At first glance, metabolism appears intimidating because of the sheer number of reactants and reactions. Nevertheless, there are unifying themes that make the comprehension of this complexity more manageable. These unifying themes include common metabolites, reactions, and regulatory schemes that stem from a common evolutionary heritage.

We have seen that phosphoryl transfer can be used to drive otherwise endergonic reactions, alter the energy of conformation of a protein, or serve as a signal to alter the activity of a protein. The phosphoryl-group donor in all of these reactions is ATP. In other words, ATP is an activated carrier of phosphoryl groups because phosphoryl transfer from ATP is an exergonic process. The use of activated carriers is a recurring motif in biochemistry, and we will consider several such carriers here. Many such activated carriers function as coenzymes (Section 8.1):

1. Activated Carriers of Electrons for Fuel Oxidation. In aerobic organisms, the ultimate electron acceptor in the oxidation of fuel molecules is O₂. However, electrons are not transferred directly to O₂. Instead, fuel molecules transfer electrons to special carriers, which are either pyridine nucleotides or flavins. The reduced forms of these carriers then transfer their high-potential electrons to O₂.

Nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules (Figure 15.13). The reactive part of NAD⁺ is its nicotinamide ring, a pyridine derivative synthesized from the vitamin niacin. In the oxidation of a substrate, the nicotinamide ring of NAD⁺ accepts a hydrogen ion and two electrons, which are equivalent to a hydride ion (H:⁻). The reduced form of this carrier is called NADH. In the oxidized form, the nitrogen atom carries a positive charge, as indicated by NAD⁺. NAD⁺ is the electron acceptor in many reactions of the type

In this dehydrogenation, one hydrogen atom of the substrate is directly transferred to NAD⁺, whereas the other appears in the solvent as a proton. Both electrons lost by the substrate are transferred to the nicotinamide ring.

The other major electron carrier in the oxidation of fuel molecules is the coenzyme flavin adenine dinucleotide (Figure 15.14). The abbreviations for the oxidized and reduced forms of this carrier are FAD and FADH₂, respectively. FAD is the electron acceptor in reactions of the type

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The reactive part of FAD is its isoalloxazine ring, a derivative of the vitamin riboflavin (Figure 15.15). FAD, like NAD⁺, can accept two electrons. In doing so, FAD, unlike NAD⁺, takes up two protons. These carriers of high-potential electrons as well as flavin mononucleotide (FMN), an electron carrier similar to FAD but lacking the adenine nucleotide, will be considered further in Chapter 18.

2. An Activated Carrier of Electrons for Reductive Biosynthesis. High-potential electrons are required in most biosyntheses because the precursors are more oxidized than the products. Hence, reducing power is needed in addition to ATP. For example, in the biosynthesis of fatty acids, a keto group is reduced to a methylene group in several steps. This sequence of reactions requires an input of four electrons.

The electron donor in most reductive biosyntheses is NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate (NADP⁺; Figure 15.13). NADPH differs from NADH in that the 2′ -hydroxyl group of its adenosine moiety is esterified with phosphate. NADPH carries electrons in the same way as NADH. However, NADPH is used almost exclusively for reductive biosyntheses, whereas NADH is used primarily for the generation of ATP. The extra phosphoryl group on NADPH is a tag that enables enzymes to distinguish between high-potential electrons to be used in anabolism and those to be used in catabolism.

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3. An Activated Carrier of Two-Carbon Fragments. Coenzyme A, another central molecule in metabolism, is a carrier of acyl groups derived from the vitamin pantothenate (Figure 15.16). Acyl groups are important constituents both in catabolism, as in the oxidation of fatty acids, and in anabolism, as in the synthesis of membrane lipids. The terminal sulfhydryl group in CoA is the reactive site. Acyl groups are linked to CoA by thioester bonds. The resulting derivative is called an acyl CoA. An acyl group often linked to CoA is the acetyl unit; this derivative is called acetyl CoA. The ΔG°′ for the hydrolysis of acetyl CoA has a large negative value:

A thioester is thermodynamically more unstable than an oxygen ester because the electrons of the C O bond cannot form resonance structures with the C S bond that are as stable as those that they can form with the C O bond. Consequently, acetyl CoA has a high acetyl-group-transfer potential because transfer of the acetyl group is exergonic. Acetyl CoA carries an activated acetyl group, just as ATP carries an activated phosphoryl group.

The use of activated carriers illustrates two key aspects of metabolism. First, NADH, NADPH, and FADH₂ react slowly with O₂ in the absence of a catalyst. Likewise, ATP and acetyl CoA are hydrolyzed slowly (over many hours or even days) in the absence of a catalyst. These molecules are kinetically quite stable in the face of a large thermodynamic driving force for reaction with O₂ (in regard to the electron carriers) and H₂O (for ATP and acetyl CoA). The kinetic stability of these molecules in the absence of specific catalysts is essential for their biological function because it enables enzymes to control the flow of free energy and reducing power.

Second, most interchanges of activated groups in metabolism are accomplished by a rather small set of carriers (Table 15.2). The existence of a recurring set of activated carriers in all organisms is one of the unifying motifs of biochemistry. Furthermore, it illustrates the modular design of metabolism. A small set of molecules carries out a very wide range of tasks. Metabolism is readily comprehended because of the economy and elegance of its underlying design.

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Almost all the activated carriers that act as coenzymes are derived from vitamins. Vitamins are organic molecules that are needed in small amounts in the diets of some higher animals. Table 15.3 lists the vitamins that act as coenzymes and Figure 15.17 shows the structures of some of them. This series of vitamins is known as the vitamin B group. In all cases, the vitamin must be modified before it can serve its function. We have already touched on the roles of niacin, riboflavin, and pantothenate. We will see these three and the other B vitamins many times in our study of biochemistry.

Vitamins serve the same roles in nearly all forms of life, but higher animals lost the capacity to synthesize them in the course of evolution. For instance, whereas E. coli can thrive on glucose and organic salts, human beings require at least 12 vitamins in their diet. The biosynthetic pathways for vitamins can be complex; thus, it is biologically more efficient to ingest vitamins than to synthesize the enzymes required to construct them from simple molecules. This efficiency comes at the cost of dependence on other organisms for chemicals essential for life. Indeed, vitamin deficiency can generate diseases in all organisms requiring these molecules (Tables 15.3 and 15.4).

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Not all vitamins function as coenzymes. Vitamins designated by the letters A, C, D, E, and K (Figure 15.18 and Table 15.4) have a diverse array of functions. Vitamin A (retinol) is the precursor of retinal, the light-sensitive group in rhodopsin and other visual pigments (Section 32.3), and retinoic acid, an important signaling molecule. A deficiency of this vitamin leads to night blindness. In addition, young animals require vitamin A for growth. Vitamin C, or ascorbate, acts as an antioxidant. A deficiency in vitamin C results in the formation of unstable collagen molecules and is the cause of scurvy, a disease characterized by skin lesions and blood-vessel fragility (Section 27.6). A metabolite of vitamin D is a hormone that regulates the metabolism of calcium and phosphorus. A deficiency in vitamin D impairs bone formation in growing animals. Vitamin E (α-tocopherol) deficiency causes a variety of neuromuscular pathologies. This vitamin inactivates reactive oxygen species such as hydroxyl radicals before they can oxidize unsaturated membrane lipids, damaging cell structures. Vitamin K is required for normal blood clotting (Section 10.4).

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Just as there is an economy of design in the use of activated carriers, so is there an economy of design in biochemical reactions. The thousands of metabolic reactions, bewildering at first in their variety, can be subdivided into just six types (Table 15.5). Specific reactions of each type appear repeatedly, reducing the number of reactions that a student needs to learn.

1. Oxidation– reduction reactions are essential components of many pathways. Useful energy is often derived from the oxidation of carbon compounds. Consider the following two reactions:

These two oxidation–reduction reactions are components of the citric acid cycle (Chapter 17), which completely oxidizes the activated two-carbon fragment of acetyl CoA to two molecules of CO₂. In reaction 1, FADH₂ carries the electrons, whereas, in reaction 2, electrons are carried by NADH.

2. Ligation reactions form bonds by using free energy from ATP cleavage. Reaction 3 illustrates the ATP-dependent formation of a carbon–carbon bond, necessary to combine smaller molecules to form larger ones. Oxaloacetate is formed from pyruvate and CO₂.

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The oxaloacetate can be used in the citric acid cycle, or converted into glucose or amino acids such as aspartic acid.

3. Isomerization reactions rearrange particular atoms within a molecule. Their role is often to prepare the molecule for subsequent reactions such as the oxidation–reduction reactions described in point 1.

Reaction 4 is, again, a component of the citric acid cycle. This isomerization prepares the molecule for subsequent oxidation and decarboxylation reactions by moving the hydroxyl group of citrate from a tertiary to a secondary position.

4. Group-transfer reactions play a variety of roles. Reaction 5 is representative of such a reaction. A phosphoryl group is transferred from the activated phosphoryl-group carrier, ATP, to glucose, the initial step in glycolysis, a key pathway for extracting energy from glucose (Chapter 16). This reaction traps glucose in the cell so that further catabolism can take place.

As stated earlier, group-transfer reactions are used to synthesize ATP. We also saw examples of their use in signaling pathways (Chapter 14).

5. Hydrolytic reactions cleave bonds by the addition of water. Hydrolysis is a common means of degrading large molecules, either to facilitate further metabolism or to reuse some of the components for biosynthetic purposes. Proteins are digested by hydrolytic cleavage (chapters 9 and 10). Reaction 6 illustrates the hydrolysis of a peptide to yield two smaller peptides.

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6. Carbon bonds can be cleaved by means other than hydrolysis or oxidation, with two substrates yielding one product or vice versa. When CO₂ or H₂O is released, a double bond is formed. The enzymes that catalyze these types of reaction are classified as lyases. An important example, illustrated in reaction 7, is the conversion of the six-carbon molecule fructose 1,6-bisphosphate into two three-carbon fragments: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

This reaction is a critical step in glycolysis (Chapter 16). Dehydrations to form double bonds, such as the formation of phosphoenolpyruvate (Figure 15.6) from 2-phosphoglycerate (reaction 8), are important reactions of this type.

The dehydration sets up the next step in the pathway, a group-transfer reaction that uses the high phosphoryl-transfer potential of the product PEP to form ATP from ADP.

These six fundamental reaction types are the basis of metabolism. Remember that all six types can proceed in either direction, depending on the standard free energy for the specific reaction and the concentrations of the reactants and products inside the cell. An effective way to learn is to look for commonalities in the diverse metabolic pathways that we will be examining. There is a chemical logic that, when exposed, renders the complexity of the chemistry of living systems more manageable and reveals its elegance.

It is evident that the complex network of metabolic reactions must be rigorously regulated. The levels of available nutrients must be monitored and the activity of metabolic pathways must be altered and integrated to create homeostasis, a stable biochemical environment. At the same time, metabolic control must be flexible, able to adjust metabolic activity to the constantly changing external environments of cells. Figure 15.19 illustrates the nutrient pools and their connections that must be monitored and regulated. Metabolism is regulated through control of (1) the amounts of enzymes, (2) their catalytic activities, and (3) the accessibility of substrates.

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Controlling the amounts of enzymes. The amount of a particular enzyme depends on both its rate of synthesis and its rate of degradation. The level of many enzymes is adjusted by a change in the rate of transcription of the genes encoding them (chapters 29 and 31). In E. coli, for example, the presence of lactose induces within minutes a more than 50-fold increase in the rate of synthesis of β-galactosidase, the enzyme required for the breakdown of this disaccharide.

Controlling catalytic activity. The catalytic activity of enzymes is controlled in several ways. Allosteric control is especially important. For example, the first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate product of the pathway. The inhibition of aspartate transcarbamoylase by cytidine triphosphate (Section 10.1) is a well-understood example of feedback inhibition. This type of control can be almost instantaneous. Another recurring mechanism is reversible covalent modification (Section 10.3). For example, glycogen phosphorylase, the enzyme catalyzing the breakdown of glycogen, a storage form of sugar, is activated by the phosphorylation of a particular serine residue when glucose is scarce (Section 21.1).

Hormones coordinate metabolic relations between different tissues, often by regulating the reversible modification of key enzymes. For instance, the hormone epinephrine triggers a signal-transduction cascade in muscle, resulting in the phosphorylation and activation of key enzymes and leading to the rapid degradation of glycogen to glucose, which is then used to supply ATP for muscle contraction. As described in Chapter 14, many hormones act through intracellular messengers, such as cyclic AMP and calcium ion, that coordinate the activities of many target proteins.

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Many reactions in metabolism are controlled by the energy status of the cell. One index of the energy status is the energy charge, which is proportional to the mole fraction of ATP plus half the mole fraction of ADP, given that ATP contains two anhydride bonds, whereas ADP contains one. Hence, the energy charge is defined as

The energy charge can have a value ranging from 0 (all AMP) to 1 (all ATP). ATP-generating (catabolic) pathways are inhibited by a high energy charge, whereas ATP-utilizing (anabolic) pathways are stimulated by a high energy charge. In plots of the reaction rates of such pathways versus the energy charge, the curves are steep near an energy charge of 0.9, where they usually intersect (Figure 15.20). It is evident that the control of these pathways has evolved to maintain the energy charge within rather narrow limits. In other words, the energy charge, like the pH of a cell, is buffered. The energy charge of most cells ranges from 0.90 to 0.95, but can fall to less than 0.7 in muscle during high-intensity exercise. An alternative index of the energy status is the phosphorylation potential, which is defined as

The phosphorylation potential, in contrast with the energy charge, depends on the concentration of P_i and is directly related to the free-energy storage available from ATP.

Controlling the accessibility of substrates. Controlling the availability of substrates is another means of regulating metabolism in all organisms. For instance, glucose breakdown can take place in many cells only if insulin is present to promote the entry of glucose into the cell. In eukaryotes, metabolic regulation and flexibility are enhanced by compartmentalization. The transfer of substrates from one compartment of a cell to another can serve as a control point. For example, fatty acid oxidation takes place in mitochondria, where as fatty acid synthesis takes place in the cytoplasm. Compartmentalization segregates opposed reactions.

How did the complex pathways that constitute metabolism evolve? The current thinking is that RNA was an early biomolecule that dominated metabolism, serving both as a catalyst and an information storage molecule. This hypothetical time is called the RNA world.

Why do activated carriers such as ATP, NADH, FADH₂, and coenzyme A contain adenosine diphosphate units? A possible explanation is that these molecules evolved from the early RNA catalysts. Non-RNA units such as the isoalloxazine ring may have been recruited to serve as efficient carriers of activated electrons and chemical units, a function not readily performed by RNA itself. We can picture the adenine ring of FADH₂ binding to a uracil unit in a niche of an RNA enzyme (ribozyme) by base-pairing, whereas the isoalloxazine ring protrudes and functions as an electron carrier. When the more versatile proteins replaced RNA as the major catalysts, the ribonucleotide coenzymes stayed essentially unchanged because they were already well suited to their metabolic roles. The nicotinamide unit of NADH, for example, can readily transfer electrons irrespective of whether the adenine unit interacts with a base in an RNA enzyme or with amino acid residues in a protein enzyme. With the advent of protein enzymes, these important cofactors evolved as free molecules without losing the adenosine diphosphate vestige of their RNA-world ancestry. That molecules and motifs of metabolism are common to all forms of life testifies to their common origin and to the retention of functioning modules through billions of years of evolution. Our understanding of metabolism, like that of other biological processes, is enriched by inquiry into how these beautifully integrated patterns of reactions came into being.

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