that of hydrogen is as H₂O. A cell is therefore able to obtain energy from sugars or other organic molecules by allowing their carbon and hydrogen atoms to combine with oxygen to produce CO₂ and H₂O, respectively. The cell, however, does not oxidize organic molecules in one step, as occurs in a fire. Through the use of specific enzyme catalysts, it takes the molecules through a large number of reactions that only rarely involve the direct addition of oxygen. Before we can consider these reactions and the driving force behind them, we need to discuss what is meant by the process of oxidation.

Oxidation, in the sense used above, does not mean only the addition of oxygen atoms; rather, it applies more generally to any reaction in which electrons are transferred from one atom to another. Oxidation in this sense refers to the removal of electrons, and reduction - the converse of oxidation - means the addition of electrons. Thus, Fe²⁺ is oxidized if it loses an electron to become Fe³⁺, and a chlorine atom is reduced if it gains an electron to become Cl^-. The same terms are used when there is only a partial shift of electrons between atoms linked by a covalent bond (Figure 2-14A). When a carbon atom becomes covalently bonded to an atom with a strong affinity for electrons, such as oxygen, chlorine, or sulfur, for example, it gives up more than its equal share of electrons; it therefore acquires a partial positive charge and is said to be oxidized. Conversely, a carbon atom in a C-H linkage has more than its share of electrons, and so it is said to be reduced (Figure 2-14B).

Often, when a molecule picks up an electron (e^-), it picks up a proton (H⁺) at the same time (protons being freely available in an aqueous solution). The net effect in this case is to add a hydrogen atom to the molecule

Even though a proton plus an electron is involved (instead of just an electron), such hydrogenation reactions are reductions, and the reverse, dehydrogenation reactions, are oxidations.

The combustion of food materials in a cell converts the C and H atoms in organic molecules (where they are both in a relatively electron-rich, or reduced, state) to CO₂ and H₂O, where they have given up electrons and are therefore highly oxidized. The shift of electrons from carbon and hydrogen to oxygen allows these atoms to achieve a more stable state and hence is energetically favorable.

The Breakdown of an Organic Molecule Takes Place in a Sequence of Enzyme-catalyzed Reactions ¹¹

Although the most energetically favorable form of carbon is as CO₂ and that of hydrogen is as H₂O, a living organism does not disappear in a puff of smoke for the same reason that the book in your hands does not burst into flame: the molecules of both exist in metastable energy troughs and require activation energy (Figure 2-15) before they can pass to more stable configurations. In the case of the book, the activation energy can be provided by a lighted match. For a living cell the combustion is achieved molecule by molecule in a much more controlled way. The place of the match is taken by an unusually energetic collision of one molecule with another. Moreover, the only molecules that react are those that are bound to the surface of enzymes.

As explained in Chapter 3, enzymes are highly specific protein catalysts. Like all other types of catalysts, they speed up reactions by reducing the activation energy for a particular chemical change. Enzymes bind tightly to their substrate molecules and hold them in a way that greatly reduces the activation energy of one particular reaction that rearranges covalent bonds. By selectively lowering the activation energy of only one reaction path for the bound molecule, enzymes determine which of several alternative bond-breaking and bond-forming reactions occurs (Figure 2-16). After the product of one enzyme is released, it can bind to a second enzyme that catalyzes an additional change. In this way each of the many different molecules in a cell moves from enzyme to enzyme along a specific reaction pathway, and it is the sum of all of these pathways that determines the cell's chemistry. We discuss a few central pathways of energy metabolism later.

The success of living organisms is attributable to their cells' ability to make enzymes of many different types, each with precisely specified properties. Each enzyme has a unique shape and binds a particular set of other molecules (called substrates) in such a way as to speed up a particular chemical reaction enormously, often by a factor of as much as 10¹⁴. Like all other catalysts, enzyme molecules themselves are not changed after participating in a reaction and therefore can function over and over again.

Part of the Energy Released in Oxidation Reactions Is Coupled to the Formation of ATP ¹²

Cells derive useful energy from the "burning" of glucose only because they burn it in a very complex and controlled way. By means of enzyme-directed reaction paths, the synthetic, or anabolic, chemical reactions that create biological order are closely coupled to the degradative, or catabolic, reactions that provide the energy. The crucial difference between a coupled reaction and an uncoupled reaction is illustrated by the mechanical analogy shown in Figure 2-17, where an energetically favorable chemical reaction is represented by rocks falling from a cliff. The kinetic energy of falling rocks would normally be entirely wasted in the form of heat generated when they hit the ground (section A). But, by careful design, part of the kinetic energy could be used to drive a paddle wheel that lifts a bucket of water (section B). Because the rocks can reach the ground only by moving the paddle wheel, we say that the spontaneous reaction of rock falling has been directly coupled to the nonspontaneous reaction of lifting the bucket of water. Note that because part of the energy is now used to do work in section B, the rocks hit the ground with less velocity than in section A, and therefore correspondingly less energy is wasted as heat.

In cells enzymes play the role of paddle wheels in our analogy and couple the spontaneous burning of foodstuffs to reactions that generate ATP. Just as the energy stored in the elevated bucket of water in Figure 2-17 can be dispensed in small doses to drive a wide variety of hydraulic machines (section C), ATP serves as a convenient and versatile store, or currency, of energy to drive many different chemical reactions that the cell needs (Figure 2-18).

The Hydrolysis of ATP Generates Order in Cells ¹³

How does ATP act as a carrier of chemical energy? Under the conditions existing in the cytoplasm, the breakdown of ATP by hydrolysis to release inorganic phosphate (P_i) requires catalysis by an enzyme, but whenever it occurs, it releases a great deal of usable energy. A chemical group that is linked by such a reactive bond is readily transferred to another molecule; for this reason the terminal phosphate in ATP can be considered to exist in an activated state. The bond broken in this hydrolysis reaction is sometimes described as a high-energy bond. There is nothing special about the covalent bond itself, however; it is simply that in aqueous solution the hydrolysis of ATP creates two molecules of much lower energy (ADP and P_i).

Many of the chemical reactions in cells are energetically unfavorable. These reactions are driven by the energy released by ATP hydrolysis through enzymes that directly couple the unfavorable reaction to the favorable reaction of ATP hydrolysis. Among these reactions are those involved in the synthesis of biological molecules, in the active transport of molecules across cell membranes, and in the generation of force and movement. These processes play a vital part in establishing biological order. The macromolecules formed in biosynthetic reactions, for example, carry information, catalyze specific reactions, and are assembled into highly ordered structures. Membrane-bound pumps maintain the special internal composition of cells and permit many signals to pass within and between cells. And the production of force and movement enables the cytoplasmic contents of cells to become organized and the cells themselves to move about and assemble into tissues.

Summary

Living cells are highly ordered and must create order within themselves in order to survive and grow. This is thermodynamically possible only because of a continual input of energy, part of which is released from the cells to their environment as heat. The energy comes ultimately from the electromagnetic radiation of the sun, which drives the formation of organic molecules in photosynthetic organisms such as green plants. Animals obtain their energy by eating these organic molecules and oxidizing them in a series of enzyme-catalyzed reactions that are coupled to the formation of ATP. ATP is a common currency of energy in all cells, and its energetically favorable hydrolysis is coupled to other reactions to drive a variety of energetically unfavorable processes that create the high degree of order essential for life.

Food Molecules Are Broken Down in Three Stages to Give ATP

The proteins, lipids, and polysaccharides that make up the major part of the food we eat must be broken down into smaller molecules before our cells can use them. The enzymatic breakdown, or catabolism, of these molecules may be regarded as proceeding in three stages (Figure 2-19). We shall give a short outline of these stages before discussing the last two of them in more detail.

Stage 1, called digestion, occurs mainly in our intestine. Here, large polymeric molecules are broken down into their monomeric subunits - proteins into amino acids, polysaccharides into sugars, and fats into fatty acids and glycerol - through the action of secreted enzymes. Stage 2 occurs in the cytoplasm after the small molecules generated in stage 1 enter cells, where they are further degraded. Most of the carbon and hydrogen atoms of sugars are converted into pyruvate, which then enters mitochondria, where it is converted to the acetyl groups of the chemically reactive compound acetyl coenzyme A (acetyl CoA) (Figure 2-20). Major amounts of acetyl CoA are also produced by the oxidation of fatty acids. In stage 3 the acetyl group of acetyl CoA is completely degraded to CO₂ and H₂O in the mitochondrion. It is in this final stage that most of the ATP is generated. Through a series of coupled chemical reactions, about half of the energy theoretically derivable from the combustion of carbohydrates and fats to H₂O and CO₂ is channeled into driving the energetically unfavorable reaction P_i + ADP → ATP. Because the rest of the combustion energy is released by the cell as heat, this generation of ATP creates net disorder in the universe, in conformity with the second law of thermodynamics.

Through the production of ATP, the energy originally derived from the combustion of carbohydrates and fats is redistributed as a conveniently packaged form of chemical energy. Roughly 10⁹ molecules of ATP are in solution throughout the intracellular space in a typical cell, where their energetically favorable hydrolysis back to ADP and phosphate in coupled reactions provides the driving energy for a very large number of different coupled reactions that would otherwise not occur.

Glycolysis Can Produce ATP Even in the Absence of Oxygen

The most important process in stage 2 of catabolism is the degradation of carbohydrates in a sequence of reactions known as glycolysis - the lysis (splitting) of glucose. Glycolysis can produce ATP in the absence of oxygen, and it probably evolved early in the history of life, before the activities of photosynthetic organisms introduced oxygen into the atmosphere. In the process of glycolysis, a glucose molecule with six carbon atoms is converted into two molecules of pyruvate, each with three carbon atoms. This conversion involves a sequence of nine enzymatic steps that create phosphate-containing intermediates (Figure 2-21). The cell hydrolyzes two molecules of ATP to drive the early steps but produces four molecules of ATP in the later steps, so that there is a net gain of ATP by the end of glycolysis.

Logically, the sequence of reactions that constitute glycolysis can be divided into three parts: (1) in steps 1 to 4, glucose is converted to two molecules of the three-carbon aldehyde glyceraldehyde 3-phosphate - a conversion that requires an investment of energy in the form of ATP hydrolysis to provide the two phosphates; (2) in steps 5 and 6, the aldehyde group of each glyceraldehyde 3-phosphate molecule is oxidized to a carboxylic acid, and the energy from this reaction is coupled to the synthesis of ATP from ADP and inorganic phosphate; and (3) in steps 7, 8, and 9, the same two phosphate molecules that were added to sugars in the first reaction sequence are transferred back to ADP to form ATP, thereby repaying the original investment of two ATP molecules hydrolyzed in the first reaction sequence (see Figure 2-21).

At the end of glycolysis, therefore, the ATP balance sheet shows a net profit of the two molecules of ATP (per glucose molecule) that were produced in steps 5 and 6. As the only reactions in the sequence in which a high-energy phosphate linkage is created from inorganic phosphate, these two steps lie at the heart of glycolysis. They also provide an excellent illustration of the way in which reactions in the cell can be coupled together by enzymes to harvest the energy released by oxidations (Figure 2-22). The overall result is that an aldehyde group on a sugar is oxidized to a carboxylic acid and an inorganic phosphate group is transferred to a high-energy linkage on ATP; in addition, a molecule of NAD⁺ is reduced to NADH, a molecule with a central role in energy metabolism, as we discuss next. This elegant set of coupled reactions was probably among the earliest metabolic steps to appear in the evolving cell.

For most animal cells glycolysis is only a prelude to stage 3 of catabolism, since the pyruvic acid that is formed at the last step quickly enters the mitochondria to be completely oxidized to CO₂ and H₂O. In the case of anaerobic organisms (those that do not utilize molecular oxygen), however, and for tissues, such as skeletal muscle, that can function under anaerobic conditions, glycolysis can become a major source of the cell's ATP. Anaerobic energy-yielding reactions of this type are called fermentations. Here, instead of being degraded in mitochondria, the pyruvate molecules stay in the cytosol and, depending on the organism, can be converted into ethanol plus CO₂ (as in yeast) or into lactate (as in muscle), which is then excreted from the cell. These further reactions of pyruvate use up the reducing power produced in reaction 5 of glycolysis, thereby regenerating the NAD⁺ required for glycolysis to continue, as we discuss in Chapter 14.

NADH Is a Central Intermediate in Oxidative Catabolism

The anaerobic generation of ATP from glucose through the reactions of glycolysis is relatively inefficient. The end products of anaerobic glycolysis still contain a great deal of chemical energy that can be released by further oxidation. The evolution of oxidative catabolism (cellular respiration) became possible only after molecular oxygen had accumulated in the earth's atmosphere as a result of photosynthesis by the cyanobacteria. Earlier, anaerobic processes had dominated life on earth. The addition of an oxygen-requiring stage to the catabolic process (stage 3 in Figure 2-19) provided cells with a much more powerful and efficient method for extracting energy from food molecules. This third stage begins with a series of reactions called the citric acid cycle (also called the tricarboxylic acid cycle, or the Krebs cycle) and ends with oxidative phosphorylation, both of which occur in aerobic bacteria and the mitochondria of eucaryotic cells.

A simplified version of the two central processes of oxidative catabolism is given in Figure 2-23. First, in the citric acid cycle, the acetyl groups from acetyl CoA are oxidized to produce CO₂ and NADH. Next, in the process of oxidative phosphorylation, the NADH generated reacts with molecular oxygen (O₂) to produce ATP and H₂O in a complicated series of steps that relies on electron transport in a membrane.

NADH, which serves as a central intermediate in the above process, was previously encountered as a product of glycolysis (see Figure 2-22). It is an important carrier of reducing power in cells. As illustrated in Figure 2-24, it is formed by the addition of a hydrogen nucleus and two electrons (a hydride ion, H^-) to nicotinamide adenine dinucleotide (NAD). Because this addition occurs in a way that leaves the hydride ion held in a high-energy linkage, NADH acts as a convenient source of readily transferable electrons in cells, in much the same way that ATP acts as a convenient source of readily transferable phosphate groups.

Metabolism Is Dominated by the Citric Acid Cycle ¹⁵

The primary function of the citric acid cycle is to oxidize acetyl groups that enter the cycle in the form of acetyl CoA molecules. The reactions form a cycle because the acetyl group is not oxidized directly, but only after it has been covalently added to a larger molecule, oxaloacetate,which is regenerated at the end of one turn of the cycle. As illustrated in Figure 2-25, the cycle begins with the reaction between acetyl CoA and oxaloacetate to form the tricarboxylic acid molecule called citric acid (or citrate). A series of enzymatically catalyzed reactions then occurs in which two of the six carbons of citrate are oxidized to CO₂, forming another molecule of oxaloacetate to repeat the cycle. (Because the two carbons that are newly added in each cycle enter a different part of the citrate molecule from the part oxidized to CO₂, it is only after several cycles that their turn comes to be oxidized.) The CO₂ produced in these reactions then diffuses from the mitochondrion (or from the bacterium) and leaves the cell.

The energy made available when the C - H and C - C bonds in citrate are oxidized is captured in several ways in the course of the citric acid cycle. At one step in the cycle (succinyl CoA to succinate), a high-energy phosphate linkage is created by a mechanism resembling that described for glycolysis above. All of the remaining energy of oxidation that is captured is channeled into the conversion of hydrogen - or hydride ion - carrier molecules to their reduced forms; for each turn of the cycle, three molecules of NAD⁺ are converted to NADH and one flavin adenine nucleotide (FAD) is converted to FADH₂. The energy that is stored in the readily transferred electrons on these carrier molecules will subsequently be harnessed through the reactions of oxidative phosphorylation (considered in more detail below), which are the only reactions described here that require molecular oxygen from the atmosphere.

The additional oxygen atoms required to make CO₂ from the acetyl groups entering the citric acid cycle are supplied not by molecular oxygen but by water. Three molecules of water are split in each cycle, and their oxygen atoms are used to make CO₂. Some of their hydrogen atoms enter substrate molecules and, like the hydrogen atoms of the acetyl groups, are ultimately removed to carrier molecules such as NADH.

In the eucaryotic cell the mitochondrion is the center toward which all catabolic processes lead, whether they begin with sugars, fats, or proteins. For, in addition to pyruvate, fatty acids and some amino acids also pass from the cytosol into mitochondria, where they are converted into acetyl CoA or one of the other intermediates of the citric acid cycle. The mitochondrion also functions as the starting point for some biosynthetic reactions by producing vital carbon-containing intermediates, such as oxaloacetate and alpha-ketoglutarate. These substances are transferred back from the mitochondrion to the cytosol, where they serve as precursors for the synthesis of essential molecules, such as amino acids.

In Oxidative Phosphorylation the Transfer of Electrons to Oxygen Drives ATP Formation ^{10, 16}

Oxidative phosphorylation is the last step in catabolism and the point at which the major portion of metabolic energy is released. In this process molecules of NADH and FADH₂ transfer the electrons that they have gained from the oxidation of food molecules to molecular oxygen, O₂, forming H₂O. The reaction, which is formally equivalent to the burning of hydrogen in air to form water, releases a great deal of chemical energy. Part of this energy is used to make the major portion of the cell's ATP; the rest is liberated as heat.

Although the overall chemistry of NADH and FADH₂ oxidation involves a transfer of hydrogen to oxygen, each hydrogen atom is transferred as an electron plus a proton (the hydrogen nucleus, H⁺). This is possible because a hydrogen atom can be readily dissociated into its constituent electron and proton (H⁺). The electron can then be transferred separately to a molecule that accepts only electrons, while the proton remains in aqueous solution. Conversely, if an electron alone is donated to a molecule with a strong affinity for hydrogen, then a hydrogen atom will be reconstituted automatically by the capture of a proton from solution. In the course of oxidative phosphorylation, electrons from NADH and FADH₂ pass down a long chain of carrier molecules that are known as the electron-transport chain. The presence or absence of intact hydrogen atoms at each step of the electron-transfer process depends on the nature of the carrier.

In a eucaryotic cell this series of electron transfers along the electron-transport chain takes place on the inner membrane of the mitochondrion, in which all of the electron carrier molecules are embedded. At each step of the transfer, the electrons fall to a lower energy state, until at the end they are transferred to oxygen molecules. Each oxygen molecule (O₂) picks up four electrons from the electron-transport chain plus four protons from aqueous solution to form two molecules of water. Oxygen molecules have a high affinity for electrons, and electrons bound to oxygen are thus in a low energy state.

The electron-transport chain is important for the cell because the energy released as the electrons fall to lower energy states is harnessed in a remarkable way. As described in Chapter 14, particular electron transfers cause protons to be pumped across the membrane from the inner mitochondrial compartment to the outside (Figure 2-26). An electrochemical proton gradient is thereby generated across the inner mitochondrial membrane. This gradient in turn drives a flux of protons back through a special enzyme complex in the same membrane, causing the enzyme (ATP synthase) to add a phosphate group to ADP and thereby generating ATP inside the mitochondrion. The newly made ATP is then transferred from the mitochondrion to the rest of the cell.

Amino Acids and Nucleotides Are Part of the Nitrogen Cycle

In our discussion so far we have concentrated mainly on carbohydrate metabolism. We have not yet considered the metabolism of nitrogen or sulfur. These two elements are constituents of proteins and nucleic acids, which are the two most important classes of macromolecules in the cell and make up approximately two-thirds of its dry weight. Atoms of nitrogen and sulfur pass from compound to compound and between organisms and their environment in a series of reversible cycles.

Although molecular nitrogen is abundant in the earth's atmosphere, nitrogen is chemically unreactive as a gas. Only a few living species are able to incorporate it into organic molecules, a process called nitrogen fixation. Nitrogen fixation occurs in certain microorganisms and by some geophysical processes, such as lightning discharge. It is essential to the biosphere as a whole, for without it life would not exist on this planet. Only a small fraction of the nitrogenous compounds in today's organisms, however, represents fresh products of nitrogen fixation from the atmosphere. Most organic nitrogen has been in circulation for some time, passing from one living organism to another. Thus present-day nitrogen-fixing reactions can be said to perform a "topping-up" function for the total nitrogen supply.

Vertebrates receive virtually all of their nitrogen in their dietary intake of proteins and nucleic acids. In the body these macromolecules are broken down to component amino acids and nucleotides, which are then repolymerized into new proteins and nucleic acids or utilized to make other molecules. About half of the 20 amino acids found in proteins are essential amino acids (Figure 2-27) for vertebrates, which means that they cannot be synthesized from other ingredients of the diet. The others can be so synthesized, using a variety of raw materials, including intermediates of the citric acid cycle. The essential amino acids are made by nonvertebrate organisms, usually by long and energetically expensive pathways that have been lost in the course of vertebrate evolution.

The nucleotides needed to make RNA and DNA can be synthesized using specialized biosynthetic pathways: there are no "essential nucleotides" that must be provided in the diet. All of the nitrogens in the purine and pyrimidine bases (as well as some of the carbons) are derived from the plentiful amino acids glutamine, aspartic acid, and glycine, whereas the ribose and deoxyribose sugars are derived from glucose.

Amino acids that are not utilized in biosynthesis can be oxidized to generate metabolic energy. Most of their carbon and hydrogen atoms eventually form CO₂ or H₂O, whereas their nitrogen atoms are shuttled through various forms and eventually appear as urea, which is excreted. Each amino acid is processed differently, and a whole constellation of enzymatic reactions exists for their catabolism.

Summary

Animal cells derive energy from food in three stages. In stage 1, called digestion, proteins, polysaccharides, and fats are broken down by extracellular reactions to small molecules. In stage 2, these small molecules are degraded within cells to produce acetyl CoA and a limited amount of ATP and NADH. These are the only reactions that can yield energy in the absence of oxygen. In stage 3, the acetyl CoA molecules are degraded in mitochondria to give CO₂ and hydrogen atoms that are linked to carrier molecules such as NADH. Electrons from the hydrogen atoms are passed through a complex chain of membrane-bound carriers, finally being passed to molecular oxygen to form water. Driven by the energy released in these electron-transfer steps, protons (H⁺) are transported out of the mitochondria. The resulting electrochemical proton gradient across the inner mitochondrial membrane is harnessed to drive the synthesis of most of the cell's ATP.

Introduction

Thousands of different chemical reactions are occurring in a cell at any instant of time. The reactions are all linked together in chains and networks in which the product of one reaction becomes the substrate of the next. Most of the chemical reactions in cells can be roughly classified as being concerned either with catabolism or with biosynthesis (anabolism). Having discussed the catabolic reactions, we now turn to the reactions of biosynthesis. These begin with the intermediate products of glycolysis and the citric acid cycle (and closely related compounds) and generate the larger and more complex molecules of the cell.

The Free-Energy Change for a Reaction Determines Whether It Can Occur ¹⁸

Although enzymes speed up energetically favorable reactions, they cannot force energetically unfavorable reactions to occur. In terms of a water analogy, enzymes by themselves cannot make water run uphill. Cells, however, must do just that in order to grow and divide; they must build highly ordered and energy-rich molecules from small and simple ones. We have seen that, in a general way, this is done through enzymes that directly couple energetically favorable reactions, which consume energy (derived ultimately from the sun) and produce heat, to energetically unfavorable reactions, which produce biological order. Let us examine in greater detail how such coupling is achieved.

First, we must consider more carefully the term "energetically favorable," which we have so far used loosely without giving it a definition. As explained earlier, a chemical reaction can proceed spontaneously only if it results in a net increase in the disorder of the universe. Disorder increases when useful energy (energy that could be harnessed to do work) is dissipated as heat; and the criterion for an increase of disorder can be expressed conveniently in terms of a quantity called the free energy, G. This is defined in such a way that changes in its value, denoted by Δ- G, measure the amount of disorder created in the universe when a reaction takes place. Energetically favorable reactions, by definition, are those that release a large quantity of free energy, or, in other words, have a large negative Δ- G and create much disorder. A familiar example on a macroscopic scale would be the "reaction" by which a compressed spring relaxes to an expanded state, releasing its stored elastic energy as heat to its surroundings. Energetically favorable reactions, with Δ- G < O, have a strong tendency to occur spontaneously, although their rate will depend on other factors, such as the availability of specific enzymes (discussed below). Conversely, energetically unfavorable reactions, with a positive Δ- G, such as those in which two amino acids are joined together to form a peptide bond, by themselves create order in the universe and therefore do not occur spontaneously. Reactions of this kind can take place only if they are coupled to a second reaction with a negative Δ- G so large that the Δ- G of the entire process is negative.

The course of most reactions can be predicted quantitatively. A large body of thermodynamic data has been collected that makes it possible to calculate the change in free energy for most of the important metabolic reactions of the cell. The overall free-energy change for a pathway is then simply the sum of the free-energy changes in each of its component steps. Consider, for example, two reactions

where the Δ Gvalues are +1 and -13 kcal/mole, respectively. (Recall that a mole is 6 x 10²³ molecules of a substance.) If these two reactions can be coupled together, the Δ- Gfor the coupled reaction will be -12 kcal/mole. Thus, the unfavorable reaction X → Y, which will not occur spontaneously, can be driven by the favorable reaction C → D, provided that a mechanism exists by which the two reactions can be coupled together.

Biosynthetic Reactions Are Often Directly Coupled to ATP Hydrolysis

Consider a typical biosynthetic reaction in which two monomers, A and B, are to be joined in a dehydration (also called condensation) reaction, in which water is released:

Almost invariably the reverse reaction (called hydrolysis), in which water breaks the covalently linked compound A-B, will be the energetically favorable one. This is the case, for example, in the hydrolysis of proteins, nucleic acids, and polysaccharides into their subunits.

The general strategy that allows the cell to make A-B from A-H and B-OH involves a sequence of steps through which the energetically unfavorable synthesis of the desired compound is coupled to an even more energetically favorable reaction (see Figure 2-17). ATP hydrolysis (Figure 2-28) has a large negative Δ- G, and it is the usual source of the free energy used to drive the biosynthetic reactions in a cell. In the coupled pathway from A-H and B-OH to A-B, energy from ATP hydrolysis is first used to convert B-OH to a higher-energy intermediate compound, which then reacts directly with A-H to give A-B. The simplest mechanism involves the transfer of a phosphate from ATP to B-OH to make B-OPO₃ (or BO- ), in which case the reaction pathway contains only two steps:

1. B-OH + ATP → B-O- + ADP

2. A-H + B-O- → A-B + P_i

Since the intermediate B-O- is formed only transiently, the overall reactions that occur are

The first reaction, which by itself is energetically unfavorable, is forced to occur by being directly coupled to the second energetically favorable reaction (ATP hydrolysis). An example of a coupled biosynthetic reaction of this kind, the synthesis of the amino acid glutamine, is shown in Figure 2-29.

The Δ- G for the hydrolysis of ATP to ADP and inorganic phosphate (P_i) depends on the concentrations of all of the reactants, and under the usual conditions in a cell it is between -11 and -13 kcal/mole. In principle, this hydrolysis reaction can be used to drive an unfavorable reaction with a Δ- G of, perhaps, +10 kcal/mole, provided that a suitable reaction path is available. For some biosynthetic reactions, however, even -13 kcal/mole may not be enough. In these cases the path of ATP hydrolysis can be altered so that it initially produces AMP and pyrophosphate (PP_i), which is itself then hydrolyzed in a subsequent step (Figure 2-30). The whole process makes available a total free-energy change of about -26 kcal/mole.

How is the energy of pyrophosphate hydrolysis coupled to a biosynthetic reaction? One way can be illustrated by considering again the synthesis of compound A-B from A-H and B-OH. By an appropriate enzyme, B-OH can be converted to the higher-energy intermediate B-O- - by its reaction with ATP. The complete reaction now contains three steps:

1. B-OH + ATP → B-O- - + AMP

2. A-H + B-O- - → A-B + PP_i

3. PP_i + H₂O → 2P_i

And the overall reactions are

Since an enzyme always facilitates equally the forward and backward directions of the reaction it catalyzes, the compound AB can be destroyed by recombining it with pyrophosphate (a reversal of step 2). But the energetically favorable reaction of pyrophosphate hydrolysis (step 3) greatly stabilizes compound A-B by keeping the concentration of pyrophosphate very low, essentially preventing the reversal of step 2. In this way the energy of pyrophosphate hydrolysis is used to drive this reaction in the forward direction. An example of an important biosynthetic reaction of this kind, polynucleotide synthesis, is illustrated in Figure 2-31.

Coenzymes Are Involved in the Transfer of Specific Chemical Groups

Because the terminal phosphate linkage in ATP is easily cleaved, with release of free energy, ATP acts as an efficient donor of a phosphate group in a large number of phosphorylation reactions. A wide variety of other chemically labile linkages also function in this way, and molecules bearing them often bind tightly to the surface of enzymes so that they can be used efficiently as donors of their reactive group in enzymatically catalyzed reactions. Such molecules are called coenzymes because they are essential for the activity of the enzyme; the same coenzyme can participate in many different biosynthetic reactions in which its group is needed.

Some examples of coenzymes are listed in Table 2-2. Among them is acetyl coenzyme A (acetyl CoA), which we encountered earlier. It carries an acetyl group linked to CoA through a reactive thioester bond (see Figure 2-20). This acetyl group is readily transferred to another molecule, such as a growing fatty acid. Other important coenzymes are NADH, which carries a hydride ion (see Figure 2-24), and biotin, which transfers a carboxyl group in many biosynthetic reactions (Figure 2-32).

Many coenzymes cannot be synthesized by animals and must be obtained from plants or microorganisms in the diet. Vitamins - essential nutritional factors that animals need in trace amounts - are often the precursors of required coenzymes.

The Structure of Coenzymes Suggests That They May Have Originated in an RNA World

As discussed in Chapter 1, the recent discovery that RNA molecules can fold up to form highly specific catalytic surfaces has led to the view that RNAs (or their close relatives) were probably the main catalysts for early life forms and that proteins were a later evolutionary addition. To allow efficient catalysis of the many types of reactions needed in cells, it seems likely that these RNAs would have required a large variety of coenzymes to compensate for their own chemical monotony (being made from only four different nucleotide subunits). Some present-day RNAs contain highly specific binding sites for nucleotides, which utilize non-Watson-Crick, hydrogen-bonded base-base contacts (see Figure 3-21). It is conceivable that coenzymes bound to early RNAs by means of the same type of nucleotide "handle" that is present on the back side of many present-day coenzymes, such as ATP, acetyl CoA, and NADH. According to this view, these molecules evolved with a covalently attached nucleotide because of the nucleotide's earlier usefulness for coenzyme binding in an RNA world.

Biosynthesis Requires Reducing Power

We have seen that oxidation and reduction reactions occur continuously in cells. The chemical energy in food molecules is released by catabolic oxidation reactions, while, in order to make biological molecules, the cell needs (among other things) to carry out a series of reduction reactions that require an input of chemical energy. By using the principle of coupled reactions described previously, cells directly channel chemical energy derived from catabolism into the synthesis of NADH (see Figure 2-22, for example). The high-energy bond between hydrogen and the nicotinamide ring in NADH then provides the energy for otherwise unfavorable enzyme reactions that transfer two electrons plus a proton (as a hydride ion) to another molecule. NADH, and the closely related NADPH to which it can be readily converted, are therefore said to carry "reducing power"; both are used as coenzymes in many types of reduction reactions.

To see how this hydrogen transfer works in practice, consider just one biosynthetic step: the last reaction in a pathway for the synthesis of the lipid molecule cholesterol. In this reaction two hydrogen atoms are added to the polycyclic steroid ring in order to reduce a carbon-carbon double bond. As in most biosynthetic reactions, the constituents of the two hydrogen atoms required in this reaction are supplied as a hydride ion from NADPH and a proton (H⁺) from the solution (H^- + H⁺ = 2H) (Figure 2-33). As in NADH, the hydride ion to be transferred from NADPH is part of a nicotinamide ring and is easily lost because the ring can achieve a more stable aromatic state without it (see Figure 2-24). Therefore, NADH and NADPH both hold a hydride ion in a high-energy linkage from which it can be transferred to another molecule when a suitable enzyme is available to catalyze the transfer.

The difference between NADH and NADPH is trivial in chemical terms, but it is crucial for their distinctive functions. The extra phosphate group on NADPH is far from the active region (Figure 2-34) and is of no importance to the hydride ion transfer reaction; but it determines the enzymes to which NADPH can bind as a coenzyme. As a general rule, NADH operates with enzymes catalyzing catabolic reactions, whereas NADPH operates with enzymes that catalyze biosynetic reactions. By having the two coenzymes act in different pathways, the cell can keep NADPH:NADP⁺ ratios high to provide the reducing power necessary for biosynthetic pathways, while at the same time it can keep NADH:NAD⁺ ratios low to provide the NAD⁺ required to accept electrons during catabolism.

Biological Polymers Are Synthesized by Repetition of Elementary Dehydration Reactions

The principal macromolecules synthesized by cells are polynucleotides (DNA and RNA), polysaccharides, and proteins. They are enormously diverse in structure and include the most complex molecules known. Despite this, they are synthesized from relatively few kinds of small molecules (referred to as either monomers or subunits) by a restricted repertoire of chemical reactions.

An oversimplified outline of the mechanism of addition of monomers to proteins, polynucleotides, and polysaccharides is shown in Figure 2-35. Although the synthetic reactions for each polymer involve a different kind of covalent bond and different enzymes and cofactors, there are strong underlying similarities. As indicated by the red shading, the addition of subunits in each case occurs by a dehydration reaction, involving the removal of a molecule of water from the two reactants.

As in the general case discussed on page 76, the formation of these polymers requires the input of chemical energy, which is achieved by the standard strategy of coupling the biosynthetic reaction to the energetically favorable hydrolysis of a nucleoside triphosphate. For all three types of macromolecules, at least one nucleoside triphosphate is cleaved to produce pyrophosphate, which is subsequently hydrolyzed so that the driving force for the reaction is large. The mechanism used for polynucleotide synthesis was illustrated earlier in Figure 2-31.

The activated intermediates in the polymerization reactions can be oriented in one of two ways, giving rise to either the head polymerization or the tail polymerization of monomers. In head polymerization the reactive bond is carried on the end of the growing polymer and must therefore be regenerated each time a monomer is added. In this case each monomer brings with it the reactive bond that will be used to react with the next monomer in the series (Figure 2-36). In tail polymerization the reactive bond carried by each monomer is used instead for its own addition. Both types of polymerization are used for the synthesis of biological macromolecules. The synthesis of polynucleotides and some simple polysaccharides occurs by tail polymerization, for example, whereas the synthesis of proteins occurs by head polymerization.

Summary

The hydrolysis of ATP is commonly coupled to energetically unfavorable reactions, such as the biosynthesis of macromolecules, by the transfer of phosphate groups to form reactive phosphorylated intermediates. Because the energetically unfavorable reaction now becomes energetically favorable, ATP hydrolysis is said to drive the reaction. Polymeric molecules such as proteins, nucleic acids, and polysaccharides are assembled from small activated precursor molecules by repetitive dehydration reactions that are driven in this way. Other reactive molecules, called coenzymes, transfer other chemical groups in the course of biosynthesis: NADPH transfers hydrogen as a proton plus two electrons (a hydride ion), for example, whereas acetyl CoA transfers acetyl groups.

Metabolism Is Organized and Regulated

Some idea of how intricate a cell is when viewed as a chemical machine can be obtained from Figure 2-37, which is a chart showing only some of the enzymatic pathways in a cell. All of these reactions occur in a cell that is less than 0.1 mm in diameter, and each requires an enzyme that is itself the product of a whole series of information-transfer and protein-synthesis reactions. For a typical small molecule - the amino acid serine, for example - there are half a dozen or more enzymes that can modify it chemically in different ways: it can be linked to AMP (adenylated) in preparation for protein synthesis, or degraded to glycine, or converted to pyruvate in preparation for oxidation; it can be acetylated by acetyl CoA or transferred to a fatty acid to make phosphatidyl serine. All of these different pathways compete for the same serine molecule, and similar competitions for thousands of other small molecules go on at the same time. One might think that the whole system would need to be so finely balanced that any minor upset, such as a temporary change in dietary intake, would be disastrous.

In fact, the cell is amazingly stable. Whenever it is perturbed, the cell reacts so as to restore its initial state. It can adapt and continue to function during starvation or disease. Mutations of many kinds can eliminate particular reaction pathways, and yet - provided that certain minimum requirements are met - the cell survives. It does so because an elaborate network of control mechanisms regulates and coordinates the rates of its reactions. Some of the higher levels of control will be considered in later chapters. Here we are concerned only with the simplest mechanisms that regulate the flow of small molecules through the various metabolic pathways.

Metabolic Pathways Are Regulated by Changes in Enzyme Activity ²⁰

The concentrations of the various small molecules in a cell are buffered against major changes by a process known as feedback regulation, which fine-tunes the flux of metabolites through a particular pathway by temporarily increasing or decreasing the activity of crucial enzymes. The first enzyme of a series of reactions, for example, is usually inhibited by a negative feedback effect of the final product of that pathway: if large quantities of the final product accumulate, further entry of precursors into the reaction pathway is automatically inhibited (Figure 2-38). Where pathways branch or intersect, as they often do, there are usually multiple points of control by different final products. The complexity of such feedback control processes is illustrated in Figure 2-39, which shows the pattern of enzyme regulation observed in a set of related amino acid pathways.

Feedback regulation can work almost instantaneously and is reversible; in addition, a given end product may activate enzymes leading along other pathways, as well as inhibit enzymes that cause its own synthesis. The molecular basis for this type of control in cells is well understood, but since an explanation requires some knowledge of protein structure, it will be deferred until Chapter 5.

Catabolic Reactions Can Be Reversed by an Input of Energy ²¹

By regulating a few enzymes at key points in a metabolic network, a cell can effect large-scale changes in its general metabolism. A special pattern of feedback regulation enables a cell to switch, for example, from glucose degradation to glucose biosynthesis (denoted gluconeogenesis). The need for gluconeogenesis is especially acute in periods of violent exercise, when the glucose needed for muscle contraction is generated from lactic acid by liver cells, and also in periods of starvation, when glucose must be formed from the glycerol portion of fats and from amino acids for survival.

The normal breakdown of glucose to pyruvate during glycolysis is catalyzed by a number of enzymes acting in series. The reactions catalyzed by most of these enzymes are readily reversible, but three reaction steps (numbers 1, 3, and 9 in the sequence of Figure 2-21) are effectively irreversible. In fact, it is the large negative free-energy change that occurs in these reactions that normally drives the sequence in the direction of glucose breakdown. For the reactions to proceed in the opposite direction and make glucose from pyruvate, each of these three reactions must be bypassed. This is achieved by substituting three enzyme-catalyzed bypass reactions that are driven in the uphill direction by an input of chemical energy (Figure 2-40). Thus, whereas two ATP molecules are generated as each molecule of glucose is degraded to two molecules of pyruvate, the reverse reaction during gluconeogenesis requires the hydrolysis of four ATP and two GTP molecules. This is equivalent, in total, to the hydrolysis of six molecules of ATP for every molecule of glucose synthesized.

The bypass reactions in Figure 2-40 must be controlled so that glucose is broken down rapidly when energy is needed but synthesized when the cell is nutritionally replete. If both forward and reverse reactions were allowed to proceed at the same time without restraint, they would shuttle large quantities of metabolites backward and forward in futile cycles that would consume large amounts of ATP and generate heat for no purpose.

The elegance of the control mechanisms involved can be illustrated by a single example. Step 3 of glycolysis is one of the reactions that must be bypassed during glucose formation (see Figure 2-40). Normally, this step involves the addition of a second phosphate group to fructose 6-phosphate from ATP and is catalyzed by the enzyme phosphofructokinase. This enzyme is activated by AMP, ADP, and inorganic phosphate, whereas it is inhibited by ATP, citrate, and fatty acids. Therefore, the enzyme is activated by the accumulation of the products of ATP hydrolysis when energy supplies are low, and it is inactivated when energy (in the form of ATP) or food supplies such as fatty acids or citrate (derived from amino acids) are abundant. Fructose bisphosphatase is the enzyme that catalyzes the reverse bypass reaction (the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate, leading to the formation of glucose); this enzyme is regulated in the opposite way by the same feedback control molecules so that it is stimulated when the phosphofructokinase is inhibited.

Enzymes Can Be Switched On and Off by Covalent Modification ²²

The types of feedback control just described permit the rates of reaction sequences to be regulated continuously and automatically in response to second-by-second fluctuations in metabolism. Cells have different devices for regulating enzymes when longer-lasting changes in activity, occurring over minutes or hours, are required. These involve reversible covalent modification of enzymes. This modification is usually, but not always, accomplished by the addition of a phosphate group to a specific serine, threonine, or tyrosine residue in the enzyme. The phosphate comes from ATP, and its transfer is catalyzed by a family of enzymes known as protein kinases.

In Chapter 5 we describe how phosphorylation can alter the shape of an enzyme in such a way as to increase or inhibit its activity. The subsequent removal of the phosphate group, which reverses the effect of the phosphorylation, is achieved by a second type of enzyme, called a protein phosphatase. Covalent modification of enzymes adds another dimension to metabolic control because it allows specific reaction pathways to be regulated by extracellular signals (such as hormones and growth factors) that are unrelated to the metabolic intermediates themselves.

Reactions Are Compartmentalized Both Within Cells and Within Organisms ²³

Not all of a cell's metabolic reactions occur within the same subcellular compartment. Because different enzymes are found in different parts of the cell, the flow of chemical components is channeled physically as well as chemically.

The simplest form of such spatial segregation occurs when two enzymes that catalyze sequential reactions form an enzyme complex and the product of the first enzyme does not have to diffuse through the cytosol to encounter the second enzyme. The second reaction begins as soon as the first is over. Some large enzyme aggregates carry out whole series of reactions without losing contact with the substrate. The conversion of pyruvate to acetyl CoA, for example, proceeds in three chemical steps, all of which take place on the same large enzyme complex (Figure 2-41). In fatty acid synthesis an even longer sequence of reactions is catalyzed by a single enzyme assembly. Not surprisingly, some of the largest enzyme complexes are concerned with the synthesis of macromolecules such as proteins and DNA.

The next level of spatial segregation in cells involves the confinement of functionally related enzymes within the same membrane or within the aqueous compartment of an organelle that is bounded by a membrane. The oxidative metabolism of glucose is a good example. After glycolysis, pyruvate is actively taken up from the cytosol into the inner compartment of the mitochondrion, which contains all of the enzymes and metabolites involved in the citric acid cycle (Figure 2-42). Moreover, the inner mitochondrial membrane itself contains all of the enzymes that catalyze the subsequent reactions of oxidative phosphorylation, including those involved in the transfer of electrons from NADH to O₂ and in the synthesis of ATP. The entire mitochondrion can therefore be regarded as a small ATP-producing factory. In the same way other cellular organelles, such as the nucleus, the Golgi apparatus, and the lysosomes, can be viewed as specialized compartments where functionally related enzymes are confined to perform a specific task. In a sense, the living cell is like a city, with many specialized services concentrated in different areas that are extensively interconnected by various paths of communication.

Spatial organization in a multicellular organism extends beyond the individual cell. The different tissues of the body have different sets of enzymes and make distinct contributions to the chemistry of the organism as a whole. In addition to differences in specialized products such as hormones or antibodies, there are significant differences in the "common" metabolic pathways among various types of cells in the same organism. Although virtually all cells contain the enzymes of glycolysis, the citric acid cycle, lipid synthesis and breakdown, and amino acid metabolism, the levels of these processes in different tissues are differently regulated. Nerve cells, which are probably the most fastidious cells in the body, maintain almost no reserves of glycogen or fatty acids and rely almost entirely on a supply of glucose from the bloodstream. Liver cells supply glucose to actively contracting muscle cells and recycle the lactic acid produced by muscle cells back into glucose (Figure 2-43). All types of cells have their distinctive metabolic traits and cooperate extensively in the normal state as well as in response to stress and starvation.

Summary

The many thousands of different chemical reactions carried out simultaneously by a cell are closely coordinated. A variety of control mechanisms regulate the activities of key enzymes in response to the changing conditions in the cell. One very common form of regulation is a rapidly reversible feedback inhibition exerted on the first enzyme of a pathway by the final product of that pathway. A longer-lasting form of regulation involves the chemical modification of one enzyme by another, usually by phosphorylation. Combinations of regulatory mechanisms can produce major and long-lasting changes in the metabolism of the cell. Not all cellular reactions occur within the same intracellular compartment, and spatial segregation by internal membranes permits organelles to specialize in their biochemical tasks.