Bioenergetics and Metabolism

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PART II

BIOENERGETICS AND METABOLISM

13 Principles of Bioenergetics 480 Although metabolism embraces hundreds of differ- 14 Glycolysis, Gluconeogenesis, and the Pentose ent enzyme-catalyzed reactions, our major concern in Phosphate Pathway 521 Part II is the central metabolic pathways, which are few in number and remarkably similar in all forms of life. 15 Principles of Metabolic Regulation, Illustrated with Living organisms can be divided into two large groups the Metabolism of Glucose and Glycogen 560 according to the chemical form in which they obtain 16 The Citric Acid Cycle 601 carbon from the environment. Autotrophs (such as 17 Fatty Acid Catabolism 631 photosynthetic bacteria and vascular plants) can use carbon dioxide from the atmosphere as their sole source 18 Amino Acid Oxidation and the Production of carbon, from which they construct all their carbon- of Urea 666 containing biomolecules (see Fig. 1–5). Some auto- 19 Oxidative Phosphorylation and trophic organisms, such as cyanobacteria, can also use Photophosphorylation 700 atmospheric nitrogen to generate all their nitrogenous 20 Carbohydrate Biosynthesis in Plants components. Heterotrophs cannot use atmospheric and Bacteria 761 carbon dioxide and must obtain carbon from their environment in the form of relatively complex organic mol- 21 Lipid Biosynthesis 797 ecules such as glucose. Multicellular animals and most 22 Biosynthesis of Amino Acids, Nucleotides, and microorganisms are heterotrophic. Autotrophic cells Related Molecules 843 and organisms are relatively self-sufficient, whereas het- 23 Integration and Hormonal Regulation of Mammalian erotrophic cells and organisms, with their requirements Metabolism 891 for carbon in more complex forms, must subsist on the products of other organisms. Many autotrophic organisms are photosynthetic and obtain their energy from sunlight, whereas heterotrophic organisms obtain their energy from the etabolism is a highly coordinated cellular activity degradation of organic nutrients produced by auto- Min which many multienzyme systems (metabolic trophs. In our biosphere, autotrophs and heterotrophs pathways) cooperate to (1) obtain chemical energy by live together in a vast, interdependent cycle in which capturing solar energy or degrading energy-rich nutrients autotrophic organisms use atmospheric carbon dioxide from the environment; (2) convert nutrient molecules to build their organic biomolecules, some of them gen- into the cell’s own characteristic molecules, including erating oxygen from water in the process. Heterotrophs precursors of macromolecules; (3) polymerize mono- in turn use the organic products of autotrophs as nu- meric precursors into macromolecules: proteins, nucleic trients and return carbon dioxide to the atmosphere. acids, and polysaccharides; and (4) synthesize and Some of the oxidation reactions that produce carbon degrade biomolecules required for specialized cellular dioxide also consume oxygen, converting it to water. functions, such as membrane lipids, intracellular mes- Thus carbon, oxygen, and water are constantly cycled sengers, and pigments. between the heterotrophic and autotrophic worlds, with

481 482 Part II Bioenergetics and Metabolism solar energy as the driving force for this global process Atmospheric (Fig. 1). N2 All living organisms also require a source of nitrogen, which is necessary for the synthesis of amino acids, nucleotides, and other compounds. Plants can generally Nitrogen- use either ammonia or nitrate as their sole source of ni- Denitrifying fixing bacteria trogen, but vertebrates must obtain nitrogen in the form bacteria of amino acids or other organic compounds. Only a few organisms—the cyanobacteria and many species of soil bacteria that live symbiotically on the roots of some plants—are capable of converting (“fixing”) atmos- Ammonia pheric nitrogen (N2) into ammonia. Other bacteria (the nitrifying bacteria) oxidize ammonia to nitrites and nitrates; yet others convert nitrate to N2. Thus, in addi- Animals Nitrifying tion to the global carbon and oxygen cycle, a nitrogen bacteria cycle operates in the biosphere, turning over huge amounts of nitrogen (Fig. 2). The cycling of carbon, oxygen, and nitrogen, which ultimately involves all species, depends on a proper balance between the activities of Amino Nitrates, the producers (autotrophs) and consumers (het- acids nitrites erotrophs) in our biosphere. These cycles of matter are driven by an enormous flow of energy into and through the biosphere, begin- Plants ning with the capture of solar energy by photosynthetic organisms and use of this energy to generate energy- FIGURE 2 Cycling of nitrogen in the biosphere. Gaseous nitrogen rich carbohydrates and other organic nutrients; these (N ) makes up 80% of the earth’s atmosphere. nutrients are then used as energy sources by het- 2 erotrophic organisms. In metabolic processes, and in all energy transformations, there is a loss of useful energy (free energy) and an inevitable increase in the amount through the biosphere; organisms cannot regenerate of unusable energy (heat and entropy). In contrast useful energy from energy dissipated as heat and to the cycling of matter, therefore, energy flows one way entropy. Carbon, oxygen, and nitrogen recycle continuously, but energy is constantly transformed into unusable forms such as heat. Metabolism, the sum of all the chemical transformations taking place in a cell or organism, occurs through a series of enzyme-catalyzed reactions that constitute metabolic pathways. Each of the consecutive steps in a metabolic pathway brings about a specific, small chemical change, usually the removal, transfer, or addition of a particular atom or functional group. The precursor is converted into a product through a series

O2 of metabolic intermediates called metabolites. The term intermediary metabolism is often applied to the p nic rodu ga c combined activities of all the metabolic pathways that r ts O interconvert precursors, metabolites, and products of Photosynthetic Heterotrophs low molecular weight (generally, Mr 1,000). autotrophs Catabolism is the degradative phase of metabolism in which organic nutrient molecules (carbohydrates,

CO2 fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO2, NH3). Catabolic H2O pathways release energy, some of which is conserved in FIGURE 1 Cycling of carbon dioxide and oxygen between the auto- the formation of ATP and reduced electron carriers trophic (photosynthetic) and heterotrophic domains in the biosphere. (NADH, NADPH, and FADH2); the rest is lost as heat. The flow of mass through this cycle is enormous; about 4 1011 met- In anabolism, also called biosynthesis, small, simple ric tons of carbon are turned over in the biosphere annually. precursors are built up into larger and more complex Part II Bioenergetics and Metabolism 483 molecules, including lipids, polysaccharides, proteins, simultaneous synthesis and degradation of fatty acids and nucleic acids. Anabolic reactions require an input would be wasteful, however, and this is prevented by of energy, generally in the form of the phosphoryl group reciprocally regulating the anabolic and catabolic reac- transfer potential of ATP and the reducing power of tion sequences: when one sequence is active, the other

NADH, NADPH, and FADH2 (Fig. 3). is suppressed. Such regulation could not occur if ana- Some metabolic pathways are linear, and some are bolic and catabolic pathways were catalyzed by exactly branched, yielding multiple useful end products from a the same set of enzymes, operating in one direction for single precursor or converting several starting materi- anabolism, the opposite direction for catabolism: inhi- als into a single product. In general, catabolic pathways bition of an enzyme involved in catabolism would also are convergent and anabolic pathways divergent (Fig. inhibit the reaction sequence in the anabolic direction. 4). Some pathways are cyclic: one starting component Catabolic and anabolic pathways that connect the same of the pathway is regenerated in a series of reactions two end points (glucose nnpyruvate and pyruvate that converts another starting component into a prod- nnglucose, for example) may employ many of the uct. We shall see examples of each type of pathway in same enzymes, but invariably at least one of the steps the following chapters. is catalyzed by different enzymes in the catabolic and Most cells have the enzymes to carry out both the anabolic directions, and these enzymes are the sites of degradation and the synthesis of the important cate- separate regulation. Moreover, for both anabolic and gories of biomolecules—fatty acids, for example. The catabolic pathways to be essentially irreversible, the reactions unique to each direction must include at least one that is thermodynamically very favorable—in other words, a reaction for which the reverse reaction is very Cell Energy- unfavorable. As a further contribution to the separate macromolecules containing regulation of catabolic and anabolic reaction sequences, nutrients Proteins paired catabolic and anabolic pathways commonly take Polysaccharides Carbohydrates Lipids Fats place in different cellular compartments: for example, Nucleic acids Proteins fatty acid catabolism in mitochondria, fatty acid synthesis in the cytosol. The concentrations of intermediates, enzymes, and regulators can be maintained at different levels in these different compartments. Be- cause metabolic pathways are subject to kinetic con- 2 ADP HPO4 trol by substrate concentration, separate pools of NAD anabolic and catabolic intermediates also contribute to NADP FAD the control of metabolic rates. Devices that separate anabolic and catabolic processes will be of particular interest in our discussions of metabolism. Anabolism ATP Catabolism Metabolic pathways are regulated at several levels, NADH NADPH from within the cell and from outside. The most imme- FADH2 diate regulation is by the availability of substrate; when the intracellular concentration of an enzyme’s substrate

is near or below Km (as is commonly the case), the rate Chemical of the reaction depends strongly upon substrate con- energy centration (see Fig. 6–11). A second type of rapid control from within is allosteric regulation (p. 225) by a metabolic intermediate or coenzyme—an amino acid or Precursor Energy- ATP, for example—that signals the cell’s internal meta- molecules depleted bolic state. When the cell contains an amount of, say, end products Amino acids aspartate sufficient for its immediate needs, or when the Sugars CO2 cellular level of ATP indicates that further fuel con- H O Fatty acids 2 sumption is unnecessary at the moment, these signals Nitrogenous bases NH3 allosterically inhibit the activity of one or more enzymes in the relevant pathway. In multicellular organisms the FIGURE 3 Energy relationships between catabolic and anabolic metabolic activities of different tissues are regulated and pathways. Catabolic pathways deliver chemical energy in the form integrated by growth factors and hormones that act from of ATP, NADH, NADPH, and FADH2. These energy carriers are used outside the cell. In some cases this regulation occurs in anabolic pathways to convert small precursor molecules into cell virtually instantaneously (sometimes in less than a mil- macromolecules. lisecond) through changes in the levels of intracellular 484 Part II Bioenergetics and Metabolism

Rubber Carotenoid Steroid pigments hormones

Phospholipids Isopentenyl- Bile Cholesterol pyrophosphate acids Triacylglycerols Fatty acids

Mevalonate Vitamin K Cholesteryl Phenyl- Starch Alanine esters alanine Acetate Glycogen Glucose Pyruvate (acetyl-CoA) Acetoacetyl-CoA Eicosanoids

Sucrose Serine Leucine Isoleucine Fatty acids Triacylglycerols (a) Converging catabolism

Citrate CDP-diacylglycerol Phospholipids Oxaloacetate (b) Diverging anabolism

CO2

CO2 (c) Cyclic pathway

FIGURE 4 Three types of nonlinear metabolic pathways. (a) Con- the breakdown product of a variety of fuels (a), serves as the precur- verging, catabolic; (b) diverging, anabolic; and (c) cyclic, in which sor for an array of products (b), and is consumed in the catabolic path- one of the starting materials (oxaloacetate in this case) is regenerated way known as the citric acid cycle (c). and reenters the pathway. Acetate, a key metabolic intermediate, is

messengers that modify the activity of existing enzyme Before reviewing the five main reaction classes of molecules by allosteric mechanisms or by covalent mod- biochemistry, let’s consider two basic chemical princi- ification such as phosphorylation. In other cases, the ex- ples. First, a covalent bond consists of a shared pair of tracellular signal changes the cellular concentration of electrons, and the bond can be broken in two general an enzyme by altering the rate of its synthesis or degra- ways (Fig. 5). In homolytic cleavage, each atom leaves dation, so the effect is seen only after minutes or hours. the bond as a radical, carrying one of the two electrons The number of metabolic transformations taking (now unpaired) that held the bonded atoms together. place in a typical cell can seem overwhelming to a be- In the more common, heterolytic cleavage, one atom re- ginning student. Most cells have the capacity to carry tains both bonding electrons. The species generated out thousands of specific, enzyme-catalyzed reactions: when COC and COH bonds are cleaved are illustrated for example, transformation of a simple nutrient such in Figure 5. Carbanions, carbocations, and hydride ions as glucose into amino acids, nucleotides, or lipids; ex- are highly unstable; this instability shapes the chemistry traction of energy from fuels by oxidation; or polymer- of these ions, as described further below. ization of monomeric subunits into macromolecules. The second chemical principle of interest here is that Fortunately for the student of biochemistry, there are many biochemical reactions involve interactions between patterns within this multitude of reactions; you do not nucleophiles (functional groups rich in electrons and need to learn all these reactions to comprehend the capable of donating them) and electrophiles (electron- molecular logic of biochemistry. Most of the reactions deficient functional groups that seek electrons). Nucle- in living cells fall into one of five general categories: ophiles combine with, and give up electrons to, elec- (1) oxidation-reductions; (2) reactions that make or trophiles. Common nucleophiles and electrophiles are break carbon–carbon bonds; (3) internal rearrangements, listed in Figure 6–21. Note that a carbon atom can act isomerizations, and eliminations; (4) group transfers; as either a nucleophile or an electrophile, depending on and (5) free radical reactions. Reactions within each which bonds and functional groups surround it. general category usually proceed by a limited set of We now consider the five main reaction classes you mechanisms and often employ characteristic cofactors. will encounter in upcoming chapters. Part II Bioenergetics and Metabolism 485

catalyze these oxidations are generally called oxidases Homolytic CH C H cleavage or, if the oxygen atom is derived directly from molecular oxygen (O ), oxygenases. Carbon H atom 2 radical Every oxidation must be accompanied by a reduction, in which an electron acceptor acquires the electrons CC C C removed by oxidation. Oxidation reactions generally release energy (think of camp fires: the compounds in Carbon radicals wood are oxidized by oxygen molecules in the air). Most living cells obtain the energy needed for cellular work by oxidizing metabolic fuels such as carbohydrates or fat; Heterolytic CH C H photosynthetic organisms can also trap and use the en- cleavage ergy of sunlight. The catabolic (energy-yielding) path- Carbanion Proton ways described in Chapters 14 through 19 are oxidative reaction sequences that result in the transfer of electrons from fuel molecules, through a series of electron carri- CH C H ers, to oxygen. The high affinity of O2 for electrons makes the overall electron-transfer process highly exergonic, Carbocation Hydride providing the energy that drives ATP synthesis—the central goal of catabolism.

CC C C 2. Reactions that make or break carbon–carbon bonds Het- erolytic cleavage of a C C bond yields a carbanion and Carbanion Carbocation O a carbocation (Fig. 5). Conversely, the formation of a FIGURE 5 Two mechanisms for cleavage of a COC or COH bond. COC bond involves the combination of a nucleophilic In homolytic cleavages, each atom keeps one of the bonding elec- carbanion and an electrophilic carbocation. Groups with trons, resulting in the formation of carbon radicals (carbons having electronegative atoms play key roles in these reactions. unpaired electrons) or uncharged hydrogen atoms. In heterolytic cleav- Carbonyl groups are particularly important in the chem- ages, one of the atoms retains both bonding electrons. This can result ical transformations of metabolic pathways. As noted in the formation of carbanions, carbocations, protons, or hydride ions. above, the carbon of a carbonyl group has a partial positive charge due to the electron-withdrawing nature of 1. Oxidation-reduction reactions Carbon atoms encoun- the adjacent bonded oxygen, and thus is an electrophilic tered in biochemistry can exist in five oxidation states, carbon. The presence of a carbonyl group can also depending on the elements with which carbon shares facilitate the formation of a carbanion on an adjoining electrons (Fig. 6). In many biological oxidations, a com- carbon, because the carbonyl group can delocalize elec- pound loses two electrons and two hydrogen ions (that trons through resonance (Fig. 8a, b). The importance is, two hydrogen atoms); these reactions are commonly of a carbonyl group is evident in three major classes of called dehydrogenations and the enzymes that catalyze reactions in which COC bonds are formed or broken them are called dehydrogenases (Fig. 7). In some, but (Fig 8c): aldol condensations (such as the aldolase not all, biological oxidations, a carbon atom becomes co- reaction; see Fig. 14–5), Claisen condensations (as valently bonded to an oxygen atom. The enzymes that in the citrate synthase reaction; see Fig. 16–9), and

OH 2H 2e O CH2 CH3 Alkane O O CH CH C CH C C CH CH OH Alcohol 3 3 2 2 O O O 2H 2e Lactatelactate Pyruvate CH2 C Aldehyde (ketone) H(R) dehydrogenase O FIGURE 7 An oxidation-reduction reaction. Shown here is the oxi-

CH2 C Carboxylic acid dation of lactate to pyruvate. In this dehydrogenation, two electrons OH and two hydrogen ions (the equivalent of two hydrogen atoms) are re- O C O Carbon dioxide moved from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the reaction is catalyzed by lactate dehydrogenase and the elec- FIGURE 6 The oxidation states of carbon in biomolecules. Each com- trons are transferred to a cofactor called nicotinamide adenine dinu- pound is formed by oxidation of the red carbon in the compound cleotide. This reaction is fully reversible; pyruvate can be reduced by listed above it. Carbon dioxide is the most highly oxidized form of electrons from the cofactor. In Chapter 13 we discuss the factors that carbon found in living systems. determine the direction of a reaction. 486 Part II Bioenergetics and Metabolism decarboxylations (as in the acetoacetate decarboxylase electrons results in isomerization, transposition of dou- reaction; see Fig. 17–18). Entire metabolic pathways are ble bonds, or cis-trans rearrangements of double bonds. organized around the introduction of a carbonyl group An example of isomerization is the formation of fruc- in a particular location so that a nearby carbon–carbon tose 6-phosphate from glucose 6-phosphate during bond can be formed or cleaved. In some reactions, this sugar metabolism (Fig 9a; this reaction is discussed in role is played by an imine group or a specialized cofac- detail in Chapter 14). Carbon-1 is reduced (from alde- tor such as pyridoxal phosphate, rather than by a car- hyde to alcohol) and C-2 is oxidized (from alcohol to bonyl group. ketone). Figure 9b shows the details of the electron movements that result in isomerization. 3. Internal rearrangements, isomerizations, and eliminations A simple transposition of a CUC bond occurs dur- Another common type of cellular reaction is an in- ing metabolism of the common fatty acid oleic acid (see tramolecular rearrangement, in which redistribution of Fig. 17–9), and you will encounter some spectacular examples of double-bond repositioning in the synthesis of cholesterol (see Fig. 21–35). Elimination of water introduces a C C bond be- O U tween two carbons that previously were saturated (as (a) C in the enolase reaction; see Fig. 6–23). Similar reactions can result in the elimination of alcohols and amines. O O H H (b) CC CC H2O R H

RCC R1 C C H R H OH H2O 1 O R R O R R 2 3 H 2 3 (c) R C C C O R C C C OH 1 1 4. Group transfer reactions The transfer of acyl, glycosyl, H R4 H R4 and phosphoryl groups from one nucleophile to another Aldol condensation is common in living cells. Acyl group transfer generally involves the addition of a nucleophile to the carbonyl O H R O H R 1 H 1 carbon of an acyl group to form a tetrahedral interme- CoA-S C C C O CoA-S C C C OH diate. H R H R 2 2 O O O Claisen ester condensation C R C X C R X R Y O H O H Y O H Y X Tetrahedral RCC C RCC H CO2 intermediate H O H Decarboxylation of a -keto acid The chymotrypsin reaction is one example of acyl group transfer (see Fig. 6–21). Glycosyl group transfers in- FIGURE 8 Carbon–carbon bond formation reactions. (a) The carbon volve nucleophilic substitution at C-1 of a sugar ring, atom of a carbonyl group is an electrophile by virtue of the electron- which is the central atom of an acetal. In principle, the withdrawing capacity of the electronegative oxygen atom, which results substitution could proceed by an SN1 or SN2 path, as in a resonance hybrid structure in which the carbon has a partial pos- described for the enzyme lysozyme (see Fig. 6–25). itive charge. (b) Within a molecule, delocalization of electrons into a Phosphoryl group transfers play a special role in carbonyl group facilitates the transient formation of a carbanion on an metabolic pathways. A general theme in metabolism is adjacent carbon. (c) Some of the major reactions involved in the for- the attachment of a good leaving group to a metabolic mation and breakage of C C bonds in biological systems. For both the O intermediate to “activate” the intermediate for subse- aldol condensation and the Claisen condensation, a carbanion serves quent reaction. Among the better leaving groups in as nucleophile and the carbon of a carbonyl group serves as elec- nucleophilic substitution reactions are inorganic or- trophile. The carbanion is stabilized in each case by another carbonyl thophosphate (the ionized form of H3PO4 at neutral pH, at the carbon adjoining the carbanion carbon. In the decarboxylation 2 reaction, a carbanion is formed on the carbon shaded blue as the CO a mixture of H2PO4 and HPO4 , commonly abbreviated 2 4 leaves. The reaction would not occur at an appreciable rate but for Pi) and inorganic pyrophosphate (P2O7 , abbreviated the stabilizing effect of the carbonyl adjacent to the carbanion car- PPi); esters and anhydrides of phosphoric acid are bon. Wherever a carbanion is shown, a stabilizing resonance with the effectively activated for reaction. Nucleophilic substi- adjacent carbonyl, as shown in (a), is assumed. The formation of the tution is made more favorable by the attachment of a carbanion is highly disfavored unless the stabilizing carbonyl group, phosphoryl group to an otherwise poor leaving group or a group of similar function such as an imine, is present. such as OOH. Nucleophilic substitutions in which the Part II Bioenergetics and Metabolism 487

(a) H OH H H H O H OH H H H O H 1C 2C C C C C O P O H 1C 2C C C C C O P O phosphohexose OOHH OH OH H O isomerase OH O H OH OH H O Glucose 6-phosphate Fructose 6-phosphate

(b) B B B 1 1 B1 abstracts a 1 1 proton. H 5 An electron leaves the C C bond to form H H 2 This allows the a CH bond with C formation of a C C C the proton donated C C CC double bond. by B . O O 1 OH O OOH HH 3 Electrons from 4 B2 abstracts a H carbonyl form an proton, allowing H O H bond with B2 the formation of O bond. B2 the hydrogen ion a C B2 donated by B . 2 Enediol intermediate

FIGURE 9 Isomerization and elimination reactions. (a) The conver- rows represent the movement of bonding electrons from nucleophile sion of glucose 6-phosphate to fructose 6-phosphate, a reaction of (pink) to electrophile (blue). B1 and B2 are basic groups on the sugar metabolism catalyzed by phosphohexose isomerase. (b) This re- enzyme; they are capable of donating and accepting hydrogen ions action proceeds through an enediol intermediate. The curved blue ar- (protons) as the reaction progresses.

2 charge and can therefore act as an electrophile. In a very phosphoryl group (OPO3 ) serves as a leaving group occur in hundreds of metabolic reactions. large number of metabolic reactions, a phosphoryl group 2 Phosphorus can form five covalent bonds. The con- (OPO3 ) is transferred from ATP to an alcohol (form- ventional representation of Pi (Fig. 10a), with three ing a phosphate ester) (Fig. 10c) or to a carboxylic acid POO bonds and one PUO bond, is not an accurate pic- (forming a mixed anhydride). When a nucleophile at- ture. In Pi, four equivalent phosphorus–oxygen bonds tacks the electrophilic phosphorus atom in ATP, a rela- share some double-bond character, and the anion has a tively stable pentacovalent structure is formed as a re- tetrahedral structure (Fig. 10b). As oxygen is more elec- action intermediate (Fig. 10d). With departure of the tronegative than phosphorus, the sharing of electrons is leaving group (ADP), the transfer of a phosphoryl group unequal: the central phosphorus bears a partial positive is complete. The large family of enzymes that catalyze

(a) O O (c) O O O O P O O P O Adenine Ribose O P O POP O HO R O O O O O Glucose ATP O O O O O O P O O P O Adenine Ribose O P O P O O P O R OO O O O (b) ADP Glucose 6-phosphate, 3 a phosphate ester O O O P O (d) O O O O P Z P W Z R OH O O W ADP O

FIGURE 10 Alternative ways of showing the structure of inorganic all four phosphorus–oxygen bonds with some double-bond character; orthophosphate. (a) In one (inadequate) representation, three oxygens the hybrid orbitals so represented are arranged in a tetrahedron with are single-bonded to phosphorus, and the fourth is double-bonded, P at its center. (c) When a nucleophile Z (in this case, the OOH on allowing the four different resonance structures shown. (b) The four C-6 of glucose) attacks ATP, it displaces ADP (W). In this SN2 reac- resonance structures can be represented more accurately by showing tion, a pentacovalent intermediate (d) forms transiently. 488 Part II Bioenergetics and Metabolism phosphoryl group transfers with ATP as donor are called chemiosmotic energy coupling, a universal mechanism kinases (Greek kinein, “to move”). Hexokinase, for ex- in which a transmembrane electrochemical potential, ample, “moves” a phosphoryl group from ATP to glucose. produced either by substrate oxidation or by light ab- Phosphoryl groups are not the only activators of this sorption, drives the synthesis of ATP. type. Thioalcohols (thiols), in which the oxygen atom Chapters 20 through 22 describe the major anabolic of an alcohol is replaced with a sulfur atom, are also pathways by which cells use the energy in ATP to pro- good leaving groups. Thiols activate carboxylic acids by duce carbohydrates, lipids, amino acids, and nucleotides forming thioesters (thiol esters) with them. We will dis- from simpler precursors. In Chapter 23 we step back cuss a number of cases, including the reactions cat- from our detailed look at the metabolic pathways—as alyzed by the fatty acyl transferases in lipid synthesis they occur in all organisms, from Escherichia coli to (see Fig. 21–2), in which nucleophilic substitution at the humans—and consider how they are regulated and in- carbonyl carbon of a thioester results in transfer of the tegrated in mammals by hormonal mechanisms. acyl group to another moiety. As we undertake our study of intermediary metabolism, a final word. Keep in mind that the myriad re- 5. Free radical reactions Once thought to be rare, the actions described in these pages take place in, and play homolytic cleavage of covalent bonds to generate free crucial roles in, living organisms. As you encounter each radicals has now been found in a range of biochemical reaction and each pathway ask, What does this chemi- processes. Some examples are the reactions of methyl- cal transformation do for the organism? How does this malonyl-CoA mutase (see Box 17–2), ribonucleotide pathway interconnect with the other pathways operat- reductase (see Fig. 22–41), and DNA photolyase (see ing simultaneously in the same cell to produce the en- Fig. 25–25). ergy and products required for cell maintenance and growth? How do the multilayered regulatory mecha- We begin Part II with a discussion of the basic en- nisms cooperate to balance metabolic and energy in- ergetic principles that govern all metabolism (Chapter puts and outputs, achieving the dynamic steady state 13). We then consider the major catabolic pathways by of life? Studied with this perspective, metabolism pro- which cells obtain energy from the oxidation of various vides fascinating and revealing insights into life, with fuels (Chapters 14 through 19). Chapter 19 is the piv- countless applications in medicine, agriculture, and otal point of our discussion of metabolism; it concerns biotechnology. chapter13 PRINCIPLES OF BIOENERGETICS

13.1 Bioenergetics and Thermodynamics 490 heat and that this process of 13.2 Phosphoryl Group Transfers and ATP 496 respiration is essential to life. He observed that 13.3 Biological Oxidation-Reduction Reactions 507 ...in general, respiration is nothing but a slow com- The total energy of the universe is constant; the total bustion of carbon and hy- entropy is continually increasing. drogen, which is entirely similar to that which oc- —Rudolf Clausius, The Mechanical Theory of Heat with Its curs in a lighted lamp or Applications to the Steam-Engine and to the Physical candle, and that, from this Properties of Bodies, 1865 (trans. 1867) point of view, animals that Antoine Lavoisier, respire are true com- 1743–1794 The isomorphism of entropy and information establishes a bustible bodies that burn link between the two forms of power: the power to do and and consume themselves . . . One may say that this the power to direct what is done. analogy between combustion and respiration has —François Jacob, La logique du vivant: une histoire de l’hérédité not escaped the notice of the poets, or rather the (The Logic of Life: A History of Heredity), 1970 philosophers of antiquity, and which they had ex- pounded and interpreted. This fire stolen from heaven, this torch of Prometheus, does not only rep- iving cells and organisms must perform work to stay resent an ingenious and poetic idea, it is a faithful L alive, to grow, and to reproduce. The ability to har- picture of the operations of nature, at least for an- ness energy and to channel it into biological work is a imals that breathe; one may therefore say, with the fundamental property of all living organisms; it must ancients, that the torch of life lights itself at the mo- have been acquired very early in cellular evolution. Mod- ment the infant breathes for the first time, and it * ern organisms carry out a remarkable variety of energy does not extinguish itself except at death. transductions, conversions of one form of energy to an- In this century, biochemical studies have revealed other. They use the chemical energy in fuels to bring much of the chemistry underlying that “torch of life.” about the synthesis of complex, highly ordered macro- Biological energy transductions obey the same physical molecules from simple precursors. They also convert the laws that govern all other natural processes. It is there- chemical energy of fuels into concentration gradients fore essential for a student of biochemistry to under- and electrical gradients, into motion and heat, and, in a stand these laws and how they apply to the flow of few organisms such as fireflies and some deep-sea fish, energy in the biosphere. In this chapter we first review into light. Photosynthetic organisms transduce light en- the laws of thermodynamics and the quantitative rela- ergy into all these other forms of energy. tionships among free energy, enthalpy, and entropy. We The chemical mechanisms that underlie biological then describe the special role of ATP in biological energy transductions have fascinated and challenged biologists for centuries. Antoine Lavoisier, before he lost *From a memoir by Armand Seguin and Antoine Lavoisier, dated 1789, his head in the French Revolution, recognized that an- quoted in Lavoisier, A. (1862) Oeuvres de Lavoisier, Imprimerie imals somehow transform chemical fuels (foods) into Impériale, Paris.

489 490 Chapter 13 Principles of Bioenergetics energy exchanges. Finally, we consider the importance not violate the second law; they operate strictly within of oxidation-reduction reactions in living cells, the en- it. To discuss the application of the second law to bio- ergetics of electron-transfer reactions, and the electron logical systems, we must first define those systems and carriers commonly employed as cofactors of the en- their surroundings. zymes that catalyze these reactions. The reacting system is the collection of matter that is undergoing a particular chemical or physical process; it may be an organism, a cell, or two reacting com- 13.1 Bioenergetics and Thermodynamics pounds. The reacting system and its surroundings together constitute the universe. In the laboratory, some Bioenergetics is the quantitative study of the energy chemical or physical processes can be carried out in iso- transductions that occur in living cells and of the nature lated or closed systems, in which no material or energy and function of the chemical processes underlying these is exchanged with the surroundings. Living cells and or- transductions. Although many of the principles of ther- ganisms, however, are open systems, exchanging both modynamics have been introduced in earlier chapters material and energy with their surroundings; living sys- and may be familiar to you, a review of the quantitative tems are never at equilibrium with their surroundings, aspects of these principles is useful here. and the constant transactions between system and surroundings explain how organisms can create order Biological Energy Transformations Obey the Laws within themselves while operating within the second law of Thermodynamics of thermodynamics. Many quantitative observations made by physicists and In Chapter 1 (p. 23) we defined three thermody- chemists on the interconversion of different forms of namic quantities that describe the energy changes oc- energy led, in the nineteenth century, to the formula- curring in a chemical reaction: tion of two fundamental laws of thermodynamics. The first law is the principle of the conservation of energy: Gibbs free energy, G, expresses the amount of for any physical or chemical change, the total energy capable of doing work during a reaction amount of energy in the universe remains constant; at constant temperature and pressure. When a energy may change form or it may be transported reaction proceeds with the release of free energy from one region to another, but it cannot be created (that is, when the system changes so as to or destroyed. The second law of thermodynamics, which possess less free energy), the free-energy change, can be stated in several forms, says that the universe G, has a negative value and the reaction is said always tends toward increasing disorder: in all natu- to be exergonic. In endergonic reactions, the ral processes, the entropy of the universe increases. system gains free energy and G is positive. Living organisms consist of collections of molecules Enthalpy, H, is the heat content of the reacting much more highly organized than the surrounding ma- system. It reflects the number and kinds of terials from which they are constructed, and organisms chemical bonds in the reactants and products. maintain and produce order, seemingly oblivious to the When a chemical reaction releases heat, it is second law of thermodynamics. But living organisms do said to be exothermic; the heat content of the products is less than that of the reactants and H has, by convention, a negative value. Reacting systems that take up heat from their surroundings are endothermic and have positive values of H. Entropy, S, is a quantitative expression for the randomness or disorder in a system (see Box 1–3). When the products of a reaction are less complex and more disordered than the reactants, the reaction is said to proceed with a gain in entropy.

The units of G and H are joules/mole or calories/mole (recall that 1 cal 4.184 J); units of entropy are joules/mole Kelvin (J/mol K) (Table 13–1). Under the conditions existing in biological systems (including constant temperature and pressure), changes in free energy, enthalpy, and entropy are related to each other quantitatively by the equation

G H T S (13–1) 13.1 Bioenergetics and Thermodynamics 491

free energy into ATP and other energy-rich compounds TABLE 13–1 Some Physical Constants and capable of providing energy for biological work at con- Units Used in Thermodynamics stant temperature. Boltzmann constant, k 1.381 1023 J/K Avogadro’s number, N 6.022 1023 mol1 The Standard Free-Energy Change Is Directly Related Faraday constant, 96,480 J/V mol to the Equilibrium Constant Gas constant, R 8.315 J/mol K ( 1.987 cal/mol K) The composition of a reacting system (a mixture of chemical reactants and products) tends to continue Units of G and H are J/mol (or cal/mol) changing until equilibrium is reached. At the equilibrium Units of S are J/mol K (or cal/mol K) concentration of reactants and products, the rates of the 1 cal 4.184 J forward and reverse reactions are exactly equal and no Units of absolute temperature, T, are Kelvin, K further net change occurs in the system. The concen- 25 C 298 K trations of reactants and products at equilibrium At 25 C, RT 2.479 kJ/mol define the equilibrium constant, Keq (p. 26). In the ( 0.592 kcal/mol) general reaction aA bB yz cC dD, where a, b, c, and d are the number of molecules of A, B, C, and D participating, the equilibrium constant is given by in which G is the change in Gibbs free energy of the [C]c[D]d K (13–2) reacting system, H is the change in enthalpy of the eq [A]a[B]b system, T is the absolute temperature, and S is the where [A], [B], [C], and [D] are the molar concentrations change in entropy of the system. By convention, S has of the reaction components at the point of equilibrium. a positive sign when entropy increases and H, as noted When a reacting system is not at equilibrium, the above, has a negative sign when heat is released by the tendency to move toward equilibrium represents a driv- system to its surroundings. Either of these conditions, ing force, the magnitude of which can be expressed as which are typical of favorable processes, tend to make the free-energy change for the reaction, G. Under stan- G negative. In fact, G of a spontaneously reacting sys- dard conditions (298 K 25 C), when reactants and tem is always negative. products are initially present at 1 M concentrations or, The second law of thermodynamics states that the for gases, at partial pressures of 101.3 kilopascals (kPa), entropy of the universe increases during all chemical or 1 atm, the force driving the system toward equilib- and physical processes, but it does not require that the rium is defined as the standard free-energy change, G. entropy increase take place in the reacting system it- By this definition, the standard state for reactions that self. The order produced within cells as they grow and involve hydrogen ions is [H ] 1 M, or pH 0. Most bio- divide is more than compensated for by the disorder chemical reactions, however, occur in well-buffered they create in their surroundings in the course of growth aqueous solutions near pH 7; both the pH and the con- and division (see Box 1–3, case 2). In short, living or- centration of water (55.5 M) are essentially constant. ganisms preserve their internal order by taking from the For convenience of calculations, biochemists therefore surroundings free energy in the form of nutrients or sun- define a different standard state, in which the concen- light, and returning to their surroundings an equal 7 tration of H is 10 M (pH 7) and that of water is amount of energy as heat and entropy. 2 55.5 M; for reactions that involve Mg (including most Cells Require Sources of Free Energy in which ATP is a reactant), its concentration in solution is commonly taken to be constant at 1 mM. Physi- Cells are isothermal systems—they function at essen- cal constants based on this biochemical standard state tially constant temperature (they also function at con- are called standard transformed constants and are stant pressure). Heat flow is not a source of energy for written with a prime (such as G and Keq) to distin- cells, because heat can do work only as it passes to a guish them from the untransformed constants used by zone or object at a lower temperature. The energy that chemists and physicists. (Notice that most other text- cells can and must use is free energy, described by the books use the symbol G rather than G. Our use of Gibbs free-energy function G, which allows prediction G, recommended by an international committee of of the direction of chemical reactions, their exact equi- chemists and biochemists, is intended to emphasize that librium position, and the amount of work they can in the transformed free energy G is the criterion for equi- 2 theory perform at constant temperature and pressure. librium.) By convention, when H2O, H , and/or Mg Heterotrophic cells acquire free energy from nutrient are reactants or products, their concentrations are not molecules, and photosynthetic cells acquire it from ab- included in equations such as Equation 13–2 but are in- sorbed solar radiation. Both kinds of cells transform this stead incorporated into the constants Keq and G. 492 Chapter 13 Principles of Bioenergetics

Just as Keq is a physical constant characteristic for each reaction, so too is G a constant. As we noted in TABLE 13–3 Relationships among Keq, G , and the Direction of Chemical Reactions under Chapter 6, there is a simple relationship between Keq and G: Standard Conditions

G RT ln Keq Starting with all components at 1 M, The standard free-energy change of a chemical re- When K is . . . G is . . . the reaction . . . action is simply an alternative mathematical way of eq expressing its equilibrium constant. Table 13–2 1.0 negative proceeds forward shows the relationship between G and Keq. If the 1.0 zero is at equilibrium equilibrium constant for a given chemical reaction is 1.0, 1.0 positive proceeds in reverse the standard free-energy change of that reaction is 0.0

(the natural logarithm of 1.0 is zero). If Keq of a reaction is greater than 1.0, its G is negative. If Keq is less than 1.0, G is positive. Because the relationship be- As an example, let’s make a simple calculation of tween G and K is exponential, relatively small the standard free-energy change of the reaction cat- eq alyzed by the enzyme phosphoglucomutase: changes in G correspond to large changes in Keq. It may be helpful to think of the standard free- Glucose 1-phosphate 34 glucose 6-phosphate energy change in another way. G is the difference between the free-energy content of the products and the Chemical analysis shows that whether we start with, say, free-energy content of the reactants, under standard 20 mM glucose 1-phosphate (but no glucose 6-phosphate) conditions. When G is negative, the products contain or with 20 mM glucose 6-phosphate (but no glucose less free energy than the reactants and the reaction will 1-phosphate), the final equilibrium mixture at 25 C and proceed spontaneously under standard conditions; all pH 7.0 will be the same: 1 mM glucose 1-phosphate and chemical reactions tend to go in the direction that re- 19 mM glucose 6-phosphate. (Remember that enzymes do sults in a decrease in the free energy of the system. A not affect the point of equilibrium of a reaction; they positive value of G means that the products of the merely hasten its attainment.) From these data we can reaction contain more free energy than the reactants, calculate the equilibrium constant: and this reaction will tend to go in the reverse direction [glucose 6-phosphate] 19 mM Keq 19 if we start with 1.0 M concentrations of all components [glucose 1-phosphate] 1 mM (standard conditions). Table 13–3 summarizes these points. From this value of Keq we can calculate the standard free-energy change: TABLE 13–2 Relationship between the G RT ln Keq Equilibrium Constants and Standard Free-Energy (8.315 J/mol K)(298 K)(ln 19) Changes of Chemical Reactions 7.3 kJ/mol Because the standard free-energy change is negative, G when the reaction starts with 1.0 M glucose 1-phosphate and 1.0 M glucose 6-phosphate, the conversion of glu- Keq (kJ/mol) (kcal/mol)* cose 1-phosphate to glucose 6-phosphate proceeds with 103 17.1 4.1 a loss (release) of free energy. For the reverse reaction 102 11.4 2.7 (the conversion of glucose 6-phosphate to glucose 101 5.7 1.4 1-phosphate), G has the same magnitude but the op- 1 0.0 0.0 posite sign. 101 5.7 1.4 Table 13–4 gives the standard free-energy changes 102 11.4 2.7 for some representative chemical reactions. Note that 103 17.1 4.1 hydrolysis of simple esters, amides, peptides, and gly- 104 22.8 5.5 cosides, as well as rearrangements and eliminations, 105 28.5 6.8 proceed with relatively small standard free-energy 106 34.2 8.2 changes, whereas hydrolysis of acid anhydrides is accompanied by relatively large decreases in standard free *Although joules and kilojoules are the standard units of energy and are used energy. The complete oxidation of organic compounds throughout this text, biochemists sometimes express G values in kilocalories per such as glucose or palmitate to CO2 and H2O, which in mole. We have therefore included values in both kilojoules and kilocalories in this table and in Tables 13–4 and 13–6. To convert kilojoules to kilocalories, divide the number cells requires many steps, results in very large decreases of kilojoules by 4.184. in standard free energy. However, standard free-energy 13.1 Bioenergetics and Thermodynamics 493

TABLE 13–4 Standard Free-Energy Changes of Some Chemical Reactions at pH 7.0 and 25 C (298 K)

G Reaction type (kJ/mol) (kcal/mol) Hydrolysis reactions Acid anhydrides Acetic anhydride H2O On 2 acetate 91.1 21.8 ATP H2O 88n ADP Pi 30.5 7.3 ATP H2O 88n AMP PPi 45.6 10.9 PPi H2O 88n 2Pi 19.2 4.6 UDP-glucose H2O 88n UMP glucose 1-phosphate 43.0 10.3 Esters

Ethyl acetate H2O 88n ethanol acetate 19.6 4.7 Glucose 6-phosphate H2O 88n glucose Pi 13.8 3.3 Amides and peptides Glutamine H2O 88n glutamate NH4 14.2 3.4 Glycylglycine H2O 88n 2 glycine 9.2 2.2 Glycosides

Maltose H2O 88n 2 glucose 15.5 3.7 Lactose H2O 88n glucose galactose 15.9 3.8 Rearrangements

Glucose 1-phosphate 88n glucose 6-phosphate 7.3 1.7 Fructose 6-phosphate 88n glucose 6-phosphate 1.7 0.4 Elimination of water

Malate 88n fumarate H2O 3.1 0.8 Oxidations with molecular oxygen

Glucose 6O2 88n 6CO2 6H2O 2,840 686 Palmitate 23O2 88n 16CO2 16H2O 9,770 2,338

changes such as those in Table 13–4 indicate how much energy change tells us in which direction and how far a free energy is available from a reaction under standard given reaction must go to reach equilibrium when the conditions. To describe the energy released under the initial concentration of each component is 1.0 M, the conditions existing in cells, an expression for the actual pH is 7.0, the temperature is 25 C, and the pressure is free-energy change is essential. 101.3 kPa. Thus G is a constant: it has a characteristic, unchanging value for a given reaction. But the ac- Actual Free-Energy Changes Depend on Reactant tual free-energy change, G, is a function of reactant and Product Concentrations and product concentrations and of the temperature prevailing during the reaction, which will not necessarily We must be careful to distinguish between two differ- match the standard conditions as defined above. More- ent quantities: the free-energy change, G, and the stan- over, the G of any reaction proceeding spontaneously dard free-energy change, G. Each chemical reaction toward its equilibrium is always negative, becomes less has a characteristic standard free-energy change, which negative as the reaction proceeds, and is zero at the may be positive, negative, or zero, depending on the point of equilibrium, indicating that no more work can equilibrium constant of the reaction. The standard free- be done by the reaction. 494 Chapter 13 Principles of Bioenergetics

DG and DG for any reaction A B 34 C D are urable rates. For example, combustion of firewood to related by the equation CO2 and H2O is very favorable thermodynamically, but firewood remains stable for years because the activation [C][D] G G RT ln (13–3) energy (see Figs 6–2 and 6–3) for the combustion re- [A][B] action is higher than the energy available at room tem- in which the terms in red are those actually prevail- perature. If the necessary activation energy is provided ing in the system under observation. The concentration (with a lighted match, for example), combustion will be- terms in this equation express the effects commonly gin, converting the wood to the more stable products called mass action, and the term [C][D]/[A][B] is called CO2 and H2O and releasing energy as heat and light. The the mass-action ratio, Q. As an example, let us sup- heat released by this exothermic reaction provides the pose that the reaction A B 34 C D is taking place activation energy for combustion of neighboring regions at the standard conditions of temperature (25 C) and of the firewood; the process is self-perpetuating. pressure (101.3 kPa) but that the concentrations of A, In living cells, reactions that would be extremely B, C, and D are not equal and none of the components slow if uncatalyzed are caused to proceed, not by sup- is present at the standard concentration of 1.0 M. To de- plying additional heat but by lowering the activation en- termine the actual free-energy change, G, under these ergy with an enzyme. An enzyme provides an alternative nonstandard conditions of concentration as the reaction reaction pathway with a lower activation energy than the proceeds from left to right, we simply enter the actual uncatalyzed reaction, so that at room temperature a large concentrations of A, B, C, and D in Equation 13–3; the fraction of the substrate molecules have enough thermal values of R, T, and G are the standard values. G is energy to overcome the activation barrier, and the re- negative and approaches zero as the reaction proceeds action rate increases dramatically. The free-energy because the actual concentrations of A and B decrease change for a reaction is independent of the pathway and the concentrations of C and D increase. Notice that by which the reaction occurs; it depends only on the when a reaction is at equilibrium—when there is no nature and concentration of the initial reactants and the force driving the reaction in either direction and G is final products. Enzymes cannot, therefore, change equi- zero—Equation 13–3 reduces to librium constants; but they can and do increase the rate at which a reaction proceeds in the direction dictated by [C]eq[D]eq thermodynamics. 0 G G RT ln [A]eq[B]eq or Standard Free-Energy Changes Are Additive G RT ln Keq In the case of two sequential chemical reactions, A 34 B and B C, each reaction has its own equilibrium which is the equation relating the standard free-energy 34 constant and each has its characteristic standard free- change and equilibrium constant given earlier. energy change, G and G. As the two reactions are The criterion for spontaneity of a reaction is the 1 2 sequential, B cancels out to give the overall reaction value of G, not G. A reaction with a positive G A C, which has its own equilibrium constant and thus can go in the forward direction if G is negative. This 34 its own standard free-energy change, G . The G is possible if the term RT ln ([products]/[reactants]) in total values of sequential chemical reactions are additive. Equation 13–3 is negative and has a larger absolute For the overall reaction A C, G is the sum of value than G. For example, the immediate removal 34 total the individual standard free-energy changes, G and of the products of a reaction can keep the ratio [prod- 1 G, of the two reactions: G G G. ucts]/[reactants] well below 1, such that the term RT ln 2 total 1 2 ([products]/[reactants]) has a large, negative value. (1) A88nB G1 G and G are expressions of the maximum (2) B88nC G2 amount of free energy that a given reaction can theo- Sum: A88nC G1 G2 retically deliver—an amount of energy that could be This principle of bioenergetics explains how a ther- realized only if a perfectly efficient device were avail- modynamically unfavorable (endergonic) reaction can able to trap or harness it. Given that no such device is be driven in the forward direction by coupling it to possible (some free energy is always lost to entropy dur- a highly exergonic reaction through a common inter- ing any process), the amount of work done by the re- mediate. For example, the synthesis of glucose 6- action at constant temperature and pressure is always phosphate is the first step in the utilization of glucose less than the theoretical amount. by many organisms: Another important point is that some thermodynamically favorable reactions (that is, reactions for Glucose Pi 88n glucose 6-phosphate H2O which G is large and negative) do not occur at meas- G 13.8 kJ/mol 13.1 Bioenergetics and Thermodynamics 495

The positive value of G predicts that under standard cose 6-phosphate synthesis, the Keq for formation of conditions the reaction will tend not to proceed spon- glucose 6-phosphate has been raised by a factor of about taneously in the direction written. Another cellular re- 2 105. action, the hydrolysis of ATP to ADP and Pi, is very This common-intermediate strategy is employed by exergonic: all living cells in the synthesis of metabolic intermediates and cellular components. Obviously, the strategy works ATP H O ADP P G 30.5 kJ/mol 2 88n i only if compounds such as ATP are continuously avail- These two reactions share the common intermediates able. In the following chapters we consider several of the

Pi and H2O and may be expressed as sequential reac- most important cellular pathways for producing ATP. tions: SUMMARY 13.1 Bioenergetics and Thermodynamics (1) Glucose Pi 88n glucose 6-phosphate H2O (2) ATP H2O 88n ADP Pi Sum: ATP glucose 88n ADP glucose 6-phosphate ■ Living cells constantly perform work. They require energy for maintaining their highly The overall standard free-energy change is obtained by organized structures, synthesizing cellular adding the G values for individual reactions: components, generating electric currents, and G 13.8 kJ/mol (30.5 kJ/mol) 16.7 kJ/mol many other processes. The overall reaction is exergonic. In this case, energy ■ Bioenergetics is the quantitative study of stored in ATP is used to drive the synthesis of glucose energy relationships and energy conversions in 6-phosphate, even though its formation from glucose biological systems. Biological energy transformations obey the laws of and inorganic phosphate (Pi) is endergonic. The pathway of glucose 6-phosphate formation by phosphoryl thermodynamics. transfer from ATP is different from reactions (1) and ■ All chemical reactions are influenced by two (2) above, but the net result is the same as the sum of forces: the tendency to achieve the most stable the two reactions. In thermodynamic calculations, all bonding state (for which enthalpy, H, is a that matters is the state of the system at the beginning useful expression) and the tendency to achieve of the process and its state at the end; the route be- the highest degree of randomness, expressed tween the initial and final states is immaterial. as entropy, S. The net driving force in a We have said that G is a way of expressing the reaction is G, the free-energy change, which equilibrium constant for a reaction. For reaction (1) represents the net effect of these two factors: above, G H T S. ■ The standard transformed free-energy change, [glucose 6-phosphate] 3 1 Keq 3.9 10 M 1 G, is a physical constant that is [glucose][Pi] characteristic for a given reaction and can be Notice that H2O is not included in this expression, as its calculated from the equilibrium constant for concentration (55.5 M) is assumed to remain unchanged the reaction: G RT ln Keq. by the reaction. The equilibrium constant for the hydrolysis of ATP is ■ The actual free-energy change, G, is a variable that depends on G and on the

[ADP][Pi] 5 concentrations of reactants and products: Keq 2.0 10 M 2 [ATP] G G RT ln ([products]/[reactants]). The equilibrium constant for the two coupled reactions ■ When G is large and negative, the reaction is tends to go in the forward direction; when G is large and positive, the reaction tends to go in [glucose 6-phosphate][ADP][Pi] Keq the reverse direction; and when G 0, the 3 [glucose][P ][ATP] i system is at equilibrium. 3 1 5 (Keq )(Keq ) (3.9 10 M ) (2.0 10 M) 1 2 ■ The free-energy change for a reaction is 2 7.8 10 independent of the pathway by which the This calculation illustrates an important point about reaction occurs. Free-energy changes are equilibrium constants: although the G values for two additive; the net chemical reaction that results reactions that sum to a third are additive, the Keq for from successive reactions sharing a common a reaction that is the sum of two reactions is the prod- intermediate has an overall free-energy change uct of their individual Keq values. Equilibrium constants that is the sum of the G values for the are multiplicative. By coupling ATP hydrolysis to glu- individual reactions. 496 Chapter 13 Principles of Bioenergetics

13.2 Phosphoryl Group Transfers and ATP O O O B B B OOPOOOPOOOPOOO Rib O Adenine Having developed some fundamental principles of en- A A A 4 ergy changes in chemical systems, we can now exam- H O O O O ATP ine the energy cycle in cells and the special role of ATP H as the energy currency that links catabolism and an- 1 abolism (see Fig. 1–28). Heterotrophic cells obtain free O hydrolysis, B with relief energy in a chemical form by the catabolism of nutrient OOPOOH A of charge molecules, and they use that energy to make ATP from repulsion Pi O ADP and Pi. ATP then donates some of its chemical en- resonance ergy to endergonic processes such as the synthesis of 2 stabilization metabolic intermediates and macromolecules from smaller precursors, the transport of substances across 3 O membranes against concentration gradients, and me- A chanical motion. This donation of energy from ATP gen- OOPOO H A erally involves the covalent participation of ATP in the O reaction that is to be driven, with the eventual result that ATP is converted to ADP and Pi or, in some reac- O O B B tions, to AMP and 2 Pi. We discuss here the chemical HOOPOOOPOOO Rib O Adenine basis for the large free-energy changes that accompany A A O O ADP2 hydrolysis of ATP and other high-energy phosphate compounds, and we show that most cases of energy donation by ATP involve group transfer, not simple hy- 3 ionization drolysis of ATP. To illustrate the range of energy transductions in which ATP provides the energy, we consider O O B B the synthesis of information-rich macromolecules, the H OOPOOOPOOO Rib O Adenine A A transport of solutes across membranes, and motion pro- O O ADP3 duced by muscle contraction. 4 3 2 ATP H2O ADP P i H The Free-Energy Change for ATP Hydrolysis G 30.5 kJ/mol Is Large and Negative FIGURE 13–1 Chemical basis for the large free-energy change asso- Figure 13–1 summarizes the chemical basis for the rel- ciated with ATP hydrolysis. 1The charge separation that results from atively large, negative, standard free energy of hydrol- hydrolysis relieves electrostatic repulsion among the four negative ysis of ATP. The hydrolytic cleavage of the terminal charges on ATP. 2 The product inorganic phosphate (Pi) is stabilized phosphoric acid anhydride (phosphoanhydride) bond in by formation of a resonance hybrid, in which each of the four phos- ATP separates one of the three negatively charged phorus–oxygen bonds has the same degree of double-bond character phosphates and thus relieves some of the electrostatic and the hydrogen ion is not permanently associated with any one of 2 repulsion in ATP; the Pi (HPO4 ) released is stabilized the oxygens. (Some degree of resonance stabilization also occurs in by the formation of several resonance forms not possi- phosphates involved in ester or anhydride linkages, but fewer reso- 2 2 ble in ATP; and ADP , the other direct product of nance forms are possible than for Pi.) 3 The product ADP imme- hydrolysis, immediately ionizes, releasing H into a diately ionizes, releasing a proton into a medium of very low [H ] 7 medium of very low [H ] (~10 M). Because the con- (pH 7). A fourth factor (not shown) that favors ATP hydrolysis is the centrations of the direct products of ATP hydrolysis are, greater degree of solvation (hydration) of the products Pi and ADP rel- in the cell, far below the concentrations at equilibrium ative to ATP, which further stabilizes the products relative to the re- (Table 13–5), mass action favors the hydrolysis reaction actants. in the cell.

Although the hydrolysis of ATP is highly exergonic and Pi are not identical and are much lower than the (G 30.5 kJ/mol), the molecule is kinetically sta- 1.0 M of standard conditions (Table 13–5). Furthermore, ble at pH 7 because the activation energy for ATP Mg2 in the cytosol binds to ATP and ADP (Fig. 13–2), hydrolysis is relatively high. Rapid cleavage of the phos- and for most enzymatic reactions that involve ATP as phoanhydride bonds occurs only when catalyzed by an phosphoryl group donor, the true substrate is MgATP2. enzyme. The relevant G is therefore that for MgATP2 hy- The free-energy change for ATP hydrolysis is drolysis. Box 13–1 shows how G for ATP hydrolysis in 30.5 kJ/mol under standard conditions, but the actual the intact erythrocyte can be calculated from the data free energy of hydrolysis (G) of ATP in living cells is in Table 13–5. In intact cells, G for ATP hydrolysis, very different: the cellular concentrations of ATP, ADP, usually designated Gp, is much more negative than 13.2 Phosphoryl Group Transfers and ATP 497

TABLE 13–5 Adenine Nucleotide, Inorganic Phosphate, and Phosphocreatine Concentrations in Some Cells

Concentration (mM)*

† ATP ADP AMP Pi PCr Rat hepatocyte 3.38 1.32 0.29 4.8 0 Rat myocyte 8.05 0.93 0.04 8.05 28 Rat neuron 2.59 0.73 0.06 2.72 4.7 Human erythrocyte 2.25 0.25 0.02 1.65 0 E. coli cell 7.90 1.04 0.82 7.9 0

*For erythrocytes the concentrations are those of the cytosol (human erythrocytes lack a nucleus and mitochondria). In the other types of cells the data are for the entire cell contents, although the cytosol and the mitochondria have very different concentrations of ADP.PCr is phosphocreatine, discussed on p. 489. †This value reflects total concentration; the true value for free ADP may be much lower (see Box 13–1).

G, ranging from 50 to 65 kJ/mol. Gp is often called the phosphorylation potential. In the follow- O O O B B B ing discussions we use the standard free-energy change OOPOOOPOOOPOOO Rib O Adenine A A A for ATP hydrolysis, because this allows comparison, on 2 O O O MgATP the same basis, with the energetics of other cellular ø ø Mg2 reactions. Remember, however, that in living cells G is the relevant quantity—for ATP hydrolysis and all other O O B B reactions—and may be quite different from G. OOPOOOPOOO Rib O Adenine A A Other Phosphorylated Compounds and Thioesters O O MgADP ø 2ø Also Have Large Free Energies of Hydrolysis Mg Phosphoenolpyruvate (Fig. 13–3) contains a phosphate FIGURE 13–2 Mg2 and ATP. Formation of Mg2 complexes partially ester bond that undergoes hydrolysis to yield the enol shields the negative charges and influences the conformation of the form of pyruvate, and this direct product can immedi- phosphate groups in nucleotides such as ATP and ADP. ately tautomerize to the more stable keto form of pyruvate. Because the reactant (phosphoenolpyruvate) has only one form (enol) and the product (pyruvate) has two possible forms, the product is stabilized relative to the 49.3 kJ/mol), which can, again, be explained in terms reactant. This is the greatest contributing factor to of the structure of reactant and products. When H2O is the high standard free energy of hydrolysis of phospho- added across the anhydride bond of 1,3-bisphospho- enolpyruvate: G 61.9 kJ/mol. glycerate, one of the direct products, 3-phosphoglyceric Another three-carbon compound, 1,3-bisphospho- acid, can immediately lose a proton to give the car- glycerate (Fig. 13–4), contains an anhydride bond be- boxylate ion, 3-phosphoglycerate, which has two equally tween the carboxyl group at C-1 and phosphoric acid. probable resonance forms (Fig. 13–4). Removal of the Hydrolysis of this acyl phosphate is accompanied by a direct product (3-phosphoglyceric acid) and formation of large, negative, standard free-energy change (G the resonance-stabilized ion favor the forward reaction.

O O GJ O P H2O O O J D G J J FIGURE 13–3 Hydrolysis of phosphoenol- OOC O O OOC OH tautomerization OOC O G D hydrolysis G D G J pyruvate (PEP). Catalyzed by pyruvate kinase, C P C C B i B A this reaction is followed by spontaneous CH2 CH2 CH3 tautomerization of the product, pyruvate. PEP Pyruvate Pyruvate Tautomerization is not possible in PEP, and (enol form) (keto form) thus the products of hydrolysis are stabilized relative to the reactants. Resonance PEP3 H O pyruvate P2 2 i stabilization of P also occurs, as shown i G 61.9 kJ/mol in Figure 13–1. 498 Chapter 13 Principles of Bioenergetics

O O FIGURE 13–4 Hydrolysis of 1,3- G J bisphosphoglycerate. The direct P D G O O O O OH O O resonance product of hydrolysis is 3-phospho- M D M D G D stabilization glyceric acid, with an undissociated 1 C C C A A A carboxylic acid group, but Pi H 2 CHOH CHOH CHOH dissociation occurs immediately. A A A This ionization and the resonance 3 CH2 CH2 CH2 A H O A A structures it makes possible stabilize O 2 O O the product relative to the reactants. hydrolysis ionization A A A OOPPO OOPPO OOPPO Resonance stabilization of Pi further A A A O O O contributes to the negative free- energy change. 1,3-Bisphosphoglycerate3-Phosphoglyceric acid 3-Phosphoglycerate

4 3 2 1,3-Bisphosphoglycerate H2O 3-phosphoglycerate P i H G 49.3 kJ/mol

BOX 13–1 WORKING IN BIOCHEMISTRY

The Free Energy of Hydrolysis of ATP within Cells: much larger than the standard free-energy change The Real Cost of Doing Metabolic Business (30.5 kJ/mol). By the same token, the free energy required to synthesize ATP from ADP and P under The standard free energy of hydrolysis of ATP is i the conditions prevailing in the erythrocyte would be 30.5 kJ/mol. In the cell, however, the concentrations 52 kJ/mol. of ATP, ADP, and P are not only unequal but much i Because the concentrations of ATP, ADP, and P lower than the standard 1 M concentrations (see Table i differ from one cell type to another (see Table 13–5), 13–5). Moreover, the cellular pH may differ somewhat G for ATP hydrolysis likewise differs among cells. from the standard pH of 7.0. Thus the actual free p Moreover, in any given cell, G can vary from time energy of hydrolysis of ATP under intracellular con- p to time, depending on the metabolic conditions in the ditions (G ) differs from the standard free-energy p cell and how they influence the concentrations of ATP, change, G. We can easily calculate G . p ADP, P , and H (pH). We can calculate the actual In human erythrocytes, for example, the concentra- i free-energy change for any given metabolic reaction tions of ATP, ADP, and P are 2.25, 0.25, and 1.65 mM, i as it occurs in the cell, providing we know the con- respectively. Let us assume for simplicity that the pH centrations of all the reactants and products of the re- is 7.0 and the temperature is 25 C, the standard pH action and know about the other factors (such as pH, and temperature. The actual free energy of hydrolysis temperature, and concentration of Mg2) that may af- of ATP in the erythrocyte under these conditions is fect the G and thus the calculated free-energy given by the relationship change, Gp.

[ADP][Pi] To further complicate the issue, the total concen- Gp G RT ln [ATP] trations of ATP, ADP, Pi, and H may be substantially higher than the free concentrations, which are the Substituting the appropriate values we obtain thermodynamically relevant values. The difference is due to tight binding of ATP, ADP, and P to cellular Gp 30.5 kJ/mol i proteins. For example, the concentration of free ADP (0.25 103)(1.65 103) (8.315 J/mol K)(298 K) ln in resting muscle has been variously estimated at be- 3 2.25 10 tween 1 and 37 M. Using the value 25 M in the cal-

culation outlined above, we get a G of 58 kJ/mol. 30.5 kJ/mol (2.48 kJ/mol) ln 1.8 10 4 p Calculation of the exact value of G is perhaps 30.5 kJ/mol 21 kJ/mol p less instructive than the generalization we can make 52 kJ/mol about actual free-energy changes: in vivo, the energy

Thus Gp, the actual free-energy change for ATP hy- released by ATP hydrolysis is greater than the stan- drolysis in the intact erythrocyte (52 kJ/mol), is dard free-energy change, G. 13.2 Phosphoryl Group Transfers and ATP 499

COO COO COO FIGURE 13–5 Hydrolysis of phospho- A A A creatine. Breakage of the PON bond O CH2 H O CH2 CH2 H 2 H2N in phosphocreatine produces creatine, B A A A OOPONOOOC N CH3 H2NOOOC N CH3 CN OCH3 which is stabilized by formation of a A B hydrolysis B resonance H N stabilization 2 resonance hybrid. The other product, O NH2 Pi NH2 Phosphocreatine Creatine Pi, is also resonance stabilized.

2 2 Phosphocreatine H2O creatine P i G 43.0 kJ/mol

In phosphocreatine (Fig. 13–5), the PON bond can these compounds is activated for transacylation, con- be hydrolyzed to generate free creatine and Pi. The re- densation, or oxidation-reduction reactions. Thioesters lease of Pi and the resonance stabilization of creatine undergo much less resonance stabilization than do oxy- favor the forward reaction. The standard free-energy gen esters; consequently, the difference in free energy change of phosphocreatine hydrolysis is again large, between the reactant and its hydrolysis products, which 43.0 kJ/mol. are resonance-stabilized, is greater for thioesters than In all these phosphate-releasing reactions, the sev- for comparable oxygen esters (Fig. 13–7). In both cases, eral resonance forms available to Pi (Fig. 13–1) stabi- hydrolysis of the ester generates a carboxylic acid, lize this product relative to the reactant, contributing to which can ionize and assume several resonance forms. an already negative free-energy change. Table 13–6 lists Together, these factors result in the large, negative G the standard free energies of hydrolysis for a number of (31 kJ/mol) for acetyl-CoA hydrolysis. phosphorylated compounds. To summarize, for hydrolysis reactions with large, Thioesters, in which a sulfur atom replaces the negative, standard free-energy changes, the products usual oxygen in the ester bond, also have large, nega- are more stable than the reactants for one or more of tive, standard free energies of hydrolysis. Acetyl-coen- the following reasons: (1) the bond strain in reactants zyme A, or acetyl-CoA (Fig. 13–6), is one of many due to electrostatic repulsion is relieved by charge sep- thioesters important in metabolism. The acyl group in aration, as for ATP; (2) the products are stabilized by

TABLE 13–6 Standard Free Energies of Hydrolysis of Some Phosphorylated Compounds O and Acetyl-CoA (a Thioester) J CH3OC Acetyl-CoA G G S-CoA H O hydrolysis (kJ/mol) (kcal/mol) 2 CoASH Phosphoenolpyruvate 61.9 14.8 O 1,3-bisphosphoglycerate J CH3OC Acetic acid G (n 3-phosphoglycerate Pi) 49.3 11.8 OH Phosphocreatine 43.0 10.3 ionization ADP (n AMP Pi) 32.8 7.8 H ATP (n ADP Pi) 30.5 7.3 ATP ( AMP PP ) 45.6 10.9 n i O AMP (n adenosine Pi) 14.2 3.4 D CH3OC Acetate PP ( 2P ) 19.2 4.0 G i n i O Glucose 1-phosphate 20.9 5.0 resonance Fructose 6-phosphate 15.9 3.8 stabilization Glucose 6-phosphate 13.8 3.3 Acetyl-CoA H O acetate CoA H Glycerol 1-phosphate 9.2 2.2 2 G 31.4 kJ/mol Acetyl-CoA 31.4 7.5 FIGURE 13–6 Hydrolysis of acetyl-coenzyme A. Acetyl-CoA is a Source: Data mostly from Jencks, W.P.(1976) in Handbook of Biochemistry and Molecular thioester with a large, negative, standard free energy of hydrolysis. Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. I, pp. 296–304, Thioesters contain a sulfur atom in the position occupied by an oxy- CRC Press, Boca Raton, FL. The value for the free energy of hydrolysis of PPi is from Frey, P.A. & Arabshahi, A. (1995) Standard free-energy change for the hydrolysis of the –- gen atom in oxygen esters. The complete structure of coenzyme A phosphoanhydride bridge in ATP. Biochemistry 34, 11,307–11,310. (CoA, or CoASH) is shown in Figure 8–41. 500 Chapter 13 Principles of Bioenergetics

Thioester Extra stabilization of O oxygen ester by resonance J CH3OC G Oxygen SOR FIGURE 13–7 Free energy of hydrolysis ester G O resonance O for thioesters and oxygen esters. The J stabilization products of both types of hydrolysis CH3OC CH3 C G for G reaction have about the same free-energy thioester O R OOR O hydrolysis content (G), but the thioester has a higher ree energy, ree energy, G for oxygen free-energy content than the oxygen ester. F ester hydrolysis Orbital overlap between the O and C atoms allows resonance stabilization O O J J in oxygen esters; orbital overlap between CH3OC ROSH CH3OC ROOH G G S and C atoms is poorer and provides OH OH little resonance stabilization. ionization, as for ATP, acyl phosphates, and thioesters; chanical motion. This occurs in muscle contraction and (3) the products are stabilized by isomerization (tau- in the movement of enzymes along DNA or of ribosomes tomerization), as for phosphoenolpyruvate; and/or (4) along messenger RNA. The energy-dependent reactions the products are stabilized by resonance, as for creatine catalyzed by helicases, RecA protein, and some topo- released from phosphocreatine, carboxylate ion re- isomerases (Chapter 25) also involve direct hydrolysis leased from acyl phosphates and thioesters, and phos- of phosphoanhydride bonds. GTP-binding proteins that phate (Pi) released from anhydride or ester linkages. act in signaling pathways directly hydrolyze GTP to drive conformational changes that terminate signals ATP Provides Energy by Group Transfers, Not by Simple Hydrolysis (a) Written as a one-step reaction

Throughout this book you will encounter reactions or COO COO processes for which ATP supplies energy, and the con- A A H3NOCH ATP ADP Pi H3NOCH tribution of ATP to these reactions is commonly indi- A A CH CH cated as in Figure 13–8a, with a single arrow showing 2 2 A NH3 A the conversion of ATP to ADP and Pi (or, in some cases, CH2 CH2 of ATP to AMP and pyrophosphate, PPi). When written A A C C this way, these reactions of ATP appear to be simple hy- J G J G O O O NH2 drolysis reactions in which water displaces Pi (or PPi), and one is tempted to say that an ATP-dependent re- Glutamate Glutamine action is “driven by the hydrolysis of ATP.” This is not ATP NH3 the case. ATP hydrolysis per se usually accomplishes ADP COO nothing but the liberation of heat, which cannot drive a A H3NOCH chemical process in an isothermal system. A single re- 1 A 2 P action arrow such as that in Figure 13–8a almost in- CH2 i A variably represents a two-step process (Fig. 13–8b) in CH2 which part of the ATP molecule, a phosphoryl or py- A C rophosphoryl group or the adenylate moiety (AMP), is J G O O O first transferred to a substrate molecule or to an amino G J P acid residue in an enzyme, becoming covalently at- D G tached to the substrate or the enzyme and raising its O O free-energy content. Then, in a second step, the phos- Enzyme-bound glutamyl phosphate phate-containing moiety transferred in the first step is displaced, generating Pi, PPi, or AMP. Thus ATP partic- (b) Actual two-step reaction ipates covalently in the enzyme-catalyzed reaction to which it contributes free energy. FIGURE 13–8 ATP hydrolysis in two steps. (a) The contribution of Some processes do involve direct hydrolysis of ATP ATP to a reaction is often shown as a single step, but is almost always (or GTP), however. For example, noncovalent binding a two-step process. (b) Shown here is the reaction catalyzed by ATP- of ATP (or of GTP), followed by its hydrolysis to ADP dependent glutamine synthetase. 1 A phosphoryl group is transferred (or GDP) and Pi, can provide the energy to cycle some from ATP to glutamate, then 2 the phosphoryl group is displaced by proteins between two conformations, producing me- NH3 and released as Pi. 13.2 Phosphoryl Group Transfers and ATP 501 triggered by hormones or by other extracellular factors direct donation of a phosphoryl group from PEP to ADP (Chapter 12). is thermodynamically feasible: The phosphate compounds found in living organisms G (kJ/mol) can be divided somewhat arbitrarily into two groups, (1) PEP H O pyruvate P 61.9 based on their standard free energies of hydrolysis 2 8n i (2) ADP P ATP H O 30.5 (Fig. 13–9). “High-energy” compounds have a G of i 8n 2 Sum: PEP ADP pyruvate ATP 31.4 hydrolysis more negative than 25 kJ/mol; “low-energy” 8n compounds have a less negative G. Based on this cri- Notice that while the overall reaction above is repre- terion, ATP, with a G of hydrolysis of 30.5 kJ/mol sented as the algebraic sum of the first two reactions, (7.3 kcal/mol), is a high-energy compound; glucose the overall reaction is actually a third, distinct reaction

6-phosphate, with a G of hydrolysis of 13.8 kJ/mol that does not involve Pi; PEP donates a phosphoryl (3.3 kcal/mol), is a low-energy compound. group directly to ADP. We can describe phosphorylated The term “high-energy phosphate bond,” long used compounds as having a high or low phosphoryl group by biochemists to describe the POO bond broken in hy- transfer potential, on the basis of their standard free en- drolysis reactions, is incorrect and misleading as it ergies of hydrolysis (as listed in Table 13–6). The phos- wrongly suggests that the bond itself contains the en- phoryl group transfer potential of phosphoenolpyruvate ergy. In fact, the breaking of all chemical bonds requires is very high, that of ATP is high, and that of glucose 6- an input of energy. The free energy released by hy- phosphate is low (Fig. 13–9). drolysis of phosphate compounds does not come from Much of catabolism is directed toward the synthesis the specific bond that is broken; it results from the prod- of high-energy phosphate compounds, but their forma- ucts of the reaction having a lower free-energy content tion is not an end in itself; they are the means of acti- than the reactants. For simplicity, we will sometimes use vating a very wide variety of compounds for further the term “high-energy phosphate compound” when re- chemical transformation. The transfer of a phosphoryl ferring to ATP or other phosphate compounds with a group to a compound effectively puts free energy into large, negative, standard free energy of hydrolysis. that compound, so that it has more free energy to give As is evident from the additivity of free-energy up during subsequent metabolic transformations. We de- changes of sequential reactions, any phosphorylated scribed above how the synthesis of glucose 6-phosphate compound can be synthesized by coupling the synthe- is accomplished by phosphoryl group transfer from ATP. sis to the breakdown of another phosphorylated com- In the next chapter we see how this phosphorylation of pound with a more negative free energy of hydrolysis. glucose activates, or “primes,” the glucose for catabolic

For example, because cleavage of Pi from phospho- reactions that occur in nearly every living cell. Because enolpyruvate (PEP) releases more energy than is of its intermediate position on the scale of group trans- needed to drive the condensation of Pi with ADP, the fer potential, ATP can carry energy from high-energy

70 COO A COOO P Phosphoenolpyruvate B 60 CH OOO P 2 M D C A 50 CHOH P Creatine A O CH2OOO P Phosphocreatine 40 1,3-Bisphosphoglycerate FIGURE 13–9 Ranking of biological phosphate Adenine O Rib O P O P O P compounds by standard free energies of hydrol-

High-energy 30 ATP ysis. This shows the flow of phosphoryl groups, compounds of hydrolysis (kJ/mol) represented by P , from high-energy phosphoryl

G Low-energy donors via ATP to acceptor molecules (such as 20 compounds glucose and glycerol) to form their low-energy phosphate derivatives. This flow of phosphoryl Glucose 6- P Glycerol- P groups, catalyzed by enzymes called kinases, 10 proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of low-

energy phosphate compounds releases Pi, which

0 Pi has an even lower phosphoryl group transfer potential (as defined in the text). 502 Chapter 13 Principles of Bioenergetics phosphate compounds produced by catabolism to com- (p. 218) involves attack at the position of the ATP pounds such as glucose, converting them into more re- molecule. active species. ATP thus serves as the universal energy Attack at the phosphate of ATP displaces AMP and currency in all living cells. transfers a pyrophosphoryl (not pyrophosphate) group One more chemical feature of ATP is crucial to its to the attacking nucleophile (Fig. 13–10b). For exam- role in metabolism: although in aqueous solution ATP is ple, the formation of 5-phosphoribosyl-1-pyrophosphate thermodynamically unstable and is therefore a good (p. XXX), a key intermediate in nucleotide synthesis, phosphoryl group donor, it is kinetically stable. Because results from attack of an OOH of the ribose on the of the huge activation energies (200 to 400 kJ/mol) re- phosphate. quired for uncatalyzed cleavage of its phosphoanhydride Nucleophilic attack at the position of ATP displaces bonds, ATP does not spontaneously donate phosphoryl PPi and transfers adenylate (5-AMP) as an adenylyl groups to water or to the hundreds of other potential group (Fig. 13–10c); the reaction is an adenylylation acceptors in the cell. Only when specific enzymes are (a-den-i-li-la--shun, probably the most ungainly word present to lower the energy of activation does phos- in the biochemical language). Notice that hydrolysis of phoryl group transfer from ATP proceed. The cell is the – phosphoanhydride bond releases considerably therefore able to regulate the disposition of the energy more energy (~46 kJ/mol) than hydrolysis of the – carried by ATP by regulating the various enzymes that bond (~31 kJ/mol) (Table 13–6). Furthermore, the PPi act on it. formed as a byproduct of the adenylylation is hydrolyzed

to two Pi by the ubiquitous enzyme inorganic pyro- ATP Donates Phosphoryl, Pyrophosphoryl, phosphatase, releasing 19 kJ/mol and thereby provid- and Adenylyl Groups ing a further energy “push” for the adenylylation reaction. In effect, both phosphoanhydride bonds of ATP are

The reactions of ATP are generally SN2 nucleophilic dis- split in the overall reaction. Adenylylation reactions are placements (p. II.8), in which the nucleophile may be, therefore thermodynamically very favorable. When the for example, the oxygen of an alcohol or carboxylate, or energy of ATP is used to drive a particularly unfavor- a nitrogen of creatine or of the side chain of arginine or able metabolic reaction, adenylylation is often the mech- histidine. Each of the three phosphates of ATP is sus- anism of energy coupling. Fatty acid activation is a good ceptible to nucleophilic attack (Fig. 13–10), and each example of this energy-coupling strategy. position of attack yields a different type of product. The first step in the activation of a fatty acid— Nucleophilic attack by an alcohol on the phos- either for energy-yielding oxidation or for use in the syn- phate (Fig. 13–10a) displaces ADP and produces a new thesis of more complex lipids—is the formation of its phosphate ester. Studies with 18O-labeled reactants thiol ester (see Fig. 17–5). The direct condensation of have shown that the bridge oxygen in the new com- a fatty acid with coenzyme A is endergonic, but the for- pound is derived from the alcohol, not from ATP; the mation of fatty acyl–CoA is made exergonic by stepwise 2 group transferred from ATP is a phosphoryl (OPO3 ), removal of two phosphoryl groups from ATP. First, 2 not a phosphate (OOPO3 ). Phosphoryl group transfer adenylate (AMP) is transferred from ATP to the car- from ATP to glutamate (Fig. 13–8) or to glucose boxyl group of the fatty acid, forming a mixed anhydride

Three positions on ATP for attack by the nucleophile R18O O O O FIGURE 13–10 Nucleophilic displacement reactions of ATP. Any of the three P atoms (, , or ) O P O P O P O Rib Adenine may serve as the electrophilic target for O O O nucleophilic attack—in this case, by the labeled 18 18 18 18 nucleophile RO O:. The nucleophile may be an R ORORO alcohol (ROH), a carboxyl group (RCOO ), or a phosphoanhydride (a nucleoside mono- or diphosphate, for example). (a) When the oxygen O O O O of the nucleophile attacks the position, the bridge oxygen of the product is labeled, indicating that R18O P O R18O P O P O R18O P O Rib Adenine the group transferred from ATP is a phosphoryl 2 2 O O O O (OPO3 ), not a phosphate (OOPO3 ). (b) Attack on the position displaces AMP and leads to the ADP AMP PPi transfer of a pyrophosphoryl (not pyrophosphate) Phosphoryl Pyrophosphoryl Adenylyl group to the nucleophile. (c) Attack on the transfer transfer transfer position displaces PPi and transfers the adenylyl (a) (b) (c) group to the nucleophile. 13.2 Phosphoryl Group Transfers and ATP 503

(fatty acyl adenylate) and liberating PPi. The thiol group for the breakage of these bonds, or 45.6 kJ/mol of coenzyme A then displaces the adenylate group and (19.2) kJ/mol: forms a thioester with the fatty acid. The sum of these two reactions is energetically equivalent to the exer- ATP 2H2O 88n AMP 2Pi G 64.8 kJ/mol gonic hydrolysis of ATP to AMP and PPi (G 45.6 The activation of amino acids before their polymer- kJ/mol) and the endergonic formation of fatty acyl–CoA ization into proteins (see Fig. 27–14) is accomplished (G 31.4 kJ/mol). The formation of fatty acyl–CoA by an analogous set of reactions in which a transfer RNA is made energetically favorable by hydrolysis of the PPi molecule takes the place of coenzyme A. An interesting by inorganic pyrophosphatase. Thus, in the activation use of the cleavage of ATP to AMP and PPi occurs in of a fatty acid, both phosphoanhydride bonds of ATP are the firefly, which uses ATP as an energy source to pro- broken. The resulting G is the sum of the G values duce light flashes (Box 13–2).

BOX 13–2 THE WORLD OF BIOCHEMISTRY

Firefly Flashes: Glowing Reports of ATP pyrophosphate cleavage of ATP to form luciferyl Bioluminescence requires considerable amounts of adenylate. In the presence of molecular oxygen and energy. In the firefly, ATP is used in a set of reactions luciferase, the luciferin undergoes a multistep oxida- that converts chemical energy into light energy. In the tive decarboxylation to oxyluciferin. This process is 1950s, from many thousands of fireflies collected by accompanied by emission of light. The color of the children in and around Baltimore, William McElroy light flash differs with the firefly species and appears and his colleagues at The Johns Hopkins University to be determined by differences in the structure of the isolated the principal biochemical components: lu- luciferase. Luciferin is regenerated from oxyluciferin ciferin, a complex carboxylic acid, and luciferase, an in a subsequent series of reactions. enzyme. The generation of a light flash requires acti- In the laboratory, pure firefly luciferin and lu- vation of luciferin by an enzymatic reaction involving ciferase are used to measure minute quantities of ATP by the intensity of the light flash produced. As little as a few picomoles (1012 mol) of ATP can be measured in this way. An enlightening extension of the studies in luciferase was the cloning of the luciferase gene into tobacco plants. When watered with a solution containing luciferin, the plants glowed in the dark (see Fig. 9–29).

H O N N A C O PO Rib Adenine H HO S S O O H AMP Luciferyl adenylate

PPi O2

luciferase ATP light The firefly, a beetle of the Lampyridae family. H N N COO H CO2 AMP HO S S H Luciferin N N O

S S Important components in the HO regenerating Oxyluciferin firefly bioluminescence cycle. reactions 504 Chapter 13 Principles of Bioenergetics

Assembly of Informational Macromolecules relax into a second conformation until another molecule Requires Energy of ATP binds. The binding and subsequent hydrolysis of ATP (by myosin ATPase) provide the energy that forces When simple precursors are assembled into high mo- cyclic changes in the conformation of the myosin head. lecular weight polymers with defined sequences (DNA, The change in conformation of many individual myosin RNA, proteins), as described in detail in Part III, energy is required both for the condensation of monomeric units and for the creation of ordered sequences. The precursors for DNA and RNA synthesis are nucleoside O A triphosphates, and polymerization is accompanied by RNA OPPOO cleavage of the phosphoanhydride linkage between the chain A O and phosphates, with the release of PPi (Fig. 13–11). A The moieties transferred to the growing polymer in CH2 O Base these reactions are adenylate (AMP), guanylate (GMP), HH cytidylate (CMP), or uridylate (UMP) for RNA synthe- H H sis, and their deoxy analogs (with TMP in place of UMP) for DNA synthesis. As noted above, the activation of SOH OH amino acids for protein synthesis involves the donation O OO BB A of adenylate groups from ATP, and we shall see in Chap- OOPOOOPOOOPOP ter 27 that several steps of protein synthesis on the ri- A A A O O O bosome are also accompanied by GTP hydrolysis. In all A these cases, the exergonic breakdown of a nucleoside CH2 O Guanine GTP first anhydride triphosphate is coupled to the endergonic process of bond broken HH synthesizing a polymer of a specific sequence. H H O O ATP Energizes Active Transport OH OH BB and Muscle Contraction OOPOOOPOO A A O O ATP can supply the energy for transporting an ion or a PPi molecule across a membrane into another aqueous com- second partment where its concentration is higher (see Fig. anhydride 11–36). Transport processes are major consumers of en- bond broken ergy; in human kidney and brain, for example, as much as two-thirds of the energy consumed at rest is used to 2Pi O pump Na and K across plasma membranes via the A OPPOO Na K ATPase. The transport of Na and K is driven A by cyclic phosphorylation and dephosphorylation of the O transporter protein, with ATP as the phosphoryl group A CH2 O Base donor (see Fig. 11–37). Na-dependent phosphorylation of the NaK ATPase forces a change in the protein’s HH H H conformation, and K-dependent dephosphorylation favors return to the original conformation. Each cycle in O OH A the transport process results in the conversion of ATP OOPPO to ADP and Pi, and it is the free-energy change of ATP A hydrolysis that drives the cyclic changes in protein con- RNA chain O lengthened A CH formation that result in the electrogenic pumping of Na by one 2 O Guanine and K . Note that in this case ATP interacts covalently nucleotide HH by phosphoryl group transfer to the enzyme, not the H H substrate. In the contractile system of skeletal muscle cells, OH OH myosin and actin are specialized to transduce the chem- FIGURE 13–11 Nucleoside triphosphates in RNA synthesis. With ical energy of ATP into motion (see Fig. 5–33). ATP each nucleoside monophosphate added to the growing chain, one PPi binds tightly but noncovalently to one conformation of is released and hydrolyzed to two Pi. The hydrolysis of two phospho- myosin, holding the protein in that conformation. When anhydride bonds for each nucleotide added provides the energy for myosin catalyzes the hydrolysis of its bound ATP, the forming the bonds in the RNA polymer and for assembling a specific ADP and Pi dissociate from the protein, allowing it to sequence of nucleotides. 13.2 Phosphoryl Group Transfers and ATP 505 molecules results in the sliding of myosin fibrils along intermediate; then the phosphoryl group is transferred actin filaments (see Fig. 5–32), which translates into from the P –His residue to an NDP acceptor. Because macroscopic contraction of the muscle fiber. the enzyme is nonspecific for the base in the NDP and As we noted earlier, this production of mechanical works equally well on dNDPs and NDPs, it can synthe- motion at the expense of ATP is one of the few cases in size all NTPs and dNTPs, given the corresponding NDPs which ATP hydrolysis per se, rather than group trans- and a supply of ATP. fer from ATP, is the source of the chemical energy in a Phosphoryl group transfers from ATP result in an coupled process. accumulation of ADP; for example, when muscle is con- tracting vigorously, ADP accumulates and interferes Transphosphorylations between Nucleotides with ATP-dependent contraction. During periods of in- Occur in All Cell Types tense demand for ATP, the cell lowers the ADP concentration, and at the same time acquires ATP, by the Although we have focused on ATP as the cell’s energy action of adenylate kinase: currency and donor of phosphoryl groups, all other nucleoside triphosphates (GTP, UTP, and CTP) and all the Mg2 2ADP3:::4 ATP AMP DG 0 deoxynucleoside triphosphates (dATP, dGTP, dTTP, and dCTP) are energetically equivalent to ATP. The free- This reaction is fully reversible, so after the intense de- energy changes associated with hydrolysis of their mand for ATP ends, the enzyme can recycle AMP by phosphoanhydride linkages are very nearly identical converting it to ADP, which can then be phosphorylated with those shown in Table 13–6 for ATP. In preparation to ATP in mitochondria. A similar enzyme, guanylate ki- for their various biological roles, these other nucleotides nase, converts GMP to GDP at the expense of ATP. By are generated and maintained as the nucleoside triphos- pathways such as these, energy conserved in the cata- phate (NTP) forms by phosphoryl group transfer to the bolic production of ATP is used to supply the cell with corresponding nucleoside diphosphates (NDPs) and all required NTPs and dNTPs. monophosphates (NMPs). Phosphocreatine (Fig. 13–5), also called creatine ATP is the primary high-energy phosphate com- phosphate, serves as a ready source of phosphoryl pound produced by catabolism, in the processes of gly- groups for the quick synthesis of ATP from ADP. The colysis, oxidative phosphorylation, and, in photosyn- phosphocreatine (PCr) concentration in skeletal mus- thetic cells, photophosphorylation. Several enzymes cle is approximately 30 mM, nearly ten times the con- then carry phosphoryl groups from ATP to the other nu- centration of ATP, and in other tissues such as smooth cleotides. Nucleoside diphosphate kinase, found in muscle, brain, and kidney [PCr] is 5 to 10 mM. The en- all cells, catalyzes the reaction zyme creatine kinase catalyzes the reversible reaction

Mg2 Mg2 ATP NDP (or dNDP)3:::4 ADP NTP (or dNTP) ADP PCr3:::4 ATP Cr DG 12.5 kJ/mol DG 0 When a sudden demand for energy depletes ATP, the Although this reaction is fully reversible, the relatively PCr reservoir is used to replenish ATP at a rate consid- high [ATP]/[ADP] ratio in cells normally drives the re- erably faster than ATP can be synthesized by catabolic action to the right, with the net formation of NTPs and pathways. When the demand for energy slackens, ATP dNTPs. The enzyme actually catalyzes a two-step phos- produced by catabolism is used to replenish the PCr phoryl transfer, which is a classic case of a double-dis- reservoir by reversal of the creatine kinase reaction. Or- placement (Ping-Pong) mechanism (Fig. 13–12; see also ganisms in the lower phyla employ other PCr-like mole- Fig. 6–13b). First, phosphoryl group transfer from ATP cules (collectively called phosphagens) as phosphoryl to an active-site His residue produces a phosphoenzyme reservoirs.

Adenosine P P P Enz His Nucleoside P P P (ATP) (any NTP or dNTP) Ping Pong

Adenosine P P Enz His P Nucleoside P P (ADP) (any NDP or dNDP)

FIGURE 13–12 Ping-Pong mechanism of nucleoside diphosphate phate replaces it, and this is converted to the corresponding triphos- kinase. The enzyme binds its first substrate (ATP in our example), and phate by transfer of the phosphoryl group from the phosphohistidine a phosphoryl group is transferred to the side chain of a His residue. residue. ADP departs, and another nucleoside (or deoxynucleoside) diphos- 506 Chapter 13 Principles of Bioenergetics

Inorganic Polyphosphate Is a Potential fers a survival advantage. Deletion of the genes for Phosphoryl Group Donor polyphosphate kinases diminishes the ability of certain pathogenic bacteria to invade animal tissues. The en- Inorganic polyphosphate (polyP) is a linear polymer zymes may therefore prove to be vulnerable targets in composed of many tens or hundreds of Pi residues the development of new antimicrobial drugs. linked through phosphoanhydride bonds. This polymer, No gene in yeast encodes a PPK-like protein, but present in all organisms, may accumulate to high levels four genes—unrelated to bacterial PPK genes—are nec- in some cells. In yeast, for example, the amount of polyP essary for the synthesis of polyphosphate. The mecha- that accumulates in the vacuoles would represent, if dis- nism for polyphosphate synthesis in eukaryotes seems tributed uniformly throughout the cell, a concentration to be quite different from that in prokaryotes. of 200 mM! (Compare this with the concentrations of other phosphoryl donors listed in Table 13–5.) Biochemical and Chemical Equations Are Not Identical O O O O O O P O P O P O P O P O Biochemists write metabolic equations in a simplified way, and this is particularly evident for reactions in- O O O O O volving ATP. Phosphorylated compounds can exist in Inorganic polyphosphate (polyP) several ionization states and, as we have noted, the different species can bind Mg2. For example, at pH 7 and One potential role for polyP is to serve as a phos- 2 4 3 2mM Mg , ATP exists in the forms ATP , HATP , phagen, a reservoir of phosphoryl groups that can be H ATP2 , MgHATP , and Mg ATP. In thinking about the used to generate ATP, as creatine phosphate is used in 2 2 biological role of ATP, however, we are not always in- muscle. PolyP has about the same phosphoryl group terested in all this detail, and so we consider ATP as an transfer potential as PP . The shortest polyphosphate, i entity made up of a sum of species, and we write its hy- PP (n 2), can serve as the energy source for active i drolysis as the biochemical equation transport of H in plant vacuoles. For at least one form of the enzyme phosphofructokinase in plants, PPi is the ATP H2O 8n ADP Pi phosphoryl group donor, a role played by ATP in ani- where ATP, ADP, and P are sums of species. The mals and microbes (p. XXX). The finding of high con- i corresponding apparent equilibrium constant, K centrations of polyP in volcanic condensates and steam eq [ADP][Pi]/[ATP], depends on the pH and the concentra- vents suggests that it could have served as an energy tion of free Mg2 . Note that H and Mg2 do not ap- source in prebiotic and early cellular evolution. pear in the biochemical equation because they are held In prokaryotes, the enzyme polyphosphate ki- constant. Thus a biochemical equation does not balance nase-1 (PPK-1) catalyzes the reversible reaction H, Mg, or charge, although it does balance all other el- Mg2 ements involved in the reaction (C, N, O, and P in the ATP polyPn 3:::4 ADP polyPn1 equation above). DG 20 kJ/mol We can write a chemical equation that does balance by a mechanism involving an enzyme-bound phospho- for all elements and for charge. For example, when ATP 2 histidine intermediate (recall the mechanism of nucle- is hydrolyzed at a pH above 8.5 in the absence of Mg , oside diphosphate kinase, described above). A second the chemical reaction is represented by enzyme, polyphosphate kinase-2 (PPK-2), catalyzes 4 3 2 ATP H2O 8n ADP HPO4 H the reversible synthesis of GTP (or ATP) from polyphosphate and GDP (or ADP): The corresponding equilibrium constant, Keq 3 2 4 [ADP ][HPO4 ][H ]/[ATP ], depends only on tem- Mn2 perature, pressure, and ionic strength. GDP polyP 3:::4 GTP polyP n 1 n Both ways of writing a metabolic reaction have value PPK-2 is believed to act primarily in the direction of in biochemistry. Chemical equations are needed when GTP and ATP synthesis, and PPK-1 in the direction of we want to account for all atoms and charges in a re- polyphosphate synthesis. PPK-1 and PPK-2 are present action, as when we are considering the mechanism of a in a wide variety of prokaryotes, including many patho- chemical reaction. Biochemical equations are used to genic bacteria. determine in which direction a reaction will proceed In prokaryotes, elevated levels of polyP have been spontaneously, given a specified pH and [Mg2], or to shown to promote expression of a number of genes in- calculate the equilibrium constant of such a reaction. volved in adaptation of the organism to conditions of Throughout this book we use biochemical equa- starvation or other threats to survival. In Escherichia tions, unless the focus is on chemical mechanism, and coli, for example, polyP accumulates when cells are we use values of G and Keq as determined at pH 7 2 starved for amino acids or Pi, and this accumulation con- and 1 mM Mg . 13.3 Biological Oxidation-Reduction Reactions 507

SUMMARY 13.2 Phosphoryl Group Transfers The carriers in turn donate electrons to acceptors with and ATP higher electron affinities, with the release of energy. Cells contain a variety of molecular energy transducers, ■ ATP is the chemical link between catabolism which convert the energy of electron flow into useful and anabolism. It is the energy currency of the work. living cell. The exergonic conversion of ATP to We begin our discussion with a description of the general types of metabolic reactions in which electrons ADP and Pi, or to AMP and PPi, is coupled to many endergonic reactions and processes. are transferred. After considering the theoretical and experimental basis for measuring the energy changes in ■ Direct hydrolysis of ATP is the source of oxidation reactions in terms of electromotive force, we energy in the conformational changes that discuss the relationship between this force, expressed produce muscle contraction but, in general, it in volts, and the free-energy change, expressed in joules. is not ATP hydrolysis but the transfer of a We conclude by describing the structures and oxidation- phosphoryl, pyrophosphoryl, or adenylyl group reduction chemistry of the most common of the spe- from ATP to a substrate or enzyme molecule cialized electron carriers, which you will encounter that couples the energy of ATP breakdown to repeatedly in later chapters. endergonic transformations of substrates. ■ Through these group transfer reactions, ATP The Flow of Electrons Can Do Biological Work provides the energy for anabolic reactions, including the synthesis of informational Every time we use a motor, an electric light or heater, molecules, and for the transport of molecules or a spark to ignite gasoline in a car engine, we use the and ions across membranes against flow of electrons to accomplish work. In the circuit that concentration gradients and electrical potential powers a motor, the source of electrons can be a bat- gradients. tery containing two chemical species that differ in affinity for electrons. Electrical wires provide a pathway for ■ Cells contain other metabolites with large, electron flow from the chemical species at one pole of negative, free energies of hydrolysis, including the battery, through the motor, to the chemical species phosphoenolpyruvate, 1,3-bisphosphoglycerate, at the other pole of the battery. Because the two chem- and phosphocreatine. These high-energy ical species differ in their affinity for electrons, electrons compounds, like ATP, have a high phosphoryl flow spontaneously through the circuit, driven by a force group transfer potential; they are good donors proportional to the difference in electron affinity, the of the phosphoryl group. Thioesters also have electromotive force (emf). The electromotive force high free energies of hydrolysis. (typically a few volts) can accomplish work if an ap- ■ Inorganic polyphosphate, present in all cells, propriate energy transducer—in this case a motor—is may serve as a reservoir of phosphoryl groups placed in the circuit. The motor can be coupled to a va- with high group transfer potential. riety of mechanical devices to accomplish useful work. Living cells have an analogous biological “circuit,” with a relatively reduced compound such as glucose as the source of electrons. As glucose is enzymatically ox- 13.3 Biological Oxidation-Reduction idized, the released electrons flow spontaneously Reactions through a series of electron-carrier intermediates to another chemical species, such as O2. This electron flow The transfer of phosphoryl groups is a central feature is exergonic, because O2 has a higher affinity for elec- of metabolism. Equally important is another kind of trons than do the electron-carrier intermediates. The transfer, electron transfer in oxidation-reduction reac- resulting electromotive force provides energy to a vari- tions. These reactions involve the loss of electrons by ety of molecular energy transducers (enzymes and other one chemical species, which is thereby oxidized, and the proteins) that do biological work. In the mitochondrion, gain of electrons by another, which is reduced. The flow for example, membrane-bound enzymes couple electron of electrons in oxidation-reduction reactions is respon- flow to the production of a transmembrane pH differ- sible, directly or indirectly, for all work done by living ence, accomplishing osmotic and electrical work. The organisms. In nonphotosynthetic organisms, the sources proton gradient thus formed has potential energy, some- of electrons are reduced compounds (foods); in photo- times called the proton-motive force by analogy with synthetic organisms, the initial electron donor is a chem- electromotive force. Another enzyme, ATP synthase in ical species excited by the absorption of light. The path the inner mitochondrial membrane, uses the proton- of electron flow in metabolism is complex. Electrons motive force to do chemical work: synthesis of ATP from move from various metabolic intermediates to special- ADP and Pi as protons flow spontaneously across the ized electron carriers in enzyme-catalyzed reactions. membrane. Similarly, membrane-localized enzymes in 508 Chapter 13 Principles of Bioenergetics

E. coli convert electromotive force to proton-motive Biological Oxidations Often Involve Dehydrogenation force, which is then used to power flagellar motion. The carbon in living cells exists in a range of oxidation The principles of electrochemistry that govern en- states (Fig. 13–13). When a carbon atom shares an elec- ergy changes in the macroscopic circuit with a motor tron pair with another atom (typically H, C, S, N, or O), and battery apply with equal validity to the molecular the sharing is unequal in favor of the more electroneg- processes accompanying electron flow in living cells. We ative atom. The order of increasing electronegativity is turn now to a discussion of those principles. H C S N O. In oversimplified but useful terms, Oxidation-Reductions Can Be Described the more electronegative atom “owns” the bonding electrons it shares with another atom. For example, in as Half-Reactions methane (CH4), carbon is more electronegative than the Although oxidation and reduction must occur together, four hydrogens bonded to it, and the C atom therefore it is convenient when describing electron transfers to “owns” all eight bonding electrons (Fig. 13–13). In consider the two halves of an oxidation-reduction reac- ethane, the electrons in the COC bond are shared tion separately. For example, the oxidation of ferrous equally, so each C atom owns only seven of its eight ion by cupric ion, bonding electrons. In ethanol, C-1 is less electronegative than the oxygen to which it is bonded, and the O Fe2 Cu2 Fe3 Cu 34 atom therefore “owns” both electrons of the COO bond, can be described in terms of two half-reactions: leaving C-1 with only five bonding electrons. With each

formal loss of electrons, the carbon atom has undergone (1) Fe2 34 Fe3 e oxidation—even when no oxygen is involved, as in the (2) Cu2 e 34 Cu conversion of an alkane (OCH2OCH2O) to an alkene The electron-donating molecule in an oxidation- (OCHUCHO). In this case, oxidation (loss of elec- reduction reaction is called the reducing agent or reduc- trons) is coincident with the loss of hydrogen. In bio- tant; the electron-accepting molecule is the oxidizing logical systems, oxidation is often synonymous with de- agent or oxidant. A given agent, such as an iron cation hydrogenation, and many enzymes that catalyze existing in the ferrous (Fe2) or ferric (Fe3) state, func- oxidation reactions are dehydrogenases. Notice that tions as a conjugate reductant-oxidant pair (redox pair), the more reduced compounds in Figure 13–13 (top) are just as an acid and corresponding base function as a con- richer in hydrogen than in oxygen, whereas the more jugate acid-base pair. Recall from Chapter 2 that in acid- oxidized compounds (bottom) have more oxygen and base reactions we can write a general equation: proton less hydrogen. donor 34 H proton acceptor. In redox reactions we Not all biological oxidation-reduction reactions in- can write a similar general equation: electron donor 34 volve carbon. For example, in the conversion of molec- e electron acceptor. In the reversible half-reaction (1) ular nitrogen to ammonia, 6H 6e N2 n 2NH3, above, Fe2 is the electron donor and Fe3 is the elec- the nitrogen atoms are reduced. tron acceptor; together, Fe2 and Fe3 constitute a con- Electrons are transferred from one molecule (elec- jugate redox pair. tron donor) to another (electron acceptor) in one of The electron transfers in the oxidation-reduction four different ways: reactions of organic compounds are not fundamentally 2 3 different from those of inorganic species. In Chapter 7 1. Directly as electrons. For example, the Fe /Fe redox pair can transfer an electron to the we considered the oxidation of a reducing sugar (an 2 aldehyde or ketone) by cupric ion (see Fig. 7–10a): Cu /Cu redox pair: Fe2 Cu2 34 Fe3 Cu O O 2 R C 4OH 2Cu R C Cu2O2H2O 2. As hydrogen atoms. Recall that a hydrogen atom H OH consists of a proton (H) and a single electron (e). In this case we can write the general equation This overall reaction can be expressed as two half- reactions: AH2 34 A 2e 2H

O O where AH2 is the hydrogen/electron donor. (1) R C 2OH R C 2e H2O (Do not mistake the above reaction for an acid H OH dissociation; the H arises from the removal of a 2 (2) 2Cu 2e 2OH 34 Cu2O H2O hydrogen atom, H e .) AH2 and A together constitute a conjugate redox pair (A/AH ), which Because two electrons are removed from the aldehyde 2 can reduce another compound B (or redox pair, carbon, the second half-reaction (the one-electron re- B/BH ) by transfer of hydrogen atoms: duction of cupric to cuprous ion) must be doubled to 2 balance the overall equation. AH2 B 34 A BH2 13.3 Biological Oxidation-Reduction Reactions 509

3. As a hydride ion (:H), which has two electrons. potential of 0.00 V. When this hydrogen electrode is con- This occurs in the case of NAD-linked dehydroge- nected through an external circuit to another half-cell nases, described below. in which an oxidized species and its corresponding re- 4. Through direct combination with oxygen. In this duced species are present at standard concentrations case, oxygen combines with an organic reductant (each solute at 1 M, each gas at 101.3 kPa), electrons tend and is covalently incorporated in the product, as to flow through the external circuit from the half-cell of in the oxidation of a hydrocarbon to an alcohol:

1 RXCH3 2O2 88n RXCH2XOH

The hydrocarbon is the electron donor and the H oxygen atom is the electron acceptor. MethaneHHC 8 All four types of electron transfer occur in cells. The H neutral term reducing equivalent is commonly used to designate a single electron equivalent participating in an H H oxidation-reduction reaction, no matter whether this Ethane HCC H 7 equivalent is an electron per se, a hydrogen atom, or a hy- (alkane) H H dride ion, or whether the electron transfer takes place in a reaction with oxygen to yield an oxygenated product. HH Because biological fuel molecules are usually enzymati- Ethene C C 6 cally dehydrogenated to lose two reducing equivalents at (alkene) HH a time, and because each oxygen atom can accept two reducing equivalents, biochemists by convention regard the H H unit of biological oxidations as two reducing equivalents Ethanol HCC O H 5 (alcohol) passing from substrate to oxygen. H H

Reduction Potentials Measure Affinity for Electrons Acetylene HHC C 5 When two conjugate redox pairs are together in solu- (alkyne) tion, electron transfer from the electron donor of one pair to the electron acceptor of the other may proceed H spontaneously. The tendency for such a reaction de- FormaldehydeC O 4 H pends on the relative affinity of the electron acceptor of each redox pair for electrons. The standard reduc- H H tion potential, E , a measure (in volts) of this affin- Acetaldehyde H C C 3 ity, can be determined in an experiment such as that (aldehyde) described in Figure 13–14. Electrochemists have cho- H O sen as a standard of reference the half-reaction

1 O H e 88n 2 H2 H H Acetone HCC C H 2 The electrode at which this half-reaction occurs (called (ketone) a half-cell) is arbitrarily assigned a standard reduction H H

O Formic acid (carboxylic H C 2 FIGURE 13–13 Oxidation states of carbon in the biosphere. The acid) O oxidation states are illustrated with some representative compounds. H Focus on the red carbon atom and its bonding electrons. When this carbon is bonded to the less electronegative H atom, both bonding Carbon C O 2 electrons (red) are assigned to the carbon. When carbon is bonded to monoxide another carbon, bonding electrons are shared equally, so one of the two electrons is assigned to the red carbon. When the red carbon is H Acetic acid O bonded to the more electronegative O atom, the bonding electrons (carboxylic H C C 1 are assigned to the oxygen. The number to the right of each compound O acid) H is the number of electrons “owned” by the red carbon, a rough ex- H pression of the oxidation state of that carbon. When the red carbon undergoes oxidation (loses electrons), the number gets smaller. Thus Carbon O C O 0 the oxidation state increases from top to bottom of the list. dioxide 510 Chapter 13 Principles of Bioenergetics

Device for where R and T have their usual meanings, n is the num- measuring emf ber of electrons transferred per molecule, and is the Faraday constant (Table 13–1). At 298 K (25 C), this expression reduces to

0.026V [electron acceptor] E E ln (13–5) n [electron donor] H2 gas Many half-reactions of interest to biochemists in- (standard Salt bridge volve protons. As in the definition of G, biochemists pressure) (KCl solution) define the standard state for oxidation-reduction reactions as pH 7 and express reduction potential as E, the standard reduction potential at pH 7. The standard reduction potentials given in Table 13–7 and used throughout this book are values for E and are therefore valid only for systems at neutral pH. Each value represents the potential difference when the conjugate redox pair, at 1 M concentrations and pH 7, is connected with the standard (pH 0) hydrogen electrode. Notice in Table 13–7 that when the conjugate pair 2H /H2 at pH 7 is connected with the standard hydrogen electrode (pH Reference cell of Test cell containing 0), electrons tend to flow from the pH 7 cell to the stan- known emf: the 1 M concentrations hydrogen electrode of the oxidized and dard (pH 0) cell; the measured E for the 2H /H2 pair in which H2 gas reduced species of is 0.414 V. at 101.3 kPa is the redox pair to equilibrated at be examined the electrode Standard Reduction Potentials Can Be Used with 1 M H to Calculate the Free-Energy Change The usefulness of reduction potentials stems from the FIGURE 13–14 Measurement of the standard reduction potential fact that when E values have been determined for any (E) of a redox pair. Electrons flow from the test electrode to the ref- two half-cells, relative to the standard hydrogen elec- erence electrode, or vice versa. The ultimate reference half-cell is the trode, their reduction potentials relative to each other hydrogen electrode, as shown here, at pH 0. The electromotive force are also known. We can then predict the direction in (emf) of this electrode is designated 0.00 V. At pH 7 in the test cell, which electrons will tend to flow when the two half-cells E for the hydrogen electrode is 0.414 V. The direction of electron are connected through an external circuit or when com- flow depends on the relative electron “pressure” or potential of the ponents of both half-cells are present in the same solu- two cells. A salt bridge containing a saturated KCl solution provides tion. Electrons tend to flow to the half-cell with the more a path for counter-ion movement between the test cell and the refer- positive E, and the strength of that tendency is pro- ence cell. From the observed emf and the known emf of the reference portional to the difference in reduction potentials, E. cell, the experimenter can find the emf of the test cell containing the The energy made available by this spontaneous redox pair. The cell that gains electrons has, by convention, the more positive reduction potential. electron flow (the free-energy change for the oxidation- reduction reaction) is proportional to E:

G n E or G n E (13–6) lower standard reduction potential to the half-cell of Here n represents the number of electrons transferred higher standard reduction potential. By convention, the in the reaction. With this equation we can calculate the half-cell with the stronger tendency to acquire electrons free-energy change for any oxidation-reduction reaction is assigned a positive value of E. from the values of E in a table of reduction potentials The reduction potential of a half-cell depends not (Table 13–7) and the concentrations of the species par- only on the chemical species present but also on their ticipating in the reaction. activities, approximated by their concentrations. About Consider the reaction in which acetaldehyde is a century ago, Walther Nernst derived an equation that reduced by the biological electron carrier NADH: relates standard reduction potential (E) to the reduction potential (E) at any concentration of oxidized and Acetaldehyde NADH H 88n ethanol NAD reduced species in the cell: The relevant half-reactions and their E values are:

RT [electron acceptor] (1) Acetaldehyde 2H 2e ethanol E E ln (13–4) 88n nℑ [electron donor] E 0.197 V 13.3 Biological Oxidation-Reduction Reactions 511

(2) NAD 2H 2e 88n NADH H the values of E for both reductants are determined E 0.320 V (Eqn 13–4):

By convention, E is expressed as E of the electron RT [acetaldehyde] E E ln acceptor minus E of the electron donor. Because ac- acetaldehyde nℑ [ethanol] etaldehyde is accepting electrons from NADH in our 0.026V 1.00 0.197 V ln 0.167 V example, E 0.197 V (0.320 V) 0.123 V, 2 0.100 and n is 2. Therefore, RT [NAD] E E ln NADH nℑ [NADH] G n E 2(96.5 kJ/V mol)(0.123 V) 0.026V 1.00 0.320 V ln 0.350 V 23.7 kJ/mol 2 0.100 This is the free-energy change for the oxidation- Then E is used to calculate G (Eqn 13–5): reduction reaction at pH 7, when acetaldehyde, ethanol, NAD , and NADH are all present at 1.00 M concentra- E 0.167 V (0.350) V 0.183 V tions. If, instead, acetaldehyde and NADH were present G n E at 1.00 M but ethanol and NAD were present at 0.100 M, 2(96.5 kJ/V mol)(0.183 V) the value for G would be calculated as follows. First, 35.3 kJ/mol

TABLE 13–7 Standard Reduction Potentials of Some Biologically Important Half-Reactions, at pH 7.0 and 25 C (298 K)

Half-reaction E (V)

1 2O2 2H 2e 88n H2O 0.816 Fe3 e 88n Fe2 0.771 NO3 2H 2e 88n NO2 H2O 0.421 Cytochrome f (Fe3 ) e 88n cytochrome f (Fe2 ) 0.365 3 4 Fe(CN)6 (ferricyanide) e 88n Fe(CN)6 0.36 3 2 Cytochrome a3 (Fe ) e 88n cytochrome a3 (Fe ) 0.35 O2 2H 2e 88n H2O2 0.295 Cytochrome a (Fe3 ) e 88n cytochrome a (Fe2 ) 0.29 Cytochrome c (Fe3 ) e 88n cytochrome c (Fe2 ) 0.254 3 2 Cytochrome c1 (Fe ) e 88n cytochrome c1 (Fe ) 0.22 Cytochrome b (Fe3 ) e 88n cytochrome b (Fe2 ) 0.077 Ubiquinone 2H 2e 88n ubiquinol H2 0.045 Fumarate2 2H 2e 88n succinate2 0.031 2H 2e 88n H2 (at standard conditions, pH 0) 0.000 Crotonyl-CoA 2H 2e 88n butyryl-CoA 0.015 Oxaloacetate2 2H 2e 88n malate2 0.166 Pyruvate 2H 2e 88n lactate 0.185 Acetaldehyde 2H 2e 88n ethanol 0.197 FAD 2H 2e 88n FADH2 0.219* Glutathione 2H 2e 88n 2 reduced glutathione 0.23 S 2H 2e 88n H2S 0.243 Lipoic acid 2H 2e 88n dihydrolipoic acid 0.29 NAD H 2e 88n NADH 0.320 NADP H 2e 88n NADPH 0.324 Acetoacetate 2H 2e 88n -hydroxybutyrate 0.346 -Ketoglutarate CO2 2H 2e 88n isocitrate 0.38 2H 2e 88n H2 (at pH 7) 0.414 Ferredoxin (Fe3 ) e 88n ferredoxin (Fe2 ) 0.432

Source: Data mostly from Loach, P.A.(1976) In Handbook of Biochemistry and Molecular Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. I, pp. 122–130, CRC Press, Boca Raton, FL. * This is the value for free FAD; FAD bound to a specific flavoprotein (for example succinate dehydrogenase) has a different E that depends on its protein environments. 512 Chapter 13 Principles of Bioenergetics

It is thus possible to calculate the free-energy change NADH and NADPH Act with Dehydrogenases for any biological redox reaction at any concentrations as Soluble Electron Carriers of the redox pairs. Nicotinamide adenine dinucleotide (NAD in its oxi- Cellular Oxidation of Glucose to Carbon Dioxide dized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP) are composed of two Requires Specialized Electron Carriers nucleotides joined through their phosphate groups by a The principles of oxidation-reduction energetics de- phosphoanhydride bond (Fig. 13–15a). Because the scribed above apply to the many metabolic reactions that nicotinamide ring resembles pyridine, these compounds involve electron transfers. For example, in many organ- are sometimes called pyridine nucleotides. The vita- isms, the oxidation of glucose supplies energy for the min niacin is the source of the nicotinamide moiety in production of ATP. The complete oxidation of glucose: nicotinamide nucleotides. Both coenzymes undergo reversible reduction of C6H12O6 6O2 8n 6CO2 6H2O the nicotinamide ring (Fig. 13–15). As a substrate mol- has a G of 2,840 kJ/mol. This is a much larger re- ecule undergoes oxidation (dehydrogenation), giving up lease of free energy than is required for ATP synthesis two hydrogen atoms, the oxidized form of the nucleotide (50 to 60 kJ/mol; see Box 13–1). Cells convert glucose (NAD or NADP ) accepts a hydride ion (:H , the to CO2 not in a single, high-energy-releasing reaction, equivalent of a proton and two electrons) and is trans- but rather in a series of controlled reactions, some of formed into the reduced form (NADH or NADPH). The which are oxidations. The free energy released in these second proton removed from the substrate is released oxidation steps is of the same order of magnitude as that to the aqueous solvent. The half-reaction for each type required for ATP synthesis from ADP, with some energy of nucleotide is therefore to spare. Electrons removed in these oxidation steps are NAD 2e 2H 8n NADH H transferred to coenzymes specialized for carrying elec- NADP 2e 2H 8n NADPH H trons, such as NAD and FAD (described below). Reduction of NAD or NADP converts the benzenoid A Few Types of Coenzymes and Proteins Serve ring of the nicotinamide moiety (with a fixed positive as Universal Electron Carriers charge on the ring nitrogen) to the quinonoid form (with no charge on the nitrogen). Note that the reduced nu- The multitude of enzymes that catalyze cellular oxida- cleotides absorb light at 340 nm; the oxidized forms do tions channel electrons from their hundreds of different not (Fig. 13–15b). The plus sign in the abbreviations substrates into just a few types of universal electron car- NAD and NADP does not indicate the net charge on riers. The reduction of these carriers in catabolic these molecules (they are both negatively charged); processes results in the conservation of free energy re- rather, it indicates that the nicotinamide ring is in its leased by substrate oxidation. NAD, NADP, FMN, and oxidized form, with a positive charge on the nitrogen FAD are water-soluble coenzymes that undergo re- atom. In the abbreviations NADH and NADPH, the “H” versible oxidation and reduction in many of the electron- denotes the added hydride ion. To refer to these nu- transfer reactions of metabolism. The nucleotides NAD cleotides without specifying their oxidation state, we and NADP move readily from one enzyme to another; use NAD and NADP. the flavin nucleotides FMN and FAD are usually very The total concentration of NAD NADH in most 5 tightly bound to the enzymes, called flavoproteins, for tissues is about 10 M; that of NADP NADPH is 6 which they serve as prosthetic groups. Lipid-soluble about 10 M. In many cells and tissues, the ratio of quinones such as ubiquinone and plastoquinone act as NAD (oxidized) to NADH (reduced) is high, favoring electron carriers and proton donors in the nonaqueous hydride transfer from a substrate to NAD to form environment of membranes. Iron-sulfur proteins and cy- NADH. By contrast, NADPH (reduced) is generally pres- tochromes, which have tightly bound prosthetic groups ent in greater amounts than its oxidized form, NADP, that undergo reversible oxidation and reduction, also favoring hydride transfer from NADPH to a substrate. serve as electron carriers in many oxidation-reduction This reflects the specialized metabolic roles of the two reactions. Some of these proteins are water-soluble, but coenzymes: NAD generally functions in oxidations— others are peripheral or integral membrane proteins (see usually as part of a catabolic reaction; and NADPH is Fig. 11–6). the usual coenzyme in reductions—nearly always as We conclude this chapter by describing some chem- part of an anabolic reaction. A few enzymes can use ei- ical features of nucleotide coenzymes and some of the ther coenzyme, but most show a strong preference for enzymes (dehydrogenases and flavoproteins) that use one over the other. The processes in which these two them. The oxidation-reduction chemistry of quinones, cofactors function are also segregated in specific or- iron-sulfur proteins, and cytochromes is discussed in ganelles of eukaryotic cells: oxidations of fuels such as Chapter 19. pyruvate, fatty acids, and -keto acids derived from 13.3 Biological Oxidation-Reduction Reactions 513

O O O H B H H B H H B C 2e ? C ? C H H H NH2 NH2 or NH2 H N 2H N N O CH2 O A A R A side R B side H H OPPOO H H NADH (reduced)

O OH OH NH2

N OPPOO N Adenine 1.0 Oxidized N N (NAD) O CH2 O 0.8

H H 0.6 H H NAD Reduced

Absorbance 0.4 (oxidized) OH OH (NADH)

0.2 In NADP this hydroxyl group is esterified with phosphate. 0.0 (a) 220 240 260 280 300 320 340 360 380 Wavelength (nm) (b)

FIGURE 13–15 NAD and NADP. (a) Nicotinamide adenine dinu- tra of NAD and NADH. Reduction of the nicotinamide ring produces cleotide, NAD , and its phosphorylated analog NADP undergo re- a new, broad absorption band with a maximum at 340 nm. The production to NADH and NADPH, accepting a hydride ion (two elec- duction of NADH during an enzyme-catalyzed reaction can be con- trons and one proton) from an oxidizable substrate. The hydride ion veniently followed by observing the appearance of the absorbance at 1 1 is added to either the front (the A side) or the back (the B side) of the 340 nm (the molar extinction coefficient 340 6,200 M cm ). planar nicotinamide ring (see Table 13–8). (b) The UV absorption spec- amino acids occur in the mitochondrial matrix, whereas When NAD or NADP is reduced, the hydride ion reductive biosynthesis processes such as fatty acid syn- could in principle be transferred to either side of the thesis take place in the cytosol. This functional and spa- nicotinamide ring: the front (A side) or the back (B tial specialization allows a cell to maintain two distinct side), as represented in Figure 13–15a. Studies with iso- pools of electron carriers, with two distinct functions. topically labeled substrates have shown that a given en- More than 200 enzymes are known to catalyze re- zyme catalyzes either an A-type or a B-type transfer, but actions in which NAD (or NADP ) accepts a hydride not both. For example, yeast alcohol dehydrogenase and ion from a reduced substrate, or NADPH (or NADH) do- lactate dehydrogenase of vertebrate heart transfer a hy- nates a hydride ion to an oxidized substrate. The gen- dride ion to (or remove a hydride ion from) the A side eral reactions are of the nicotinamide ring; they are classed as type A dehydrogenases to distinguish them from another group AH2 NAD 8n A NADH H of enzymes that transfer a hydride ion to (or remove a A NADPH H AH NADP 8n 2 hydride ion from) the B side of the nicotinamide ring where AH2 is the reduced substrate and A the oxidized (Table 13–8). The specificity for one side or another can substrate. The general name for an enzyme of this type be very striking; lactate dehydrogenase, for example, is oxidoreductase; they are also commonly called de- prefers the A side over the B side by a factor of 5 107! hydrogenases. For example, alcohol dehydrogenase Most dehydrogenases that use NAD or NADP bind catalyzes the first step in the catabolism of ethanol, in the cofactor in a conserved protein domain called the which ethanol is oxidized to acetaldehyde: Rossmann fold (named for Michael Rossmann, who de- duced the structure of lactate dehydrogenase and first CH3CH2OH NAD 8n CH3CHO NADH H described this structural motif). The Rossmann fold typ- Ethanol Acetaldehyde ically consists of a six-stranded parallel sheet and four Notice that one of the carbon atoms in ethanol has lost associated helices (Fig. 13–16). a hydrogen; the compound has been oxidized from an The association between a dehydrogenase and NAD alcohol to an aldehyde (refer again to Fig. 13–13 for the or NADP is relatively loose; the coenzyme readily diffuses oxidation states of carbon). from one enzyme to another, acting as a water-soluble 514 Chapter 13 Principles of Bioenergetics

TABLE 13–8 Stereospecificity of Dehydrogenases That Employ NAD or NADP as Coenzymes Stereochemical specificity for nicotinamide Enzyme Coenzyme ring (A or B) Text page(s) Isocitrate dehydrogenase NAD A XXX–XXX -Ketoglutarate dehydrogenase NAD B XXX Glucose 6-phosphate dehydrogenase NADP B XXX Malate dehydrogenase NAD A XXX Glutamate dehydrogenase NAD or NADP B XXX Glyceraldehyde 3-phosphate dehydrogenase NAD B XXX Lactate dehydrogenase NAD A XXX Alcohol dehydrogenase NAD A XXX

carrier of electrons from one metabolite to another. For (1) Glyceraldehyde 3-phosphate NAD 8n example, in the production of alcohol during fermenta- 3-phosphoglycerate NADH H tion of glucose by yeast cells, a hydride ion is removed (2) Acetaldehyde NADH H 8n ethanol NAD from glyceraldehyde 3-phosphate by one enzyme (glyceraldehyde 3-phosphate dehydrogenase, a type B en- Sum: Glyceraldehyde 3-phosphate acetaldehyde 8n zyme) and transferred to NAD. The NADH produced 3-phosphoglycerate ethanol then leaves the enzyme surface and diffuses to another Notice that in the overall reaction there is no net pro- enzyme (alcohol dehydrogenase, a type A enzyme), duction or consumption of NAD or NADH; the coen- which transfers a hydride ion to acetaldehyde, produc- zymes function catalytically and are recycled repeatedly ing ethanol: without a net change in the concentration of NAD NADH.

Dietary Deficiency of Niacin, the Vitamin Form of NAD and NADP, Causes Pellagra The pyridine-like rings of NAD and NADP are derived from the vitamin niacin (nicotinic acid; Fig. 13–17), which is synthesized from tryptophan. Humans generally cannot synthesize niacin in sufficient quantities, and this is especially so for those with diets low in tryptophan (maize, for example, has a low tryptophan content). Niacin deficiency, which affects all the NAD(P)-dependent dehydrogenases, causes the serious human disease pellagra (Italian for “rough skin”) and a related disease in dogs, blacktongue. These diseases are characterized by the “three Ds”: dermatitis, diarrhea, and dementia, followed in many cases by death. A century ago, pellagra was a common human disease; in the south- ern United States, where maize was a dietary staple, about 100,000 people were afflicted and about 10,000 died between 1912 and 1916. In 1920 Joseph Goldberger showed pellagra to be caused by a dietary insufficiency, FIGURE 13–16 The nucleotide binding domain of the enzyme lac- and in 1937 Frank Strong, D. Wayne Wolley, and Conrad tate dehydrogenase. (a) The Rossmann fold is a structural motif found Elvehjem identified niacin as the curative agent for in the NAD-binding site of many dehydrogenases. It consists of a blacktongue. Supplementation of the human diet with six-stranded parallel sheet and four helices; inspection reveals this inexpensive compound led to the eradication of pel- the arrangement to be a pair of structurally similar ---- motifs. lagra in the populations of the developed world—with (b) The dinucleotide NAD binds in an extended conformation through one significant exception. Pellagra is still found among hydrogen bonds and salt bridges (derived from PDB ID 3LDH). alcoholics, whose intestinal absorption of niacin is much 13.3 Biological Oxidation-Reduction Reactions 515

O TABLE 13–9 Some Enzymes (Flavoproteins) C O C That Employ Flavin Nucleotide Coenzymes

CH3 Flavin Text Niacin Nicotine Enzyme nucleotide page(s) (nicotinic acid) Acyl–CoA dehydrogenase FAD XXX O NH3 Dihydrolipoyl dehydrogenase FAD XXX Succinate dehydrogenase FAD XXX C CH2 CH COO NH2 Glycerol 3-phosphate dehydrogenase FAD XXX Thioredoxin reductase FAD XXX–XXX NADH dehydrogenase (Complex I) FMN XXX Glycolate oxidase FMN XXX Nicotinamide Tryptophan

FIGURE 13–17 Structures of niacin (nicotinic acid) and its deriva- tive nicotinamide. The biosynthetic precursor of these compounds is tryptophan. In the laboratory, nicotinic acid was first produced by ox- loxazine ring is produced, abbreviated FADH• and idation of the natural product nicotine—thus the name. Both nicotinic FMNH•. Because flavoproteins can participate in either acid and nicotinamide cure pellagra, but nicotine (from cigarettes or one- or two-electron transfers, this class of proteins elsewhere) has no curative activity. is involved in a greater diversity of reactions than the NAD (P)-linked dehydrogenases. Like the nicotinamide coenzymes (Fig. 13–15), the reduced, and whose caloric needs are often met with dis- flavin nucleotides undergo a shift in a major absorption tilled spirits that are virtually devoid of vitamins, in- band on reduction. Flavoproteins that are fully reduced cluding niacin. In a few places, including the Deccan (two electrons accepted) generally have an absorption Plateau in India, pellagra still occurs, especially among maximum near 360 nm. When partially reduced (one the poor. ■ electron), they acquire another absorption maximum at Flavin Nucleotides Are Tightly Bound in Flavoproteins about 450 nm; when fully oxidized, the flavin has maxima at 370 and 440 nm. The intermediate radical form, Flavoproteins (Table 13–9) are enzymes that catalyze reduced by one electron, has absorption maxima at 380, oxidation-reduction reactions using either flavin 480, 580, and 625 nm. These changes can be used to as- mononucleotide (FMN) or flavin adenine dinucleotide say reactions involving a flavoprotein. (FAD) as coenzyme (Fig. 13–18). These coenzymes, the The flavin nucleotide in most flavoproteins is bound flavin nucleotides, are derived from the vitamin ri- rather tightly to the protein, and in some enzymes, such boflavin. The fused ring structure of flavin nucleotides as succinate dehydrogenase, it is bound covalently. Such (the isoalloxazine ring) undergoes reversible reduction, tightly bound coenzymes are properly called prosthetic accepting either one or two electrons in the form of one groups. They do not transfer electrons by diffusing from or two hydrogen atoms (each atom an electron plus a one enzyme to another; rather, they provide a means by proton) from a reduced substrate. The fully reduced which the flavoprotein can temporarily hold electrons forms are abbreviated FADH2 and FMNH2. When a fully while it catalyzes electron transfer from a reduced sub- oxidized flavin nucleotide accepts only one electron strate to an electron acceptor. One important feature of (one hydrogen atom), the semiquinone form of the isoal- the flavoproteins is the variability in the standard reduction potential (E) of the bound flavin nucleotide. Tight association between the enzyme and prosthetic group confers on the flavin ring a reduction potential typical of that particular flavoprotein, sometimes quite different from the reduction potential of the free flavin nucleotide. FAD bound to succinate dehydrogenase, for example, has an E close to 0.0 V, compared with 0.219 V for free Frank Strong, D. Wayne Woolley, Conrad Elvehjem, FAD; E for other flavoproteins 1908–1993 1914–1966 1901–1962 ranges from 0.40 V to 0.06 V. 516 Chapter 13 Principles of Bioenergetics

isoalloxazine ring H O O H O CH N H e CH H e 3 3 N CH3 N NH • NH NH

CH N N O CH N N O 3 3 CH3 N N O

CH2 R R H FADH• (FMNH•) HCOH FADH2 (FMNH2) FMN (semiquinone) (fully reduced) HCOH HCOH

CH2 FAD O O P O O NH2 O P O N N O N N CH2 O FIGURE 13–18 Structures of oxidized and reduced FAD and FMN. H H FMN consists of the structure above the dashed line on the FAD (ox- H H idized form). The flavin nucleotides accept two hydrogen atoms (two OH OH electrons and two protons), both of which appear in the flavin ring Flavin adenine dinucleotide (FAD) and system. When FAD or FMN accepts only one hydrogen atom, the semi- flavin mononucleotide (FMN) quinone, a stable free radical, forms.

Flavoproteins are often very complex; some have, in ad- ■ Biological oxidation-reduction reactions can be dition to a flavin nucleotide, tightly bound inorganic ions described in terms of two half-reactions, each (iron or molybdenum, for example) capable of partici- with a characteristic standard reduction pating in electron transfers. potential, E. Certain flavoproteins act in a quite different role as ■ When two electrochemical half-cells, each light receptors. Cryptochromes are a family of flavo- containing the components of a half-reaction, proteins, widely distributed in the eukaryotic phyla, that are connected, electrons tend to flow to the mediate the effects of blue light on plant development half-cell with the higher reduction potential. and the effects of light on mammalian circadian rhythms The strength of this tendency is proportional (oscillations in physiology and biochemistry, with a to the difference between the two reduction 24-hour period). The cryptochromes are homologs of potentials (E) and is a function of the another family of flavoproteins, the photolyases. Found concentrations of oxidized and reduced in both prokaryotes and eukaryotes, photolyases use species. the energy of absorbed light to repair chemical defects in DNA. ■ The standard free-energy change for an We examine the function of flavoproteins as elec- oxidation-reduction reaction is directly tron carriers in Chapter 19, when we consider their roles proportional to the difference in standard in oxidative phosphorylation (in mitochondria) and pho- reduction potentials of the two half-cells: tophosphorylation (in chloroplasts), and we describe G n E . the photolyase reactions in Chapter 25. ■ Many biological oxidation reactions are dehydrogenations in which one or two SUMMARY 13.3 Biological Oxidation-Reduction hydrogen atoms (H e) are transferred Reactions from a substrate to a hydrogen acceptor. Oxidation-reduction reactions in living cells ■ In many organisms, a central energy-conserving involve specialized electron carriers. process is the stepwise oxidation of glucose to ■ NAD and NADP are the freely diffusible

CO2, in which some of the energy of oxidation is coenzymes of many dehydrogenases. Both conserved in ATP as electrons are passed to O2. NAD and NADP accept two electrons and Chapter 13 Further Reading 517

one proton. NAD and NADP are bound to flavoproteins. They can accept either one or dehydrogenases in a widely conserved two electrons. Flavoproteins also serve as light structural motif called the Rossmann fold. receptors in cryptochromes and photolyases. ■ FAD and FMN, the flavin nucleotides, serve as tightly bound prosthetic groups of

Key Terms

Terms in bold are defined in the glossary. autotroph XXX thioester XXX dehydrogenation XXX heterotroph XXX adenylylation XXX dehydrogenases XXX metabolism XXX inorganic pyrophosphatase XXX reducing equivalent XXX metabolic pathways XXX nucleoside diphosphate standard reduction potential metabolite XXX kinase XXX (E) XXX intermediary metabolism XXX adenylate kinase XXX pyridine nucleotide XXX catabolism XXX creatine kinase XXX oxidoreductase XXX anabolism XXX phosphagens XXX flavoprotein XXX standard transformed constants XXX polyphosphate kinase-1, -2 XXX flavin nucleotides XXX phosphorylation potential electromotive force (emf) XXX cryptochrome XXX

(Gp) XXX conjugate redox pair XXX photolyase XXX