Biochemistry Steady State

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Introduction to Metabolism CHAPTER 16

1 Metabolic Pathways obtained from other organisms, ultimately phototrophs. 2 Organic Reaction Mechanisms This free energy is most often coupled to endergonic reac- A. Chemical Logic tions through the intermediate synthesis of “high-energy” B. Group-Transfer Reactions phosphate compounds such as adenosine triphosphate C. Oxidations and Reductions (ATP; Section 16-4). In addition to being completely oxi- D. Eliminations, Isomerizations, and Rearrangements dized, nutrients are broken down in a series of metabolic E. Reactions That Make and Break Carbon–Carbon Bonds reactions to common intermediates that are used as precur- 3 Experimental Approaches to the Study of Metabolism sors in the synthesis of other biological molecules. A. Metabolic Inhibitors, Growth Studies, A remarkable property of living systems is that, despite and Biochemical Genetics the complexity of their internal processes, they maintain a B. Isotopes in Biochemistry steady state. This is strikingly demonstrated by the observa- C. Isolated Organs, Cells, and Subcellular Organelles tion that, over a 40-year time span, a normal human adult D. Systems Biology consumes literally tons of nutrients and imbibes over 20,000 L 4 Thermodynamics of Phosphate Compounds of water, but does so without significant weight change. This A. Phosphoryl-Transfer Reactions steady state is maintained by a sophisticated set of metabolic B. Rationalizing the “Energy” in “High-Energy” Compounds regulatory systems. In this introductory chapter to metabo- C. The Role of ATP lism, we outline the general characteristics of metabolic 5 Oxidation–Reduction Reactions pathways, study the main types of chemical reactions that A. The Nernst Equation comprise these pathways, and consider the experimental B. Measurements of Redox Potentials techniques that have been most useful in their elucidation. C. Concentration Cells We then discuss the free energy changes associated with re- 6 Thermodynamics of Life actions of phosphate compounds and oxidation–reduction A. Living Systems Cannot Be at Equilibrium reactions. Finally we consider the thermodynamic nature B. Nonequilibrium Thermodynamics and the Steady State of biological processes, that is, what properties of life are C. Thermodynamics of Metabolic Control responsible for its self-sustaining character.

1 METABOLIC PATHWAYS Living organisms are not at equilibrium. Rather, they require a continuous influx of free energy to maintain order in a uni- Metabolic pathways are series of consecutive enzymatic reverse bent on maximizing disorder. Metabolism is the over- actions that produce specific products. Their reactants, inter- all process through which living systems acquire and utilize mediates, and products are referred to as metabolites. Since the free energy they need to carry out their various func- an organism utilizes many metabolites, it has many meta- tions. They do so by coupling the exergonic reactions of nutri- bolic pathways. Figure 16-1 shows a metabolic map for a ent oxidation to the endergonic processes required to main- typical cell with many of its interconnected pathways. Each tain the living state such as the performance of mechanical reaction on the map is catalyzed by a distinct enzyme, of work, the active transport of molecules against concentra- which there are ϳ4000 known. At first glance, this network tion gradients, and the biosynthesis of complex molecules. seems hopelessly complex. Yet, by focusing on its major How do living things acquire this necessary free energy? areas in the following chapters, for example, the main And what is the nature of the energy coupling process? pathways of glucose oxidation (the shaded areas of Fig. Phototrophs (plants and certain bacteria; Section 1-1A) 16-1), we shall become familiar with its most important av- acquire free energy from the sun through photosynthesis, a enues and their interrelationships. Maps of metabolic path- process in which light energy powers the endergonic reac- ways in a more readable form can be found on the Web

tion of CO2 and H2O to form carbohydrates and O2 at http://www.expasy.org/cgi-bin/search-biochem-index, (Chapter 24). Chemotrophs obtain their free energy by ox- http://www.iubmb-nicholson.org/, and http://www.genome. idizing organic compounds (carbohydrates, lipids, proteins) ad.jp/kegg/metabolism.html.

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Figure 16-1 Map of the major metabolic pathways in a typical cell. The main pathways of glucose metabolism are shaded. [Designed by Donald Nicholson. Published by BDH Ltd., Poole 2, Dorset, England.] JWCL281_c16_557-592.qxd 6/10/10 11:51 AM Page 561

Section 16-1. Metabolic Pathways 561

The reaction pathways that comprise metabolism are from the first to the second must differ from the pathway often divided into two categories: from the second back to the first: 1. Catabolism, or degradation, in which nutrients and A cell constituents are broken down exergonically to salvage 1 2 their components and/or to generate free energy. YX 2. Anabolism, or biosynthesis, in which biomolecules are synthesized from simpler components. This is because if metabolite 1 is converted to metabolite 2 by an exergonic process, the conversion of metabolite 2 to The free energy released by catabolic processes is con- metabolite 1 requires that free energy be supplied in order served through the synthesis of ATP from ADP and phos- to bring this otherwise endergonic process “back up the phate or through the reduction of the coenzyme NADP to hill.” Consequently, the two pathways must differ in at least NADPH (Fig. 13-2). ATP and NADPH are the major free energy sources for anabolic pathways (Fig. 16-2). A striking characteristic of degradative metabolism is that it converts large numbers of diverse substances (carbohydrates, lipids, and proteins) to common intermediates. These Proteins Carbohydrates Lipids intermediates are then further metabolized in a central oxidative pathway that terminates in a few end products. Figure Amino acids Glucose Fatty acids & Glycerol 16-3 outlines the breakdown of various foodstuffs, first to their monomeric units, and then to the common intermediate, acetyl-coenzyme A (acetyl-CoA) (Fig. 21-2). ADP ATP Biosynthesis carries out the opposite process. Relatively NAD+ Glycolysis NADH few metabolites, mainly pyruvate, acetyl-CoA, and the citric acid cycle intermediates, serve as starting materials for a host of varied biosynthetic products. In the next several chapters Pyruvate we discuss many degradative and biosynthetic pathways in

detail. For now, let us consider some general characteristics CO2 of these processes. Five principal characteristics of metabolic pathways Acetyl-CoA stem from their function of generating products for use by the cell: NH3 1. Metabolic pathways are irreversible. A highly exer- Citric NAD+ NADH gonic reaction (having a large negative free energy change) Acid FAD FADH is irreversible; that is, it goes to completion. If such a reaction Cycle 2 is part of a multistep pathway,it confers directionality on the pathway; that is, it makes the entire pathway irreversible. 2. Catabolic and anabolic pathways must differ. If two CO2 metabolites are metabolically interconvertible, the pathway

NAD+ Oxidative NADH FAD phosphorylation FADH Complex metabolites 2

2– ADP ADP + HPO 4 NADP + O2 H O ATP 2 Degradation Biosynthesis

Figure 16-3 Overview of catabolism. Complex metabolites ATP such as carbohydrates, proteins, and lipids are degraded first to NADPH their monomeric units, chiefly glucose, amino acids, fatty acids, and glycerol, and then to the common intermediate, Simple products acetyl-coenzyme A (acetyl-CoA). The acetyl group is then oxidized to CO2 via the citric acid cycle with the concomitant Figure 16-2 ATP and NADPH are the sources of free energy reduction of NAD and FAD. Reoxidation of these latter

for biosynthetic reactions. They are generated through the coenzymes by O2 via the electron-transport chain and oxidative degradation of complex metabolites. phosphorylation yields H2O and ATP. JWCL281_c16_557-592.qxd 6/10/10 11:51 AM Page 562

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one of their reaction steps. The existence of independent in- the presence in membranes of specific transport proteins. terconversion routes, as we shall see, is an important property The transport protein that facilitates the passage of ATP of metabolic pathways because it allows independent control through the mitochondrial membrane is discussed in of the two processes. If metabolite 2 is required by the cell, it Section 20-4C, along with the characteristics of membrane is necessary to “turn off” the pathway from 2 to 1 while transport processes in general.The synthesis and utilization “turning on” the pathway from 1 to 2. Such independent of acetyl-CoA are also compartmentalized. This metabolic control would be impossible without different pathways. intermediate is utilized in the cytosolic synthesis of fatty 3. Every metabolic pathway has a first committed step. acids but is synthesized in mitochondria. Yet there is no Although metabolic pathways are irreversible, most of transport protein for acetyl-CoA in the mitochondrial their component reactions function close to equilibrium. membrane. How cells solve this fundamental problem is Early in each pathway, however, there is an irreversible discussed in Section 25-4D. In multicellular organisms, com- (exergonic) reaction that “commits” the intermediate it partmentation is carried a step further to the level of tissues produces to continue down the pathway. and organs.The mammalian liver, for example, is largely responsible for the synthesis of glucose from noncarbohy- 4. All metabolic pathways are regulated. Metabolic drate precursors (gluconeogenesis; Section 23-1) so as to pathways are regulated by laws of supply and demand. In maintain a relatively constant level of glucose in the circula- order to exert control on the flux of metabolites through a tion, whereas adipose tissue is specialized for the storage metabolic pathway, it is necessary to regulate its rate-limiting and mobilization of triacylglycerols. The metabolic interde- step. The first committed step, being irreversible, functions pendence of the various organs is the subject of Chapter 27. too slowly to permit its substrates and products to equilibrate (if the reaction were at equilibrium, it would not be irreversible). Since most of the other reactions in a pathway function close to equilibrium, the first committed step 2 ORGANIC REACTION MECHANISMS is often one of its rate-limiting steps. Most metabolic path- Almost all of the reactions that occur in metabolic pathways are therefore controlled by regulating the enzymes ways are enzymatically catalyzed organic reactions. Section that catalyze their first committed step(s). This is an effi- 15-1 details the various mechanisms enzymes have at their cient way to exert control because it prevents the unneces- disposal for catalyzing reactions: acid–base catalysis, cova- sary synthesis of metabolites further along the pathway lent catalysis, metal ion catalysis, electrostatic catalysis, when they are not required. Specific aspects of such flux proximity and orientation effects, and transition state bind- control are discussed in Section 17-4C. ing. Few enzymes alter the chemical mechanisms of these 5. Metabolic pathways in eukaryotic cells occur in spe- reactions, so much can be learned about enzymatic mecha- cific cellular locations. The compartmentation of the eu- nisms from the study of nonenzymatic model reactions. We karyotic cell allows different metabolic pathways to operate therefore begin our study of metabolic reactions by outlin- in different locations, as is listed in Table 16-1 (these or- ing the types of reactions we shall encounter and the mech- ganelles are described in Section 1-2A). For example, ATP anisms by which they have been observed to proceed in is mainly generated in the mitochondrion but much of it is nonenzymatic systems. utilized in the cytoplasm. The synthesis of metabolites in Christopher Walsh has classified biochemical reactions specific membrane-bounded subcellular compartments into four categories: (1) group-transfer reactions; (2) oxida- makes their transport between these compartments a vital tions and reductions; (3) eliminations, isomerizations, and re- component of eukaryotic metabolism. Biological mem- arrangements; and (4) reactions that make or break carbon– branes are selectively permeable to metabolites because of carbon bonds. Much is known about the mechanisms of

Table 16-1 Metabolic Functions of Eukaryotic Organelles Organelle Function Mitochondrion Citric acid cycle, electron transport and oxidative phosphorylation, fatty acid oxidation, amino acid breakdown Cytosol Glycolysis, pentose phosphate pathway, fatty acid biosynthesis, many reactions of gluconeogenesis Lysosomes Enzymatic digestion of cell components and ingested matter Nucleus DNA replication and transcription, RNA processing Golgi apparatus Post-translational processing of membrane and secretory proteins; formation of plasma membrane and secretory vesicles Rough endoplasmic reticulum Synthesis of membrane-bound and secretory proteins Smooth endoplasmic reticulum Lipid and steroid biosynthesis Peroxisomes (glyoxisomes in plants) Oxidative reactions catalyzed by amino acid oxidases and catalase; glyoxylate cycle reactions in plants JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 563

Section 16-2. Organic Reaction Mechanisms 563

these reactions and about the enzymes that catalyze them. Homolytic: The discussions in the next several chapters focus on these homolytic CCH CH mechanisms as they apply to specific metabolic intercon- cleavage versions. In this section we outline the four reaction cate- Radicals gories and discuss how our knowledge of their reaction mechanisms derives from the study of model organic reac- Heterolytic:

tions.We begin by briefly reviewing the chemical logic used (i) CCH C H in analyzing these reactions.

Carbanion Proton A. Chemical Logic

(ii) A covalent bond consists of an electron pair shared between C H CC H two atoms. In breaking such a bond, the electron pair can either remain with one of the atoms (heterolytic bond cleav- Carbocation Hydride ion age) or separate such that one electron accompanies each of the atoms (homolytic bond cleavage) (Fig. 16-4). Homolytic Figure 16-4 Modes of C¬H bond breaking. Homolytic cleav- bond cleavage, which usually produces unstable radicals, oc- age yields radicals, whereas heterolytic cleavage yields either (i) curs mostly in oxidation–reduction reactions. Heterolytic a carbanion and a proton or (ii) a carbocation and a hydride ion. C¬H bond cleavage involves either carbanion and proton (H) formation or carbocation (carbonium ion) and hydride ion (H ) formation. Since hydride ions are highly reactive tion 15-1Ba):A compound acts as a base when it forms a co- species and carbon atoms are slightly more electronegative valent bond with H, whereas it acts as a nucleophile when than hydrogen atoms, bond cleavage in which the electron it forms a covalent bond with an electron-deficient center pair remains with the carbon atom is the predominant mode other than H, usually an electron-deficient carbon atom: of C¬H bond breaking in biochemical systems. Hydride ion abstraction occurs only if the hydride is transferred directly H Basic reaction to an acceptor such as NAD or NADP . of an amine RRNH2 H N H Compounds participating in reactions involving heterolytic bond cleavage and bond formation are categorized H Nucleophilic into two broad classes: electron rich and electron deficient. R H R Electron-rich compounds, which are called nucleophiles reaction of an amine RNH CO R N C OH (nucleus lovers), are negatively charged or contain un- 2 shared electron pairs that easily form covalent bonds with R R electron-deficient centers. Biologically important nucleophilic groups include amino, hydroxyl, imidazole, and Electron-deficient compounds are called electrophiles sulfhydryl functions (Fig. 16-5a). The nucleophilic forms of (electron lovers). They may be positively charged, contain these groups are also their basic forms. Indeed, nucle- an unfilled valence electron shell, or contain an electroneg- ophilicity and basicity are closely related properties (Sec- ative atom.The most common electrophiles in biochemical

(a) Nucleophiles (b) Electrophiles

Nucleophilic H Protons form n M ROHOQ ROOQS H Hydroxyl group Metal ions

R RSHOQ RSOQS H Sulfhydryl group C O Carbonyl carbon atom RNH 3 RNHO 2 H Amino group R

R R R H Imidazole group HN NH HN NS C NH Cationic imine (Schiff base) R Figure 16-5 Biologically important nucleophilic and imidazole groups. (b) Electrophiles contain an electron-deficient electrophilic groups. (a) Nucleophiles are the conjugate bases atom (red). of weak acids such as the hydroxyl, sulfhydryl, amino, and JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 564

564 Chapter 16. Introduction to Metabolism

systems are H, metal ions, the carbon atoms of carbonyl In the first reaction step, the amine’s unshared electron groups, and cationic imines (Fig. 16-5b). pair adds to the electron-deficient carbonyl carbon atom Reactions are best understood if the electron pair re- while one electron pair from its C“O double bond trans- arrangements involved in going from reactants to products fers to the oxygen atom. In the second step, the unshared can be traced. In illustrating these rearrangements we shall electron pair on the nitrogen atom adds to the electron- use the curved arrow convention in which the movement of deficient carbon atom with the elimination of water. At all an electron pair is symbolized by a curved arrow emanating times, the rules of chemical reason prevail: For example, from the electron pair and pointing to the electron- there are never five bonds to a carbon atom or two bonds deficient center attracting the electron pair. For example, to a hydrogen atom. imine formation, a biochemically important reaction between an amine and an aldehyde or ketone, is represented: B. Group-Transfer Reactions R R The group transfers that occur in biochemical systems in-

R ONH2 + OC R ON COH volve the transfer of an electrophilic group from one nucleophile to another: R H R Amine Aldehyde Carbinolamine or intermediate YYAAXX ketone H+ Nucleophile Electrophile– nucleophile R + They could equally well be called nucleophilic substitution R N C + H O 2 reactions. The most commonly transferred groups in bio- H R chemical reactions are acyl groups, phosphoryl groups, and Imine glycosyl groups (Fig. 16-6):

(a) (b) X X O O O O O O O R C X Y R C X R C Y X Y P P O P X O Y O O O Y Y

Tetrahedral Trigonal intermediate bipyramid intermediate (c) double O displacement O O (S 1) N X X

Y Resonance-stabilized single carbocation (oxonium ion) Y displacement (SN2) O O Y X Y

Figure 16-6 Types of metabolic group-transfer reactions. group to complete the transfer reaction results in the phosphoryl (a) Acyl group transfer involves addition of a nucleophile (Y) to group’s inversion of configuration. (c) Glycosyl group transfer the electrophilic carbon atom of an acyl compound to form a involves the substitution of one nucleophilic group for another at tetrahedral intermediate. The original acyl carrier (X) is then C1 of a sugar ring. This reaction usually occurs via a double expelled to form a new acyl compound. (b) Phosphoryl group displacement mechanism in which the elimination of the original transfer involves the in-line (with the leaving group) addition of glycosyl carrier (X) is accompanied by the intermediate formation a nucleophile (Y) to the electrophilic phosphorus atom of a of a resonance-stabilized carbocation (oxonuim ion) followed by tetrahedral phosphoryl group. This yields a trigonal bipyramidal the addition of the adding nucleophile (Y). The reaction also intermediate whose apical positions are occupied by the leaving may occur via a single displacement mechanism in which Y directly group (X) and the attacking group (Y). Elimination of the leaving displaces X with inversion of configuration. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 565

Section 16-2. Organic Reaction Mechanisms 565

1. Acyl group transfer from one nucleophile to another resonance-stabilized carbocation at C1 (an oxonium ion). almost invariably involves the addition of a nucleophile to The lysozyme-catalyzed hydrolysis of bacterial cell wall the acyl carbonyl carbon atom so as to form a tetrahedral polysaccharides (Section 15-2Bb) is such a reaction. intermediate (Fig. 16-6a). Peptide bond hydrolysis, as catalyzed, for example, by chymotrypsin (Section 15-3C), is a familiar example of such a reaction. C. Oxidations and Reductions 2. Phosphoryl group transfer proceeds via the in-line Oxidation–reduction (redox) reactions involve the loss or addition of a nucleophile to a phosphoryl phosphorus atom gain of electrons. The thermodynamics of these reactions to yield a trigonal bipyramidal intermediate whose apexes is discussed in Section 16-5. Many of the redox reactions are occupied by the adding and leaving groups (Fig. 16-6b). that occur in metabolic pathways involve C¬H bond The overall reaction results in the tetrahedral phosphoryl cleavage with the ultimate loss of two bonding electrons group’s inversion of configuration. Indeed, chiral phospho- by the carbon atom. These electrons are transferred to an ryl compounds have been shown to undergo just such an electron acceptor such as NAD (Fig. 13-2). Whether inversion. For example, Jeremy Knowles has synthesized these reactions involve homolytic or heterolytic bond ATP made chiral at its -phosphoryl group by isotopic sub- cleavage has not always been rigorously established. In stitution and demonstrated that this group is inverted on its most instances heterolytic cleavage is assumed when radi- transfer to glucose in the reaction catalyzed by hexokinase cal species are not observed. It is useful, however, to visu- (Fig. 16-7). alize redox C¬H bond cleavage reactions as hydride transfers as diagrammed below for the oxidation of an 3. Glycosyl group transfer involves the substitution of one nucleophilic group for another at C1 of a sugar ring alcohol by NAD : (Fig. 16-6c). This is the central carbon atom of an acetal. Chemical models of acetal reactions generally proceed H O via acid-catalyzed cleavage of the first bond to form a R H C NH B H O C H 2 R H N H R CH OH ؉ 2 O ADP General Alcohol NAD H O H base H Pγ OH H 17 16 18 HO OH O O O H OH O Glucose ATP H H R C NH B H OC 2 O ADP 16O R 18 N P O Trigonal bipyramid 17O R intermediate Glucose General Ketone NADH

acid

17O 18O 16O For aerobic organisms, the terminal acceptor for the P electron pairs removed from metabolites by their oxidation is molecular oxygen (O2). Recall that this molecule is a H CO 2 ground state diradical species whose unpaired electrons H O H have parallel spins. The rules of electron pairing (the Pauli H ADP OH H exclusion principle) therefore dictate that O2 can only ac- HO OH cept unpaired electrons; that is, electrons must be transferred to O one at a time (in contrast to redox processes H OH 2 in which electrons are transferred in pairs). Electrons that Glucose-6-phosphate are removed from metabolites as pairs must therefore be Figure 16-7 The phosphoryl-transfer reaction catalyzed by passed to O2 via the electron-transport chain one at a hexokinase. During its transfer to the 6-OH group of glucose, the time. This is accomplished through the use of conjugated -phosphoryl group of ATP made chiral by isotopic substitution coenzymes that have stable radical oxidation states and undergoes inversion of configuration via a trigonal bipyramidal can therefore undergo both 1e and 2e redox reactions. intermediate. One such coenzyme is flavin adenine dinucleotide (FAD; JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 566

566 Chapter 16. Introduction to Metabolism

NH2

N N

N O O N Adenosine CH2 O P O P O CH2 O HOC H O O HH H D-Ribitol HO C H HO OH HO C H Riboflavin CH2 H C NNO 3 9 10 8a 8 9a 10a 1 2 4a 7a 7 5a 3N 6 5 4 7, 8-Dimethylisoalloxazine H3C N H O

Flavin adenine dinucleotide (FAD) (oxidized or quinone form)

R R H

N N O N N O H3C H H3C

N N H3C N H H3C N H H O H O

FADH2 (reduced or hydroquinone form) Figure 16-8 The molecular formula and reactions of the cosubstrate as is, for example, NAD. Consequently, although coenzyme flavin adenine dinucleotide (FAD). The term “flavin” humans and other higher animals are unable to synthesize the is synonymous with the isoalloxazine system. The D-ribitol isoalloxazine component of flavins and hence must obtain it in

residue is derived from the alcohol of the sugar D-ribose. The their diets [for example, in the form of riboflavin (vitamin B2)], FAD may be half-reduced to the stable radical FADH or fully riboflavin deficiency is quite rare in humans. The symptoms of

reduced to FADH2 (boxes). Consequently, different riboflavin deficiency, which are associated with general FAD-containing enzymes cycle between different oxidation malnutrition or bizarre diets, include an inflamed tongue, lesions states of FAD. FAD is usually tightly bound to its enzymes, so in the corners of the mouth, and dermatitis. that this coenzyme is normally a prosthetic group rather than a

Fig. 16-8). Flavins (substances that contain the isoallox- H H R H azine ring) can undergo two sequential one-electron trans- R CCR CC H2O fers or a simultaneous two-electron transfer that bypasses H R the semiquinone state. H OH

Bond breaking and bond making in this reaction may pro- D. Eliminations, Isomerizations, and ceed via one of three mechanisms (Fig. 16-9a): (1) con- Rearrangements certed; (2) stepwise with the C¬O bond breaking first to a. Elimination Reactions Form Carbon–Carbon form a carbocation; or (3) stepwise with the C¬H bond Double Bonds breaking first to form a carbanion. Elimination reactions result in the formation of a dou- Enzymes catalyze dehydration reactions by either of ble bond between two previously single-bonded saturated two simple mechanisms: (1) protonation of the OH group

centers. The substances eliminated may be H2O, NH3,an by an acidic group (acid catalysis) or (2) abstraction of the alcohol (ROH), or a primary amine (RNH2). The dehydra- proton by a basic group (base catalysis). Moreover, in a step- tion of an alcohol, for example, is an elimination reaction: wise reaction, the charged intermediate may be stabilized JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 567

Section 16-2. Organic Reaction Mechanisms 567

(a) H O H O Concerted C C H H R H B H C O H BH C O H R CCR CC H OH R R H OH H R Aldose

Stepwise via a carbocation H H H H R H H H O H R CCR R CCR CC H C O H C H OH H H R B C O BH C O OH H R R Stepwise via a carbanion H H H R H Ketose cis-Enediolate intermediates R CCR R C C R CC Figure 16-10 Mechanism of aldose–ketose isomerization. The reaction occurs with acid–base catalysis and proceeds via H OH H OH H R H OH cis-enediolate intermediates.

(b)

H H R H only chiral center so as to invert that chiral center (e.g., the R CCR CC trans (anti) racemization of proline by proline racemase; Section 15-1Fa). H OH H R Such an isomerization is called an epimerization in a mole- H OH cule with more than one chiral center. H H H H c. Rearrangements Produce Altered R CCR CC cis (syn) Carbon Skeletons H OH R R Rearrangement reactions break and reform C¬C bonds H OH so as to rearrange a molecule’s carbon skeleton. There are Figure 16-9 Possible elimination reaction mechanisms using few such metabolic reactions. One is the conversion of dehydration as an example. Reactions may be (a) either L-methylmalonyl-CoA to succinyl-CoA by methylmalonyl- concerted, stepwise via a carbocation intermediate, or stepwise CoA mutase, an enzyme whose prosthetic group is a

via a carbanion intermediate; and may occur with (b) either trans vitamin B12 derivative: (anti) or cis (syn) stereochemistry. H COO methylmalonyl- H COO CoA mutase HHCC HHCC by an oppositely charged active site group (electrostatic H C S CoA SCoA C H catalysis). The glycolytic enzyme enolase (Section 17-2I) and O O the citric acid cycle enzyme fumarase (Section 21-3G) catalyze such dehydration reactions. L-Methylmalonyl-CoA Succinyl-CoA Elimination reactions may take one of two possible stereochemical courses (Fig. 16-9b): (1) trans (anti) elimi- Carbon skeleton rearrangement CCC CCC nations, the most prevalent biochemical mechanism, and (2) cis (syn) eliminations, which are biochemically less C C common. This reaction is involved in the oxidation of fatty acids with b. Biochemical Isomerizations Involve Intramolecular an odd number of carbon atoms (Section 25-2Ec) and sev- Hydrogen Atom Shifts eral amino acids (Section 26-3Ec). Biochemical isomerization reactions involve the intramolecular shift of a hydrogen atom so as to change the E. Reactions That Make and Break location of a double bond. In such a process, a proton is re- Carbon–Carbon Bonds moved from one carbon atom and added to another. The metabolically most prevalent isomerization reaction is the Reactions that make and break carbon–carbon bonds form aldose–ketose interconversion, a base-catalyzed reaction the basis of both degradative and biosynthetic metabolism.

that occurs via enediolate anion intermediates (Fig. 16-10). The breakdown of glucose to CO2 involves five such cleav- The glycolytic enzyme phosphoglucose isomerase cat- ages, whereas its synthesis involves the reverse process. alyzes such a reaction (Section 17-2B). Such reactions, considered from the synthetic direction, in- Racemization is an isomerization reaction in which a hy- volve addition of a nucleophilic carbanion to an elec- drogen atom shifts its stereochemical position at a molecule’s trophilic carbon atom. The most common electrophilic JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 568

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carbon atoms in such reactions are the sp2-hybridized car- drogenase, Section 21-3C; and fatty acid synthase, Section

bonyl carbon atoms of aldehydes, ketones, esters, and CO2: 25-4C). In nonenzymatic systems, both the aldol condensation and Claisen ester condensation involve the base- C C O CCOH catalyzed generation of a carbanion to a carbonyl group (Fig. 16-11a,b). The carbonyl group is electron withdrawing and thereby provides resonance stabilization by form- Stabilized carbanions must be generated to add to these ing an enolate (Fig. 16-12a). The enolate may be further electrophilic centers. Three examples are the aldol con- stabilized by neutralizing its negative charge. Enzymes do densation (catalyzed, e.g., by aldolase; Section 17-2D), so through hydrogen bonding or protonation (Fig. 16-12b), Claisen ester condensation (citrate synthase; Section 21-3A), conversion of the carbonyl group to a protonated Schiff and the decarboxylation of -keto acids (isocitrate dehy- base (covalent catalysis; Fig. 16-12c), or by its coordination

H (a) Aldol condensation R RRCC R C O H O R C O Second ketone R C H C O (electrophilic center) H RCH BB RCH H B RRCC H R H O H C O R C H

Ketone Resonance- stabilized carbanion (enolate)

(b) Claisen ester condensation H O C C SCoA

H O H Addition to electrophilic BBH C C SCoA H center [as H H O in (a)] Acetyl-CoA C C SCoA H

Resonance-stabilized enolate

O O RCCH2 Addition to electrophilic RCCH CO CO 2 2 center [as O in (a)]

RCCH2 ␤-Keto acid Resonance- stabilized enolate Figure 16-11 Examples of C—C bond formation and cleavage types of reactions involve generation of a resonance-stabilized reactions. (a) Aldol condensation, (b) Claisen ester carbanion followed by addition of this carbanion to an condensation, and (c) decarboxylation of a -keto acid. All three electrophilic center. JWCL281_c16_557-592.qxd 6/10/10 11:51 AM Page 569

Section 16-3. Experimental Approaches to the Study of Metabolism 569

(a) O O complex network of regulatory processes renders meta- CCH CCH bolic pathways remarkably sensitive to the needs of the organism; the output of a pathway is generally only as great Carbanion Enolate as required. As you might well imagine, the elucidation of a meta- (b) H B H B HB bolic pathway on all of these levels is a complex process, in- O O O volving contributions from a variety of disciplines. Most of the techniques used to do so involve somehow perturbing C CH CCHor CCH the system and observing the perturbation’s effect on growth or on the production of metabolic intermediates. Hydrogen-bonded Hydrogen-bonded carbonyl enolate or enol One such technique is the use of metabolic inhibitors that block metabolic pathways at specific enzymatic steps. Another is the study of genetic abnormalities that interrupt (c) specific metabolic pathways. Techniques have also been de- NH NH veloped for the dissection of organisms into their compo- CCHCH C CH nent organs, tissues, cells, and subcellular organelles, and for the purification and identification of metabolites as well as Schiff base Schiff base the enzymes that catalyze their interconversions.The use of carbanion (imine) (enamine) isotopic tracers to follow the paths of specific atoms and molecules through the metabolic maze has become routine. (d) Zn2 Zn2 Techniques utilizing NMR technology are able to trace metabolites noninvasively as they react in vivo. This section O O outlines the use of these various techniques. C CH CCH

2؉ Carbanion Zn –stabilized A. Metabolic Inhibitors, Growth Studies, and enolate Biochemical Genetics Figure 16-12 Stabilization of carbanions. (a) Carbanions adjacent to carbonyl groups are stabilized by the formation of a. Pathway Intermediates Accumulate in the enolates. (b) Carbanions adjacent to carbonyl groups hydrogen Presence of Metabolic Inhibitors bonded to general acids are stabilized electrostatically or by The first metabolic pathway to be completely traced was charge neutralization. (c) Carbanions adjacent to protonated the conversion of glucose to ethanol in yeast by a process imines (Schiff bases) are stabilized by the formation of enamines. known as glycolysis (Section 17-1A). In the course of these (d) Metal ions stabilize carbanions adjacent to carbonyl groups studies, certain substances, called metabolic inhibitors, were by the electrostatic stabilization of the enolate. found to block the pathway at specific points, thereby causing preceding intermediates to build up. For instance, iodoacetate causes yeast extracts to accumulate fructose- 1,6-bisphosphate, whereas fluoride causes the buildup of to a metal ion (metal ion catalysis; Fig. 16-12d). The decar- two phosphate esters, 3-phosphoglycerate and 2-phospho- boxylation of a -keto acid does not require base catalysis glycerate. The isolation and characterization of these inter- for the generation of the resonance-stabilized carbanion; mediates was vital to the elucidation of the glycolytic pathway: Chemical intuition combined with this information led the highly exergonic formation of CO2 provides its driving force (Fig. 16-11c). to the prediction of the pathway’s intervening steps. Each of the proposed reactions was eventually shown to occur in vitro as catalyzed by a purified enzyme. 3 EXPERIMENTAL APPROACHES TO THE STUDY OF METABOLISM b. Genetic Defects Also Cause Metabolic Intermediates to Accumulate A metabolic pathway can be understood at several levels: Archibald Garrod’s realization, in the early 1900s, that 1. In terms of the sequence of reactions by which a spe- human genetic diseases are the consequence of deficien- cific nutrient is converted to end products, and the energet- cies in specific enzymes (Section 1-4Cd) also contributed to ics of these conversions. the elucidation of metabolic pathways. For example, on the ingestion of either phenylalanine or tyrosine, individuals with 2. In terms of the mechanisms by which each intermedi- the largely harmless inherited condition known as alcap- ate is converted to its successor. Such an analysis requires tonuria, but not normal subjects, excrete homogentisic acid the isolation and characterization of the specific enzymes in their urine (Section 26-3Hd). This is because the liver of that catalyze each reaction. alcaptonurics lacks an enzyme that catalyzes the breakdown 3. In terms of the control mechanisms that regulate the of homogentisic acid. Another genetic disease, phenylke- flow of metabolites through the pathway. An exquisitely tonuria (Section 26-3Hd), results in the accumulation of JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 570

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phenylpyruvate in the urine (and which, if untreated, causes c. Metabolic Blocks Can Be Generated by severe mental retardation in infants). Ingested phenylala- Genetic Manipulation nine and phenylpyruvate appear as phenylpyruvate in the Early metabolic studies led to the astounding discovery urine of affected subjects, whereas tyrosine is metabolized that the basic metabolic pathways in most organisms are es- normally. The effects of these two abnormalities suggested sentially identical. This metabolic uniformity has greatly fa- the pathway for phenylalanine metabolism diagrammed in cilitated the study of metabolic reactions. A mutation that Fig. 16-13. However, the supposition that phenylpyruvate inactivates or deletes an enzyme in a pathway of interest can but not tyrosine occurs on the normal pathway of pheny- be readily generated in rapidly reproducing microorganisms lalanine metabolism because phenylpyruvate accumulates through the use of mutagens (chemical agents that induce in the urine of phenylketonurics has proved incorrect.This genetic changes; Section 32-1A), X-rays, or genetic engineer- indicates the pitfalls of relying solely on metabolic blocks ing techniques (Section 5-5). Desired mutants are identified and the consequent buildup of intermediates as indicators by their requirement of the pathway’s end product for of a metabolic pathway. In this case, phenylpyruvate for- growth. For example, George Beadle and Edward Tatum mation was later shown to arise from a normally minor proposed a pathway of arginine biosynthesis in the mold pathway that becomes significant only when the pheny- Neurospora crassa based on their analysis of three arginine- lalanine concentration is abnormally high, as it is in requiring auxotrophic mutants (mutants requiring a specific phenylketonurics. nutrient for growth), which were isolated after X-irradiation (Fig. 16-14). This landmark study also conclusively demonstrated that enzymes are specified by genes (Section 1-4Cd).

d. Genetic Manipulations of Higher Organisms H Provide Metabolic Insights – CH2 C COO Transgenic organisms (Section 5-5H) constitute valu- able resources for the study of metabolism. They can be NH+ 3 used to both create metabolic blocks and to express genes in Phenylalanine tissues where they are not normally present. For example, Originally unknown; creatine kinase catalyzes the formation of phosphocreatine defective in (Section 16-4Cd), a substance that functions to generate phenylketonuria ATP rapidly when it is in short supply. This enzyme is normally present in many tissues, including brain and muscle, secondary H but not in liver. The introduction of the gene encoding cre- pathway – atine kinase into the liver of a mouse causes the liver to HO CH C COO 2 synthesize phosphocreatine when the mouse is fed crea- + NH3 tine, as demonstrated by localized in vivo NMR techniques (Fig. 16-15; NMR is discussed below). The presence of Tyrosine

O O – – HO CH2 C COO CH2 C COO nonexistent: originally p-Hydroxyphenylpyruvate thought to Phenylpyruvate exist and be defective in phenylketonurics HO

– CH2 COO

OH Figure 16-13 Pathway for phenylalanine degradation. Homogentisate It was originally hypothesized that phenylpyruvate was a pathway intermediate based on the observation that Defective in phenylketonurics excrete ingested phenylalanine alcaptonuria and phenylpyruvate as phenylpyruvate. Further studies, however, demonstrated that phenylpyruvate is not a homogentisate precursor; rather, phenylpyruvate production is significant only when the phenylalanine concentration is abnormally high. Instead, tyrosine is the normal product of phenylalanine + CO H2O 2 degradation. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 571

Section 16-3. Experimental Approaches to the Study of Metabolism 571

NH2 NH2

CO C NH2 NH3 NH NH CH CH 2 CH2 2 CH CH CH mutant 1 2 mutant 2 2 mutant 3 2 CH CH2 CH2 2 NH H C NH3 H C NH3 H C COO COO COO

Ornithine Citrulline Arginine Figure 16-14 Pathway of arginine biosynthesis indicating the mutant 1, an enzyme leading to the production of ornithine is positions of genetic blocks. All of these mutants grow in the absent but enzymes farther along the pathway are normal. In presence of arginine, but mutant 1 also grows in the presence of mutant 2, the enzyme catalyzing citrulline production is the (nonstandard) -amino acids citrulline or ornithine and defective, whereas in mutant 3 an enzyme involved in the mutant 2 grows in the presence of citrulline. This is because in conversion of citrulline to arginine is lacking.

phosphocreatine in a transgenic mouse liver protects the ulation technique is being used to study mechanisms of animal against the sharp drop in [ATP] ordinarily caused metabolic control in vivo. by fructose overload (Section 17-5Aa).This genetic manip- Metabolic pathways are regulated both by controlling the activities of regulatory enzymes (Sections 17-4 and 18-3) and by controlling their concentrations at the level of gene ex- (a) Control liver ATP α pression (Sections 31-3, 32-4, and 34-3).The important ques- γ tion of how hormones and diet control metabolic processes

Pi at the level of gene expression is being addressed through the use of transgenic animals. Reporter genes (genes whose PME β products are easily detected; Section 5-5Gd) are placed under the influence of promoters (genetic elements that regulate transcriptional initiation; Section 5-4Aa) that control the expression of specific regulatory enzymes, and the resulting composite gene is expressed in animals. The transgenic animals can then be treated with specific hormones PCr and/or diets and the production of the reporter gene prod- (b) Creatine kinase positive liver uct measured. For instance, in an investigation by Richard Hanson, the promoter for the enzyme phosphoenolpyruvate carboxykinase (PEPCK) was attached to the structural gene encoding growth hormone (GH). PEPCK, an important regulatory enzyme in gluconeogenesis (the synthesis of glucose from noncarbohydrate precursors; Section 23-1), is normally present in liver and kidneys but not in blood. GH, however, is secreted into the blood and its presence there can be readily quantitated by an ELISA (Section 6-1Da). Mice transgenic for PEPCK/GH were fed either a high- 15 10 5 0 –5 –10 –15 –20 carbohydrate/low-protein diet or a high-protein/low-carbo- PPM hydrate diet, which are known to decrease and increase PEPCK activity,respectively.GH in high concentrations was Figure 16-15 The expression of creatine kinase in transgenic detected only in the serum of PEPCK/GH mice on a high- 31 mouse liver as demonstrated by localized in vivo P NMR. protein diet, thereby indicating that the GH was synthesized (a) The spectrum of a normal mouse liver after the mouse had under the same dietary control as that of the PEPCK ex- been fed a diet supplemented with 2% creatine. The peaks pressed by the normal gene. Thus, the activity of PEPCK in corresponding to inorganic phosphate (P ), the , , and i PEPCK/GH mice can be continuously monitored, albeit in- phosphoryl groups of ATP,and phosphomonoesters (PME) are labeled. (b) The spectrum of the liver of a mouse transgenic for directly, through serum GH assays (the direct measurement creatine kinase that had been fed a diet supplemented with 2% of PEPCK in mouse liver or kidney requires the sacrifice of creatine. The phosphocreatine peak is labeled PCr. [After the animal and hence can be done only once). Such use of re- Koretsky,A.P.,Brosnan, M.J., Chen, L., Chen, J., and Van Dyke, porter genes has proved to be of great value in the study of T.A., Proc. Natl. Acad. Sci. 87, 3114 (1990)]. the genetic control of metabolism. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 572

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Modern techniques also make it possible to insert a muta- b. NMR Can Be Used to Study Metabolism tion that inactivates or deletes an enzyme or control protein in in Whole Animals a pathway of interest in higher organisms such as mice (knock- Nuclear magnetic resonance (NMR) detects specific out mice; Section 5-5H). Knockout mice have proved useful isotopes due to their characteristic nuclear spins. Among for studying metabolic control mechanisms. For example, the isotopes that NMR can detect are 1H, 13C, and 31P. Since PEPCK activity is thought to be controlled exclusively by in- the NMR spectrum of a particular nucleus varies with its creasing or decreasing its availability. Diet affects its produc- immediate environment, it is possible to identify the peaks tion, as we have seen. However, this demand-based control is corresponding to specific atoms even in relatively complex superimposed on the developmental regulation of PEPCK mixtures. production.The enzyme is not produced at all in early embryos The development of magnets large enough to accom- and only appears near birth, when gluconeogenesis is required modate animals and humans, and to localize spectra to to supply the glucose that had been previously available in specific organs, has made it possible to study metabolic utero. One of the proteins thought to be responsible for the de- pathways noninvasively by NMR techniques. Thus, 31P velopmental regulation of PEPCK production is CCAAT/en- NMR can be used to study energy metabolism in muscle by hancer-binding protein ␣ (C/EBP␣), a transcription factor monitoring the levels of ATP, ADP, inorganic phosphate, (Section 5-4Aa; transcriptional regulation in eukaryotes is dis- and phosphocreatine (Figure 16-15). Indeed, a 31P NMR cussed in Section 34-3B). Newborn mice homozygous for system has been patented to measure the muscular meta- the targeted deletion of the c/ebp gene (c/ebp knockout bolic efficiency and maximum power of race horses while mice) do not produce C/EBP and therefore do not produce they are walking or running on a motor-driven treadmill in PEPCK. Consequently, their livers cannot synthesize the order to identify promising animals and to evaluate the glucose necessary to maintain adequate blood glucose levels efficacy of their training and nutritional programs. once they are disconnected from the maternal circulation. Isotopically labeling specific atoms of metabolites with Indeed, these mice become so hypoglycemic that they die 13C (which is only 1.10% naturally abundant) permits the within 8 hours of birth. Clearly C/EBP has an important metabolic progress of the labeled atoms to be followed by 13C role in the developmental regulation of PEPCK. NMR. Figure 16-16 shows in vivo 13C NMR spectra of a rat liver before and after an injection of D-[1-13C]glucose.The 13C can be seen entering the liver and then being converted to B. Isotopes in Biochemistry glycogen (the storage form of glucose; Chapter 18). 1H NMR The specific labeling of metabolites such that their inter- techniques are being used to determine the in vivo levels of a conversions can be traced is an indispensable technique for variety of metabolites in tissues such as brain and muscle. elucidating metabolic pathways. Franz Knoop formulated this technique in 1904 to study fatty acid oxidation. He fed c. The Detection of Radioactive Isotopes dogs fatty acids chemically labeled with phenyl groups All elements have isotopes. For example, the atomic and isolated the phenyl-substituted end products from mass of naturally occurring Cl is 35.45 D because, at least their urine. From the differences in these products when on Earth, it is a mixture of 55% 35Cl and 45% 36Cl (other the phenyl-substituted starting material contained odd and isotopes of Cl are present in only trace amounts). Stable even numbers of carbon atoms he deduced that fatty acids isotopes are generally identified and quantitated by mass

are degraded in C2 units (Section 25-2). spectrometry or NMR techniques. Many isotopes, however, are unstable; they undergo radioactive decay, a a. Isotopes Specifically Label Molecules without process that involves the emission from the radioactive Altering Their Chemical Properties nuclei of subatomic particles such as helium nuclei (␣ parti- Chemical labeling has the disadvantage that the chemi- cles), electrons (␤ particles), and/or photons (␥ radiation). cal properties of labeled metabolites differ from those of Radioactive nuclei emit radiation with characteristic ener- normal metabolites. This problem is eliminated by labeling gies. For example, 3H, 14C, and 32P all emit particles but molecules of interest with isotopes (atoms with the same with respective energies of 0.018, 0.155, and 1.71 MeV.The number of protons but a different number of neutrons in radiation from 32P is therefore highly penetrating, whereas their nuclei). Recall that the chemical properties of an that from 3H and 14C is not. (3H and 14C, as all radioactive element are a consequence of its electron configuration isotopes, must, nevertheless, be handled with great caution which, in turn, is determined by its atomic number, not its because they can cause genetic damage on ingestion.) atomic mass. The metabolic fate of a specific atom in a Radiation can be detected by a variety of techniques. metabolite can therefore be elucidated by isotopically Those most commonly used in biochemical investigations labeling that position and following its progress through the are proportional counting (known in its simplest form as metabolic pathway of interest. The advent of isotopic label- Geiger counting), liquid scintillation counting, and autora- ing and tracing techniques in the 1940s therefore revolu- diography. Proportional counters electronically detect the tionized the study of metabolism. (Isotope effects, which ionizations in a gas caused by the passage of radiation. are changes in reaction rates arising from the mass differ- Moreover, they can also discriminate between particles of ences between isotopes, are in most instances negligible. different energies and thus simultaneously determine the Where they are significant, most noticeably between hydro- amounts of two or more different isotopes present. gen and its isotopes deuterium and tritium, they have been Although proportional counters are quite simple to use, used to gain insight into enzymatic reaction mechanisms.) the radiation from two of the most widely used isotopes in JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 573

Section 16-3. Experimental Approaches to the Study of Metabolism 573

Glucose and CH2 Figure 16-16 The conversion of (a) glycogen [1-13C]glucose to glycogen as observed by localized in vivo 13C NMR. (a) The natural C6 13 C –C abundance C NMR spectrum of the liver 2 5 Choline of a live rat. Note the resonance CC N(CH3)3 corresponding to C1 of glycogen. (b) The 13C NMR spectrum of the liver of the same C1 Glycogen RCOOR′ rat ϳ5 min after it was intravenously injected with 100 mg of [1-13C]glucose (90% enriched). The resonances of the C1 atom C1–β of both the and anomers of glucose are Glucose clearly distinguishable from each other and (b) C1–α from the resonance of the C1 atom of glycogen. (c) The 13C NMR spectrum of the liver of the same rat ϳ30 min after the [1-13C]glucose injection. The C1 resonances of both the and glucose anomers are much reduced while the C1 resonance of glycogen has increased. [After Reo, N.V., Siegfried, B.A., and Acherman, J.J.H., J. (c) Biol. Chem. 259, 13665 (1984)].

180 120 60 0 ppm

biochemical analysis, 3H and 14C, have insufficient pene- technique, a radioactive sample is dissolved or suspended trating power to enter a proportional counter’s detection in a solution containing fluorescent substances that emit a chamber with reasonable efficiency. This limitation is cir- pulse of light when struck by radiation. The light is de- cumvented through liquid scintillation counting. In this tected electronically so that the number of light pulses can be counted. The emitting nucleus can also be identified because the intensity of a light pulse is proportional to the radiation energy (the number of fluorescent molecules Table 16-2 Some Trace Isotopes of Biochemical Importance excited by a radioactive particle is proportional to the particle’s energy). Stable Isotopes In autoradiography, radiation is detected by its blacken- Nucleus Natural Abundance (%) ing of photographic film. The radioactive sample is laid on, 2H 0.012 or in some cases mixed with, the photographic emulsion and, after sufficient exposure time (from minutes to months), 13C 1.07 the film is developed. Autoradiography is widely used to 15 N 0.36 locate radioactive substances in polyacrylamide gels (e.g., 18 O 0.20 Fig. 6-27). Position-sensitive radiation counters (electronic Radioactive Isotopes film) are similarly employed. Nucleus Radiation Type Half-Life d. Radioactive Isotopes Have Characteristic 3H 12.31 years Half-Lives 14C 5715 years Radioactive decay is a random process whose rate for a 22Na , 2.60 years given isotope depends only on the number of radioactive 32P 14.28 days atoms present. It is therefore a simple first-order process whose half-life, t , is a function only of the rate constant, k, 35S 87.2 days 1/2 for the decay process (Section 14-1Ba): 45Ca 162.7 days 60 Co , 5.271 years ln 2 0.693 t1 2 [14.5] 125I 59.4 days > k k 131 I , 8.02 days Because k is different for each radioactive isotope, each Source: Holden, N.E., in Lide, D.R. (Ed.), Handbook of Chemistry and has a characteristic half-life. The properties of some iso- Physics (90th ed.), pp. 11–57 to 266, CRC Press (2009–2010). topes in common biochemical use are listed in Table 16-2. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 574

574 Chapter 16. Introduction to Metabolism

COO H2C COO CH 15 2 NH3 CH2 CH3 Glycine

15 NH4 CH

N CH OOC CH2 CH2 3 OOCCH2CH2CH COO 15 NH3 N Fe N

Glutamate H3C CH2 CH2 N H 2C CH COO HC CH 15 H2C NH C

H2 CH CH3 Proline CH2

H3C Heme CHCH2CH COO H C 15 3 NH3

Leucine Figure 16-17 The metabolic origin of the nitrogen atoms in heme. Only [15N]glycine, of many 15N-labeled metabolites, is an 15N-labeled heme precursor.

e. Isotopes Are Indispensable for Establishing the question of their biosynthetic relationship: Which is the Metabolic Origins of Complex Metabolites and precursor and which is the product? Two possible modes of Precursor–Product Relationships synthesis can be envisioned (Fig. 16-18): The metabolic origins of complex molecules such as I. The starting material is converted to the vinyl ether heme, cholesterol, and phospholipids may be determined (plasmalogen), which is then reduced to yield the ether by administering isotopically labeled starting materials to (alkylacylglycerophospholipid). Accordingly, the vinyl animals and isolating the resulting products. One of the ether would be the precursor and the ether the product. early advances in metabolic understanding resulting from the use of isotopic tracers was the demonstration, by David Shemin and David Rittenberg in 1945, that the nitrogen atoms of heme are derived from glycine rather than from ammonia, glutamic acid, proline, or leucine (Section 26-4Aa). Starting materials They showed this by feeding rats these 15N-labeled nutri- Scheme I Scheme II ents, isolating the heme in their blood, and analyzing it for 15N content. Only when the rats were fed [15N]glycine did the heme contain 15N (Fig. 16-17). This technique was also CH2 CHO CH R CH2 O CH2 CH2 R Vinyl ether Ether used to demonstrate that all of cholesterol’s carbon atoms R R are derived from acetyl-CoA (Section 25-6A). reduction oxidation Isotopic tracers are also useful in establishing the order

of appearance of metabolic intermediates, their so-called CH2 O CH2 CH2 R CH2 CHO CH R precursor–product relationships. An example of such an Ether Vinyl ether analysis concerns the biosynthesis of the complex phos- R R pholipids called plasmalogens and alkylacylglycerophos- Figure 16-18 Two possible pathways for the biosynthesis of pholipids (Section 25-8Ab). Alkylacylglycerophospholipids ether– and vinyl ether–containing phospholipids. (I) The vinyl are ethers, whereas the closely related plasmalogens are ether is the precursor and the ether is the product. (II) The ether vinyl ethers.Their similar structures brings up the interesting is the precursor and the vinyl ether is the product. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 575

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II. The ether is formed first and then oxidized to yield 3. After [B*] begins to decrease, d[B*] dt 0, so [A*] > the vinyl ether. The ether would then be the precursor and [B*]; that is, after the radioactivity of a product has peaked, the vinyl ether the product. it should remain greater than that of its precursor. Precursor–product relationships can be most easily Such a determination of the precursor–product rela- sorted out through the use of radioactive tracers. A pulse of tionship between alkylacylglycerophospholipid and plas- the labeled starting material is administered to an organ- malogen, using 14C-labeled starting materials, indicated ism and the specific radioactivities of the resulting meta- that the ether is the precursor and the vinyl ether is the bolic products are followed with time (Fig. 16-19): product (Fig. 16-18, Scheme II). Starting material* ¡ A* ¡ B* ¡ later products* (here the * represents the radioactive label). Metabolic C. Isolated Organs, Cells, and Subcellular Organelles pathways, as we shall see in Section 16-6Ba, normally oper- In addition to understanding the chemistry and catalytic ate in a steady state; that is, the throughput of metabolites events that occur at each step of a metabolic pathway, it is in each of its reaction steps is equal. Moreover, the rates of important to learn where a given pathway occurs within an most metabolic reactions are first order for a given sub- organism. Early workers studied metabolism in whole ani- strate. Making these assumptions, we note that the rate of mals. For example, the role of the pancreas in diabetes was change of B’s radioactivity, [B*], is equal to the rate of pas- established by Frederick Banting and Charles Best in 1921 sage of label from A* to B* less the rate of passage of label by surgically removing that organ from dogs and observing from B* to the pathway’s next product: that these animals then developed the disease. d[B*] The metabolic products produced by a particular organ k[A*] k[B*] k([A*] [B*]) [16.1] dt can be studied by organ perfusion or in tissue slices. In organ perfusion, a specific organ is surgically removed from where k is the pseudo-first-order rate constant for both the an animal and the organ’s arteries and veins are connected conversion of A to B and the conversion of B to its prod- to an artificial circulatory system. The composition of the uct, and t is time. Inspection of this equation indicates the material entering the organ can thereby be controlled and criteria that must be met to establish that A is the precur- its metabolic products monitored. Metabolic processes can sor of B (Fig. 16-19): be similarly studied in slices of tissue thin enough to be nourished by free diffusion in an appropriate nutrient solu- 1. Before the radioactivity of the product [B*] is maxi- tion. Otto Warburg pioneered the tissue slice technique in mal, d[B*] dt 0, so [A*] [B*]; that is, while the radioac- > the early twentieth century through his studies of respiration, tivity of a product is rising, it should be less than that of its in which he used a manometer to measure the changes in precursor. gas volume above tissue slices as a consequence of their O2 2. When [B*] is maximal, d[B*] dt 0, so [A*] [B*]; > consumption. that is, when the radioactivity of a product is at its peak, it A given organ or tissue generally contains several cell should be equal to that of its precursor. This result also im- types. Cell sorters are devices that can separate cells ac- plies that the radioactivity of a product peaks after that of its cording to type once they have been treated with the en- precursor. zymes trypsin and collagenase to destroy the intercellular matrix that binds them into a tissue. This technique allows further localization of metabolic function. A single cell type may also be grown in tissue culture for study. Al- [A*] though culturing cells often results in their loss of differen- tiated function, techniques have been developed for main- taining several cell types that still express their original [B*] characteristics. As discussed in Section 16-1, metabolic pathways in eukaryotes are compartmentalized in various subcellular organelles (Table 16-1). For example, oxidative phosphoryla- 1 2 3

Specific radioactivity tion occurs in the mitochondrion, whereas glycolysis and fatty acid biosynthesis occur in the cytosol. Such observa- tions are made by breaking cells open and fractionating Time after addition of labeled starting material their components by differential centrifugation (Section 6-1B), possibly followed by zonal ultracentrifugation through Figure 16-19 The flow of a pulse of radioactivity from precursor to product. At point 1, product radioactivity (B*, purple) is a sucrose density gradient or by equilibrium density gradi- increasing and is less than that of its precursor (A*, orange); at ent ultracentrifugation in a CsCl density gradient, which, re- point 2, product radioactivity is maximal and is equal to that of spectively, separate particles according to their size and its precursor; and at point 3, product radioactivity is decreasing density (Section 6-5B). The cell fractions are then analyzed and is greater than that of its precursor. for biochemical function. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 576

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D. Systems Biology wide range of nutrients. This is consistent with the compli- cated lifestyle of V. cholerae, which can live on its own, in Metabolism has traditionally been studied by hypothesis- association with zooplankton, or in the human gastroin- based research: isolating individual enzymes and metabo- testinal tract (where it causes cholera; Section 19-2Cd). lites and assembling them into metabolic pathways as However, a simple catalog of an organism’s genes does not guided by experimentally testable hypotheses. This is a re- reveal how these genes function. Thus, some genes are ductionist approach: the explanation of the workings of a expressed continuously at high levels, whereas others are system in terms of its component parts. A different, so- expressed rarely, for example, only when the organism called integrative approach, systems biology, has recently encounters a particular metabolite. emerged with the advent of complete genome sequences, Creating an accurate picture of gene expression is the the development of rapid and sensitive techniques for ana- goal of transcriptomics, the study of a cell’s transcriptome lyzing large numbers of gene transcripts, proteins, and (its entire complement of mRNAs). Identifying and quan- metabolites all at once, and the development of new com- tifying all the transcripts from a single cell type reveals putational and mathematical tools. Systems biology is which genes are active. Cells transcribe thousands of genes discovery-based: collecting and integrating enormous at once so this study requires the use of DNA microarray amounts of data in searchable databases so that the prop- technology (Section 7-6B). For example, Fig. 7-39 shows a erties and dynamics of entire biological networks can be DNA microarray that indicates the differences in gene ex- analyzed. As a result, our understanding of the path from pression between yeast grown in the presence and absence genotype to phenotype has expanded. In addition to the of glucose. central dogma of molecular biology (Section 5-4), that a Differences in the expression of particular genes have single gene composed of DNA is transcribed to mRNA, been correlated with many developmental processes or which is translated to a single protein that influences growth patterns. For example, DNA microarrays have been metabolism, we are increasingly taking into account the used to profile the patterns of gene expression in tumor genome, transcriptome, proteome, and metabolome (the cells because different types of tumors express different complete set of a cell’s metabolites) and their interrelation- types and amounts of proteins (Section 34-3B). This infor- ships (Fig. 16-20). The term bibliome (Greek: biblion, mation is useful in choosing how best to treat a cancer. book) has even been coined to denote the systematic incorporation of pre-existing information about reaction mechanisms and metabolic pathways. In the following b. Proteomics paragraphs we discuss some of these emerging technolo- The correlation between the amount of a particular gies and new fields of study. mRNA and the amount of its protein product is imperfect. This is because the various mRNAs and their correspon- a. Transcriptomics ding proteins are synthesized and degraded at different The overall metabolic capabilities of an organism are rates. Furthermore, many proteins are post-translationally encoded by its genome (its entire complement of genes). In modified, sometimes in several different ways (e.g., by principle, it should be possible to reconstruct a cell’s meta- phosphorylation or glycosylation). Consequently, the num- bolic activities from its genomic sequence. However, at ber of unique proteins in a cell exceeds the number of present, this can be done only in a general sense. For exam- unique mRNAs. ple, the 4.0-Mb genome of Vibrio cholerae, the bacterium A more reliable way than transcriptomics to assess gene that causes cholera, contains a large repertoire of genes en- expression is to examine a cell’s proteome, the complete coding transport proteins and enzymes for catabolizing a set of proteins that the cell synthesizes. This proteomics

Genotype Genome DNA

Transcriptome mRNA

Proteome Enzyme

Substrates Metabolome

Phenotype Metabolites

Figure 16-20 The relationship between genotype and directs the synthesis of the proteome, whose various activities are phenotype. The path from genetic information (genotype) to responsible for synthesizing and degrading the components of metabolic function (phenotype) has several steps. Portions of the the metabolome. genome are transcribed to produce the transcriptome, which JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 577

Section 16-3. Experimental Approaches to the Study of Metabolism 577

approach requires that the proteins first be separated, usually contains either 8 hydrogen (light) or 8 deuterium (heavy) by two-dimensional (2D) gel electrophoresis (Section 6-4D). atoms, and biotin, a coenzyme (Section 23-1Ab) that is Individual proteins are then identified by using tandem used as a biotechnology tool because of its extremely tight mass spectrometry to obtain amino acid sequence infor- binding to the protein avidin (K 1015 M; Fig. 16-21a). mation (Section 7-1Ia) and correlating it with protein Avidin is immobilized on a chromatographic resin so that sequence databases. All the proteins that are contained in the ICAT-labeled peptides can be isolated by biotin/avidin a cell or tissue under a given set of conditions can thereby affinity chromatography (Section 6-3C). be catalogued. The ICAT procedure is illustrated in Fig. 16-21b.Two One can compare all the proteins synthesized by a cell protein mixtures representing two different growth condi- under two different sets of conditions by using different tions are treated with light (d0) or heavy (d8) versions of isotopically labeled reagents that are either contained in the ICAT reagent. The labeled protein mixtures are com- the growth medium (e.g., deuterated amino acids) or that bined and digested with trypsin to form Cys-containing la- are reacted with the cell extract. One technique for label- beled peptides, which are then isolated by biotin/avidin ing cellular proteins uses isotope-coded affinity tags affinity chromatography. Individual peptides are separated (ICATs), which are analogous to the differently fluoresc- by liquid chromatography and detected by mass spectrom- ing dyes that are used to label cDNAs. etry (LC/MS). The ratio of the intensities of the light and An ICAT contains three functional elements: an heavy peptide signals indicates the relative peptide abun- iodoacetyl group to react with Cys residues, a linker that dance in the two samples. Tandem mass spectrometry

(a) O

HN NH

X X X X O NH O I O S NH O X X X X Biotin Reactive group Linker Quantify by MS

1409 100 (b) R(d0)-biotin 1417 A Cys 1 Analyze by Digest and affinity intensity Percentage 1405.0 1426.0 2 LC/MS and 4 purify labeled peptides Mass (m/z) 3 MS/MS 5 B Cys Identify by MS/MS 1 100 R(d8)-biotin Two cell states: Reduce and label cysteines with ICAT reagent

Percentage intensity Percentage 0 200 400 600 800 Mass (m/z) Figure 16-21 The isotope-coded affinity tag (ICAT) method affinity chromatography.The purified peptides are analyzed by for quantitative proteome analysis. (a) An example of an ICAT mass spectrometry in two ways: (4) Liquid chromatography reagent that contains an iodoacetyl reactive group, a linker, and a followed by mass spectrometry (LC/MS) is used to quantitate biotin residue. X denotes the position of hydrogen (d0) or the peptides. The ratio of the signal intensities from the deuterium (d8). (b) The ICAT strategy for differential labeling of corresponding light and heavy peptides indicates the relative proteins expressed by cells under two different sets of conditions. peptide abundance in the two mixtures. (5) Tandem mass (1) Proteins from states A and B are respectively treated with spectrometry (MS/MS) is used to determine the amino acid the light (d0) and heavy (d8) forms of the ICAT reagent. (2) The sequence of each peptide and to thereby identify the protein labeled protein mixtures are combined. (3) The labeled proteins from which it is derived by comparing the peptide’s sequence to are digested with trypsin to form Cys-containing labeled those in a database of all known proteins. peptides. These peptides are then purified by biotin/avidin JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 578

578 Chapter 16. Introduction to Metabolism

(MS/MS) is then used to sequence each peptide and deter- A. Phosphoryl-Transfer Reactions mine its identity.This method was used to identify many of Phosphoryl-transfer reactions, the yeast proteins whose mRNA concentrations increased or decreased when glucose was absent from the growth R ¬O¬PO2 R ¬OH Δ R ¬OH R ¬O¬PO2 medium (Fig. 7-39). A hope for the future is that samples 1 3 2 1 2 3 from diseased and normal subjects can be compared in this are of enormous metabolic significance. Some of the most manner to find previously undetected disease markers that important reactions of this type involve the synthesis and would allow early diagnosis of various diseases. hydrolysis of ATP: Δ c. Metabolomics ATP H2O ADP Pi Δ In order to describe a cell’s functional state (its pheno- ATP H2O AMP PPi type) we need, in addition to the cell’s genome, transcrip- where Pi and PPi, respectively, represent orthophosphate tome, and proteome, a quantitative description of all of the (PO3 ) and pyrophosphate (P O4 ) in any of their ioniza- metabolites it contains under a given set of conditions, its 4 2 7 tion states. These highly exergonic reactions are coupled to metabolome. However, a cell or tissue contains thousands numerous endergonic biochemical processes so as to drive of metabolites with greatly varying properties, so that iden- them to completion. Conversely,ATP is regenerated by cou- tifying and quantifying all these substances is a daunting pling its formation to a more highly exergonic metabolic task, requiring many different analytical tools. Conse- process (the thermodynamics of coupled reactions is dis- quently, this huge undertaking is often subdivided. For ex- cussed in Section 3-4C). ample, lipidomics is the subdiscipline of metabolomics To illustrate these concepts, let us consider two exam- aimed at identifying and characterizing all lipids in a cell un- ples of phosphoryl-transfer reactions.The initial step in the der a particular set of conditions, including how these lipids metabolism of glucose is its conversion to glucose-6-phos- influence membrane structure, cell signaling, gene expres- phate (Section 17-2A). Yet the direct reaction of glucose sion, cell–cell interactions, etc., whereas glycomics similarly and P is thermodynamically unfavorable (Fig. 16-23a). In identifies and characterizes all the carbohydrates in a cell. i biological systems, however, this reaction is coupled to the A recently constructed model of the human exergonic hydrolysis of ATP,so the overall reaction is ther- metabolome—based on 1496 protein-encoding genes, 2004 modynamically favorable. ATP can be similarly rege- proteins, 2766 metabolites, and 3311 metabolic and trans- nerated by coupling its synthesis from ADP and P to the port reactions—has been used to simulate 288 known meta- i even more exergonic hydrolysis of phosphoenolpyruvate bolic functions in a variety of cell and tissue types. This in (Fig. 16-23b; Section 17-2J). silico (computerized) model is expected to provide a frame- The bioenergetic utility of phosphoryl-transfer reactions work for future advances in human systems biology. stems from their kinetic stability to hydrolysis combined with their capacity to transmit relatively large amounts of free energy. The G°¿ values of hydrolysis of several phos- 4 THERMODYNAMICS OF phorylated compounds of biochemical importance are tab- PHOSPHATE COMPOUNDS ulated in Table 16-3. The negatives of these values are often referred to as phosphate group-transfer potentials; they The endergonic processes that maintain the living state are driven by the exergonic reactions of nutrient oxidation. This coupling is most often mediated through the syntheses of a Phosphoester NH2 few types of “high-energy” intermediates whose exergonic bond consumption drives endergonic processes. These intermedi- N p N ates therefore form a sort of universal free energy “currency” Phos hoanhydride bonds through which free energy–producing reactions “pay for” the – N O– O– O N free energy–consuming processes in biological systems. Adenosine triphosphate (ATP; Fig. 16-22), which occurs –O P O P O P O CH O γ β α 2 in all known life-forms, is the “high-energy” intermediate that constitutes the most common cellular energy currency. O O O HH H H Its central role in energy metabolism was first recognized in HO OH 1941 by Fritz Lipmann and Herman Kalckar. ATP consists of an adenosine moiety to which three phosphoryl groups Adenosine ¬ 2 (PO3 ) are sequentially linked via a phosphoester bond AMP followed by two phosphoanhydride bonds. Adenosine ADP diphosphate (ADP) and 5؅-adenosine monophosphate (AMP) are similarly constituted but with only two and ATP one phosphoryl units, respectively. Figure 16-22 The structure of ATP indicating its relationship In this section we consider the nature of phosphoryl-trans- to ADP,AMP, and adenosine. The phosphoryl groups, starting with fer reactions, discuss why some of them are so exergonic, and that on AMP,are referred to as the , , and phosphates. Note the outline how the cell consumes and regenerates ATP. difference between phosphoester and phosphoanhydride bonds. JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 579

Section 16-4. Thermodynamics of Phosphate Compounds 579

–1 (a) ΔG° (kJ • mol )

Endergonic g g 8 half-reaction 1 Pi + lucose lucose-6-P + H2O +13.

Exergonic half-reaction 2 ATP + H2O ADP + Pi –30.5

Overall coupled reaction ATP + glucose ADP + glucose-6-P –16.7

–1 (b) ΔG° (kJ • mol ) COO– O Exergonic – half-reaction 1 CH2 C + H2O CH3 C COO + Pi – 61.9 2– OPO3

Phosphoenolpyruvate Pyruvate

Endergonic half-reaction 2 ADP + Pi ATP + H2O +30.5 COO– O Overall – coupled reaction CH2 C + ADP CH3 C COO + ATP –31.4 2– OPO3 Figure 16-23 Some overall coupled reactions involving ATP. has been conceptually decomposed into a direct phosphorylation (a) The phosphorylation of glucose to form glucose-6- step (half-reaction 1) and a step in which ATP is hydrolyzed phosphate and ADP.(b) The phosphorylation of ADP by (half-reaction 2). Both half-reactions proceed in the direction in phosphoenolpyruvate to form ATP and pyruvate. Each reaction which the overall reaction is exergonic (G 0).

are a measure of the tendency of phosphorylated com- spontaneously transfer a phosphoryl group to the hydroly- pounds to transfer their phosphoryl groups to water. Note sis products (ROH form) of the compounds below it. that ATP has an intermediate phosphate group-transfer potential. Under standard conditions, the compounds a. ⌬G of ATP Hydrolysis Varies with pH, Divalent above ATP in Table 16-3 can spontaneously transfer a Metal Ion Concentration, and Ionic Strength phosphoryl group to ADP to form ATP,which can, in turn, The G of a reaction varies with the total concentrations of its reactants and products and thus with their ionic states (Eq. [3.15]). The G’s of hydrolysis of phosphorylated compounds are therefore highly dependent on pH, Table 16-3 Standard Free Energies of Phosphate Hydrolysis divalent metal ion concentration (divalent metal ions such of Some Compounds of Biological Interest as Mg2 have high phosphate-binding affinities), and ionic Compound G°¿ (kJ ؒ mol1) strength. Reasonable estimates of the intracellular values of these quantities as well as of [ATP], [ADP], and [P ] i Phosphoenolpyruvate 61.9 (which are generally on the order of millimolar) indicate 1,3-Bisphosphoglycerate 49.4 that ATP hydrolysis under physiological conditions has S ؉ ؊ 1 1 ATP ( AMP PPi) 45.6 G Ϸ 50 kJ ؒ mol rather than the 30.5 kJ ؒ mol of its Acetyl phosphate 43.1 G°¿. Nevertheless, for the sake of consistency in compar- Phosphocreatine 43.1 ing reactions, we shall usually refer to the latter value. -S ؉ ؊ The above situation for ATP is not unique. It is impor ATP ( ADP Pi) 30.5 tant to keep in mind that within a given cell, the concentra- Glucose-1-phosphate 20.9 tions of most substances vary both with location and time. PPi 19.2 Indeed, the concentrations of many ions, coenzymes, and Fructose-6-phosphate 13.8 metabolites commonly vary by several orders of magnitude Glucose-6-phosphate 13.8 across membranous organelle boundaries. Unfortunately, it Glycerol-3-phosphate 9.2 is usually quite difficult to obtain an accurate measurement of the concentration of any particular chemical species in a Source: Mostly from Jencks, W.P., in Fasman, G.D. (Ed.), Handbook of Biochemistry and Molecular Biology (3rd ed.), Physical and Chemical specific cellular compartment. The G’s for most in vivo Data, Vol. I, pp. 296–304, CRC Press (1976). reactions are therefore little more than estimates. JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 580

580 Chapter 16. Introduction to Metabolism

B. Rationalizing the “Energy” suggest that this factor provides the dominant thermody- in “High-Energy” Compounds namic driving force for the hydrolysis of phosphoanhydrides. Bonds whose hydrolysis proceeds with large negative values A further property of ATP that suits it to its role as an -of G°¿ (customarily more negative than 25 kJ ؒ mol 1) energy intermediate stems from the relative kinetic stabil are often referred to as “high-energy” bonds or “energy- ity of phosphoanhydride bonds to hydrolysis. Most types of rich” bonds and are frequently symbolized by the squiggle anhydrides are rapidly hydrolyzed in aqueous solution. (ϳ).Thus ATP may be represented as AR¬PϳPϳP,where Phosphoanhydride bonds, however, have unusually large A, R, and P symbolize adenyl, ribosyl, and phosphoryl free energies of activation. Consequently, ATP is reason- groups, respectively.Yet, the phosphoester bond joining the ably stable under physiological conditions but is readily adenosyl group of ATP to its -phosphoryl group appears hydrolyzed in enzymatically mediated reactions. to be not greatly different in electronic character from the so-called “high-energy” bonds bridging its and phos- a. Other “High-Energy” Compounds phoryl groups. In fact, none of these bonds have any un- The compounds in Table 16-3 with phosphate group- usual properties, so the term “high-energy” bond is some- transfer potentials significantly greater than that of ATP what of a misnomer. (In any case, it should not be confused have additional destabilizing influences: with the term “bond energy,” which is defined as the energy required to break, not hydrolyze, a covalent bond.) Why 1. Acyl phosphates. The hydrolysis of acyl phosphates then, should the phosphoryl-transfer reactions of ATP be so (mixed phosphoric–carboxylic anhydrides), such as acetyl exergonic? The answer comes from the comparison of the phosphate and 1,3-bisphosphoglycerate, stabilities of the reactants and products of these reactions. O Several different factors appear to be responsible for CH C OPO2 the “high-energy” character of phosphoanhydride bonds 3 3 Acetyl phosphate such as those in ATP (Fig. 16-24):

1. The resonance stabilization of a phosphoanhydride OOH bond is less than that of its hydrolysis products. This is 2O POCH CH C OPO2 because a phosphoanhydride’s two strongly electron- 3 2 3 1,3-Bisphosphoglycerate withdrawing phosphoryl groups must compete for the lone pair of electrons of its bridging oxygen atom, whereas this is driven by the same competing resonance and differential competition is absent in the hydrolysis products. In other solvation influences that function in the hydrolysis of phos- words, the electronic requirements of the phosphoryl phoanhydrides. Apparently these effects are more pro- groups are less satisfied in a phosphoanhydride than in its nounced for acyl phosphates than for phosphoanhydrides. hydrolysis products. 2. Enol phosphates. The high phosphate group-transfer 2. Of perhaps greater importance is the destabilizing potential of an enol phosphate, such as phosphoenolpyru- effect of the electrostatic repulsions between the charged vate (Fig. 16-23b), derives from its enol hydrolysis product groups of a phosphoanhydride in comparison to that of its being less stable than its keto tautomer. Consider the hy- hydrolysis products. In the physiological pH range, ATP drolysis reaction of an enol phosphate as occurring in two has three to four negative charges whose mutual electro- steps (Fig. 16-25). The hydrolysis step is subject to the static repulsions are partially relieved by ATP hydrolysis. driving forces discussed above. It is therefore the highly 3. Another destabilizing influence, which is difficult to as- exergonic enol–keto conversion that provides phospho- sess, is the smaller solvation energy of a phosphoanhydride in enolpyruvate with the added thermodynamic impetus to comparison to that of its hydrolysis products. Some estimates phosphorylate ADP to form ATP. 3. Phosphoguanidines. The high phosphate group-trans- or O O fer potentials of phosphoguanidines, such as phosphocreatine and phosphoarginine, largely result from the compet- OOPPO ing resonances in their guanidino group, which are even O or O more pronounced than they are in the phosphate group of phosphoanhydrides (Fig. 16-26). Consequently, phospho- H O 2 creatine can phosphorylate ADP (see Section 16-4Cd).

O O Compounds such as glucose-6-phosphate or glycerol-3- phosphate, O PPO HH O O 2 – CH2OPO3 O O H O H Figure 16-24 Resonance and electrostatic stabilization in a H CH2OH phosphoanhydride and its hydrolysis products. The competing OH H HO HC resonances (curved arrows from the central O) and HO OH charge–charge repulsions (zigzag line) between the phosphoryl 2 – H OH CH2OPO3 groups of a phosphoanhydride decrease its stability relative to its hydrolysis products. α-D-Glucose-6-phosphate L-Glycerol-3-phosphate JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 581

Section 16-4. Thermodynamics of Phosphate Compounds 581

COO– COO– 2 – 2 – Δ ° –1 Hydrolysis C OϳPO3 + H2O C O H + HPO4 G = –16 kJ • mol C C HH HH

Phosphoenol- pyruvate

– COO– COO

Tautomerization C O H C O ΔG° = –46 kJ • mol–1

C H C H HH H

Pyruvate Pyruvate (enol form) (keto form)

– COO– COO

– – 2 2 Δ ° • –1 Overall reaction C OϳPO3 + H2O C O + HPO4 G = –61.9 kJ mol C H C H HH H Figure 16-25 Hydrolysis of phosphoenolpyruvate. The reaction is broken down into two steps, hydrolysis and tautomerization.

which are below ATP in Table 16-3, have no significantly In general, the highly exergonic phosphoryl-transfer different resonance stabilization or charge separation in reactions of nutrient degradation are coupled to the for-

comparison with their hydrolysis products.Their free ener- mation of ATP from ADP and Pi through the auspices of gies of hydrolysis are therefore much less than those of the various enzymes known as kinases, enzymes that catalyze preceding “high-energy” compounds. the transfer of phosphoryl groups between ATP and other molecules. Consider the two reactions in Fig. 16-23b. If carried out independently, these reactions would not influ- C. The Role of ATP ence each other. In the cell, however, the enzyme pyruvate As Table 16-3 indicates, in the thermodynamic hierarchy of phosphoryl-transfer agents, ATP occupies the middle rank. This enables ATP to serve as an energy conduit between Phosphoenolpyruvate “high-energy” phosphate donors and “low-energy” phos- –60 phate acceptors (Fig. 16-27). Let us examine the general 1,3-Bisphosphoglycerate biochemical scheme of how this occurs. ~P –50 Phosphocreatine

) ~P

–1 ~P –40 “High-energy” + mol • H2N or or O phosphate compounds C ONH P O– –30 ATP – N X O “Low-energy” phosphate R –20 P

of hyd r olysis (kJ compounds – X °′ P R = CH2 CO2 ; = CH3 Δ G Glucose-6-phosphate Phosphocreatine –10

+ Glycerol-3-phosphate NH 3 0 – X R = CH2 CH2 2 CHCH CO2 ; = H Phosphoarginine Figure 16-27 The flow of phosphoryl groups from “high-energy” phosphate donors, via the ATP–ADP Figure 16-26 Competing resonances in phosphoguanidines. system, to “low-energy” phosphate acceptors. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 582

582 Chapter 16. Introduction to Metabolism

2 2 2 O3PCHO 2 O CH2 OH O3PCHO 2 O CH2 O PO3 phosphofructokinase H HO ATP H HO ADP –1 H OH G = –14.2 kJ •mol H OH

HO H HO H

Fructose-6-phosphate Fructose-1,6-bisphosphate

Figure 16-28 The phosphorylation of fructose-6-phosphate by ATP to form fructose-1,6-bisphosphate and ADP.

kinase couples the two reactions by catalyzing the trans- ADP and Pi, thereby causing these processes to be unidi- fer of the phosphoryl group of phosphoenolpyruvate di- rectional (irreversible). rectly to ADP to result in an overall exergonic reaction 4. Additional phosphoanhydride cleavage in highly (Section 17-2J). endergonic reactions. Although many reactions involving ATP yield ADP and P (orthophosphate cleavage), others a. Consumption of ATP i yield AMP and PP (pyrophosphate cleavage). In these lat- In its role as the universal energy currency of living sys- i ter cases, the PPi is rapidly hydrolyzed to 2Pi by inorganic tems, ATP is consumed in a variety of ways: pyrophosphatase (G°¿ 19.2 kJ ؒ mol1) so that the pyrophosphate cleavage of ATP ultimately results in the 1. Early stages of nutrient breakdown. The exergonic hydrolysis of two “high-energy” phosphoanhydride bonds. hydrolysis of ATP to ADP may be enzymatically coupled The attachment of amino acids to tRNA molecules for pro- to an endergonic phosphorylation reaction to form “low- tein synthesis is an example of this phenomenon (Fig. 16-29 energy” phosphate compounds.We have seen one example and Section 32-2C).The two steps of the reaction involving of this in the hexokinase-catalyzed formation of glucose- the amino acid are readily reversible because the free ener- 6-phosphate (Fig. 16-23a). Another example is the phos- gies of hydrolysis of the bonds formed are comparable to phofructokinase-catalyzed phosphorylation of fructose-6- that of ATP hydrolysis. The overall reaction is driven to phosphate to form fructose-1,6-bisphosphate (Fig. 16-28). completion by the hydrolysis of PP , which is essentially ir- Both of these reactions occur in the first stage of glycolysis i reversible. Nucleic acid biosynthesis from the appropriate (Section 17-2). NTPs also releases PPi (Sections 30-1A and 31-2). The free 2. Interconversion of nucleoside triphosphates. Many energy changes of these vital reactions are around zero, so biosynthetic processes, such as the synthesis of proteins the subsequent hydrolysis of PPi is essential to drive the and nucleic acids, require nucleoside triphosphates other synthesis of nucleic acids. than ATP.These include the ribonucleoside triphosphates CTP,GTP,and UTP,which, together with ATP,are utilized, b. Formation of ATP for example, in the biosynthesis of RNA (Section 31-2) and To complete its intermediary metabolic function, ATP the deoxyribonucleoside triphosphate DNA precursors must be replenished. This is accomplished through three dATP, dCTP, dGTP, and dTTP (Section 5-4C). All these types of processes: nucleoside triphosphates (NTPs) are synthesized from ATP and the corresponding nucleoside diphosphate 1. Substrate-level phosphorylation. ATP may be (NDP) in reactions catalyzed by the nonspecific enzyme formed, as is indicated in Fig. 16-23b, from phospho- nucleoside diphosphate kinase: enolpyruvate by direct transfer of a phosphoryl group from a “high-energy” compound to ADP. Such reactions, which Δ ATP NDP ADP NTP are referred to as substrate-level phosphorylations, most The G°¿ values for these reactions are nearly zero, as commonly occur in the early stages of carbohydrate me- might be expected from the structural similarities among tabolism (Section 17-2). the NTPs. These reactions are driven by the depletion 2. Oxidative phosphorylation and photophosphoryla- of the NTPs through their exergonic hydrolysis in the biosyn- tion. Both oxidative metabolism and photosynthesis act to thetic reactions in which they participate (Section 3-4C). generate a proton (H) concentration gradient across a 3. Physiological processes. The hydrolysis of ATP to membrane (Sections 22-3 and 24-2D). Discharge of this gradient is enzymatically coupled to the formation of ATP ADP and Pi energizes many essential endergonic physiological processes such as chaperone-assisted protein fold- from ADP and Pi (the reverse of ATP hydrolysis). In oxida- ing (Section 9-2C), muscle contraction (Section 35-3B), tive metabolism, this process is called oxidative phosphory- and the transport of molecules and ions against concentra- lation, whereas in photosynthesis it is termed photophos- tion gradients (Section 20-3). In general, these processes phorylation. Most of the ATP produced by respiring and result from conformational changes in proteins (enzymes) photosynthesizing organisms is generated in this manner. that occur in response to their binding of ATP.This is fol- 3. Adenylate kinase reaction. The AMP resulting from lowed by the exergonic hydrolysis of ATP and release of pyrophosphate cleavage reactions of ATP is converted to JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 583

Section 16-5. Oxidation–Reduction Reactions 583

tRNA AMP H H O H O O R C C + AMP ϳϳP P R C CϳAMP R C C tRNA – + O + + NH3 NH3 NH3

Amino acid ATP Aminoacyl–adenylate Aminoacyl–tRNA

inorganic ϳ pyrophosphatase P P 2Pi

PPi H2O Figure 16-29 Pyrophosphate cleavage in the synthesis of an displaces the AMP moiety to form an aminoacyl–tRNA. The aminoacyl–tRNA. Here the squiggle (ϳ) represents a “high- exergonic hydrolysis of pyrophosphate (G°¿ 19.2 .energy” bond. In the first reaction step, the amino acid is kJ ؒ mol1) drives the reaction forward adenylylated by ATP.In the second step, a tRNA molecule

ADP in a reaction catalyzed by the enzyme adenylate ADP) are such that it operates close to equilibrium (G Ϸ 0). kinase (Section 17-4Fe): Accordingly, when the cell is in a resting state, so that [ATP] is relatively high, the reaction proceeds with net AMP ATP Δ 2ADP synthesis of phosphocreatine, whereas at times of high The ADP is subsequently converted to ATP through metabolic activity,when [ATP] is low,the equilibrium shifts substrate-level phosphorylation, oxidative phosphorylation, so as to yield net synthesis of ATP. Phosphocreatine thereby or photophosphorylation. acts as an ATP “buffer” in cells that contain creatine kinase. A resting vertebrate skeletal muscle normally has suffi- c. Rate of ATP Turnover cient phosphocreatine to supply its free energy needs for The cellular role of ATP is that of a free energy transmit- several minutes (but for only a few seconds at maximum ter rather than a free energy reservoir. The amount of ATP exertion). In the muscles of some invertebrates, such as in a cell is typically only enough to supply its free energy lobsters, phosphoarginine performs the same function. needs for a minute or two. Hence,ATP is continually being These phosphoguanidines are collectively named phos- hydrolyzed and regenerated. Indeed, 32P-labeling experi- phagens. ments indicate that the metabolic half-life of an ATP molecule varies from seconds to minutes depending on the cell type and its metabolic activity. For instance, brain cells have only a few seconds’ supply of ATP (which, in part, ac- 5 OXIDATION–REDUCTION REACTIONS counts for the rapid deterioration of brain tissue by oxygen Oxidation–reduction reactions, processes involving the deprivation). An average person at rest consumes and re- transfer of electrons, are of immense biochemical signifi- generates ATP at a rate of ϳ3 mol (1.5 kg) ؒ h 1 and as much cance; living things derive most of their free energy from

as an order of magnitude faster during strenuous activity. them. In photosynthesis (Chapter 24), CO2 is reduced (gains electrons) and H2O is oxidized (loses electrons) to d. Phosphocreatine Provides a “High-Energy” yield carbohydrates and O2 in an otherwise endergonic Reservoir for ATP Formation process that is powered by light energy. In aerobic metabo- Muscle and nerve cells, which have a high ATP turnover lism, which is carried out by all eukaryotes and many (a maximally exerting muscle has only a fraction of a prokaryotes, the overall photosynthetic reaction is essen- second’s ATP supply), have a free energy reservoir that tially reversed so as to harvest the free energy of oxidation functions to regenerate ATP rapidly. In vertebrates, phos- of carbohydrates and other organic compounds in the form phocreatine (Fig. 16-26) functions in this capacity. It is of ATP (Chapter 22). Anaerobic metabolism generates synthesized by the reversible phosphorylation of creatine ATP, although in lower yields, through intramolecular by ATP as catalyzed by creatine kinase: oxidation–reductions of various organic molecules, for example, glycolysis (Chapter 17). In certain anaerobic ATP creatine Δ phosphocreatine ADP bacteria,ATP is generated through the use of non-O oxidiz- G°¿ 12.6 kJ ؒ mol 1 2¢ ing agents such as sulfate or nitrate. In this section we out- Note that this reaction is endergonic under standard condi- line the thermodynamics of oxidation–reduction reactions tions. However, the intracellular concentrations of its reac- in order to understand the quantitative aspects of these tants and products (typically 4 mM ATP and 0.013 mM crucial biological processes. JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 584

584 Chapter 16. Introduction to Metabolism

A. The Nernst Equation where w¿, the non-pressure–volume work, is, in this case, w , the electrical work required to transfer the n moles of Oxidation–reduction reactions (also known as redox or ox- el electrons through the electric potential difference ¢e .This, idoreduction reactions) resemble other types of chemical according to the laws of electrostatics, is reactions in that they involve group transfer. For instance, f ¢e hydrolysis transfers a functional group to water. In oxidation– wel n [16.4] reduction reactions, the “groups” transferred are electrons, f which are passed from an electron donor (reductant or re- where , the faraday, is the electrical charge of 1 mol of f ؒ 1 ؒ 1 ؒ 1 ducing agent) to an electron acceptor (oxidant or oxidizing electrons (1 96,485 C mol 96,485 J V mol , agent). For example, in the reaction where C and V are the symbols for coulomb and volt). Thus, substituting Eq. [16.4] into Eq. [16.3], Fe3 Cu Δ Fe2 Cu2 ¢G nf ¢e [16.5] Cu, the reductant, is oxidized to Cu2 while Fe3, the oxidant, is reduced to Fe2. Combining Eqs. [16.2] and [16.5], and making the analo- Redox reactions may be divided into two half-reactions gous substitution for G°, yields the Nernst equation: or redox couples, such as n RT [Ared ][Box ] ¢e ¢e° ln [16.6] 3 2 f a n b Fe e Δ Fe (reduction) n [Aox ][Bred ] Δ 2 Cu Cu e (oxidation) which was originally formulated in 1881 by Walther Nernst. whose sum is the above whole reaction.These half-reactions Here ¢e, the electromotive force (emf) or redox potential, occur during oxidative metabolism in the vital mitochon- may be described as the “electron pressure” that the elec- drial electron transfer mediated by cytochrome c oxidase trochemical cell exerts. The quantity ¢e°, the redox poten- (Section 22-2C5). Note that for electrons to be transferred, tial when all components are in their standard states, is both half-reactions must occur simultaneously. In fact, called the standard redox potential. If these standard states the electrons are the two half-reactions’ common interme- refer to biochemical standard states (Section 3-4Ba), then diate. ¢e° is replaced by ¢e°¿. Note that a positive ¢e in Eq. [16.5] results in a negative G; in other words, a posi- a. Electrochemical Cells tive ¢e is indicative of a spontaneous reaction, one that can A half-reaction consists of an electron donor and its do work. conjugate electron acceptor; in the oxidation half-reaction 2 shown above, Cu is the electron donor and Cu is its con- B. Measurements of Redox Potentials jugate electron acceptor.Together these constitute a conjugate redox pair analogous to the conjugate acid–base pair The free energy change of a redox reaction may be deter- (HA and A) of a Brønsted acid (Section 2-2A).An impor- mined, as Eq. [16.5] indicates, by simply measuring its redox tant difference between redox pairs and acid–base pairs, potential with a voltmeter (Fig. 16-30). Consequently, however, is that the two half-reactions of a redox reaction, voltage measurements are commonly employed to charac- each consisting of a conjugate redox pair, may be physically terize the sequence of reactions comprising a metabolic separated so as to form an electrochemical cell (Fig. 16-30). In such a device, each half-reaction takes place in its separate half-cell, and electrons are passed between half-cells as an electric current in the wire connecting their two elec- trodes. A salt bridge is necessary to complete the electrical circuit by providing a conduit for ions to migrate in the maintenance of electrical neutrality. Voltmeter The free energy of an oxidation–reduction reaction is Pt Pt particularly easy to determine through a simple measurement of the voltage difference between its two half-cells. Salt bridge Consider the general redox reaction: n Δ n Aox Bred Ared Box in which n electrons per mole of reactants are transferred n from reductant (Bred) to oxidant (Aox ). The free energy of e– + Fe3+ Fe2+ Cu+ Cu2+ + e– this reaction is expressed, according to Eq. [3.15], as n Figure 16-30 [Ared ][Box ] Example of an electrochemical cell. The half-cell ¢ ¢ 2 G G° RT ln [16.2] undergoing oxidation (here Cu S Cu e ) passes the a [An ][B ] b ox red liberated electrons through the wire to the half-cell undergoing 3 S 2 Equation [3.12] indicates that, under reversible conditions, reduction (here e Fe Fe ). Electroneutrality in the two half-cells is maintained by the transfer of ions through the ¢ ¿ G w wel [16.3] electrolyte-containing salt bridge. JWCL281_c16_557-592.qxd 6/30/10 10:34 AM Page 585

Section 16-5. Oxidation–Reduction Reactions 585

electron-transport pathway (such as mediates, e.g., oxida- Reduction potentials, like free energies, must be defined tive metabolism; Chapter 22). with respect to some arbitrary standard. By convention, Any redox reaction can be divided into its component standard reduction potentials are defined with respect to half-reactions: the standard hydrogen half-reaction nϩ ϩ Ϫ Δ ϩ ϩ Ϫ Δ Aox ne Ared 2H 2e H2(g) ϩ Ϫ n ϩ Δ ϩ Box ne Bred in which H at pH 0, 25°C, and 1 atm is in equilibrium with where, by convention, both half-reactions are written as re- H2(g) that is in contact with a Pt electrode. This half-cell is e ϭ ductions. These half-reactions can be assigned reduction arbitrarily assigned a standard reduction potential of ° 0 V -e e (1 V ϭ 1 J ؒ CϪ1). For the biochemical convention, we like potentials, A and B,in accordance with the Nernst equation: wise define the standard (pH ϭ 0) hydrogen half-reaction RT [Ared ] as having e¿ ϭ 0 so that the hydrogen half-cell at the bio- e ϭ e° Ϫ ln [16.7a] A A f a nϩ b ϭ e ¿ ϭϪ n [Aox ] chemical standard state (pH 7) has ° 0.421 V (Table 16-4). When ¢e is positive, ⌬G is negative (Eq. RT [Bred ] e ϭ e° Ϫ ln [16.7b] [16.5]), indicating a spontaneous process. In combining B B f a nϩ b n [Box ] two half-reactions under standard conditions, the direction of spontaneity therefore involves the reduction of the For the redox reaction of any two half-reactions: redox couple with the more positive standard reduction

¢e ϭ e Ϫ Ϫ e Ϫ ° °(e acceptor) °(e donor) [16.8] potential. In other words, the more positive the standard reduction potential, the greater the tendency for the redox Thus, when the reaction proceeds with A as the electron ¢e ϭ e Ϫ e couple’s oxidized form to accept electrons and thus become acceptor and B as the electron donor, ° °A °B and reduced. similarly for ¢e .

Table 16-4 Standard Reduction Potentials of Some Biochemically Important Half-Reactions Half-Reaction e°¿ (V) 1 ϩ ϩ ϩ Ϫ Δ 2O2 2H 2e H2O 0.815 Ϫ ϩ ϩ ϩ Ϫ Δ Ϫ ϩ NO3 2H 2e NO2 H2O 0.42 3ϩ ϩ Ϫ Δ 2ϩ Cytochrome a3(Fe ) e cytochrome a3(Fe ) 0.385 ϩ ϩ ϩ Ϫ Δ O2(g) 2H 2e H2O2 0.295 Cytochrome a(Fe3ϩ ) ϩ eϪ Δ cytochrome a(Fe2ϩ ) 0.29 Cytochrome c(Fe3ϩ ) ϩ eϪ Δ cytochrome c(Fe2ϩ ) 0.235 3ϩ ϩ Ϫ Δ 2ϩ Cytochrome c1(Fe ) e cytochrome c1(Fe ) 0.22 Cytochrome b(Fe3ϩ ) ϩ eϪ Δ cytochrome b(Fe2ϩ ) (mitochondrial) 0.077 Ubiquinone ϩ 2Hϩ ϩ 2eϪ Δ ubiquinol 0.045 FumarateϪ ϩ 2Hϩ ϩ 2eϪ Δ succinateϪ 0.031 ϩ ϩ ϩ Ϫ Δ Ϫ FAD 2H 2e FADH2 (in flavoproteins) 0.040 OxaloacetateϪ ϩ 2Hϩ ϩ 2eϪ Δ malateϪ Ϫ0.166 PyruvateϪ ϩ 2Hϩ ϩ 2eϪ Δ lactateϪ Ϫ0.185 Acetaldehyde ϩ 2Hϩ ϩ 2eϪ Δ ethanol Ϫ0.197 ϩ ϩ ϩ Ϫ Δ Ϫ FAD 2H 2e FADH2 (free coenzyme) 0.219 ϩ ϩ ϩ Ϫ Δ Ϫ S 2H 2e H2S 0.23 Lipoic acid ϩ 2Hϩ ϩ 2eϪ Δ dihydrolipoic acid Ϫ0.29 NADϩ ϩ Hϩ ϩ 2eϪ Δ NADH Ϫ0.315 NADPϩ ϩ Hϩ ϩ 2eϪ Δ NADPH Ϫ0.320 Cystine ϩ 2Hϩ ϩ 2eϪ Δ 2 cysteine Ϫ0.340 AcetoacetateϪ ϩ 2Hϩ ϩ 2eϪ Δ ␤-hydroxybutyrateϪ Ϫ0.346 ϩ ϩ Ϫ Δ 1 Ϫ H e 2H2 0.421 2Ϫ ϩ ϩ ϩ Ϫ Δ 2Ϫ ϩ Ϫ SO4 2H 2e SO3 H2O 0.454 Ϫ ϩ ϩ ϩ Ϫ Δ ϩ Ϫ Acetate 3H 2e acetaldehyde H2O 0.581 Source: Mostly from Loach, P.A., in Fasman, G.D. (Ed.), Handbook of Biochemistry and Molecular Biology (3rd ed.), Physical and Chemical Data, Vol. I, pp. 123–130, CRC Press (1976). JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 586

586 Chapter 16. Introduction to Metabolism

a. Biochemical Half-Reactions Are which require electrical energy, are transmitted through Physiologically Significant the discharge of [Na] and [K] gradients that nerve cells The biochemical standard reduction potentials (e°¿) of generate across their cell membranes (Section 20-5B). some biochemically important half-reactions are listed in Quantitation of the free energy contained in a concentra- Table 16-4. The oxidized form of a redox couple with a tion gradient is accomplished by use of the concepts of large positive standard reduction potential has a high affin- electrochemical cells. ity for electrons and is a strong electron acceptor (oxidizing The reduction potential and free energy of a half-cell agent), whereas its conjugate reductant is a weak electron vary with the concentrations of its reactants. An electro- donor (reducing agent). For example, O2 is the strongest chemical cell may therefore be constructed from two oxidizing agent in Table 16-4, whereas H2O, which tightly half-cells that contain the same chemical species but at holds its electrons, is the table’s weakest reducing agent. different concentrations. The overall reaction for such an The converse is true of half-reactions with large negative electrochemical cell may be represented standard reduction potentials. Since electrons sponta- n Δ neously flow from low to high reduction potentials, they Aox (half-cell 1) Ared(half-cell 2) are transferred, under standard conditions, from the re- An (half-cell 2) A (half-cell 1) [16.9] duced products in any half-reaction in Table 16-4 to the ox- ox red idized reactants of any half-reaction above it (although this and, according to the Nernst equation, since ¢e° 0 when may not occur at a measurable rate in the absence of a suit- the same reaction occurs in both cells, able enzyme). Thus, in biological systems, the approximate lower limit for a standard reduction potential is 0.421 V [An (half-cell 2) ][A (half-cell 1) ] ¢e RT ox red e ¿ ln because reductants with a lesser value of ° would reduce f a n b n [Aox (half-cell 1) ] [Ared(half-cell 2) ] protons to H2. However, reducing centers in proteins that are protected from water may have lower potentials. Note Such concentration cells are capable of generating electri- that the Fe3 ions of the various cytochromes tabulated in cal work until they reach equilibrium.This occurs when the Table 16-4 have significantly different redox potentials. concentration ratios in the half-cells become equal (K 1). This indicates that the protein components of redox eq The reaction constitutes a sort of mixing of the two half- enzymes play active roles in electron-transfer reactions by cells; the free energy generated is a reflection of the en- modulating the redox potentials of their bound redox-active tropy of this mixing.The thermodynamics of concentration centers. gradients as they apply to membrane transport is discussed Electron-transfer reactions are of great biological in Section 20-1. importance. For example, in the mitochondrial electron- transport chain (Section 22-2), the primary source of ATP in eukaryotes, electrons are passed from NADH (Fig. 13-2) along a series of electron acceptors of increasing reduction 6 THERMODYNAMICS OF LIFE potential (many of which are listed in Table 16-4) to O2. ATP is generated from ADP and Pi by coupling its synthe- One of the last refuges of vitalism, the doctrine that biolog- sis to this free energy cascade. NADH thereby functions as ical processes are not bound by the physical laws that gov- an energy-rich electron-transfer coenzyme. In fact, the oxi- ern inanimate objects, was the belief that living things can dation of one NADH to NAD supplies sufficient free en- somehow evade the laws of thermodynamics.This view was ergy to generate 2.5 ATPs (Section 22-2Bb). The NAD / partially refuted by elaborate calorimetric measurements NADH redox couple functions as the electron acceptor in on living animals that are entirely consistent with the many exergonic metabolite oxidations. In serving as the energy conservation predictions of the first law of thermo- electron donor in ATP synthesis, it fulfills its cyclic role as a dynamics. However, the experimental verification of the free energy conduit in a manner analogous to ATP. The second law of thermodynamics in living systems is more metabolic roles of redox coenzymes are further discussed difficult. It has not been possible to measure the entropy of

in succeeding chapters. living matter because the heat, qp, of a reaction at a constant T and P is only equal to T S if the reaction is carried out reversibly (Eq. [3.8]). Obviously, the dismantling of a C. Concentration Cells living organism to its component molecules for such a A concentration gradient has a lower entropy (greater or- measurement would invariably result in its irreversible der) than the corresponding uniformly mixed solution and death. Consequently, the present experimentally verified therefore requires the input of free energy for its formation. state of knowledge is that the entropy of living matter is Consequently, discharge of a concentration gradient is an less than that of the products to which it decays. exergonic process that may be harnessed to drive an ender- In this section we consider the special aspects of the gonic reaction. For example, discharge of a proton concen- thermodynamics of living systems. Knowledge of these tration gradient (generated by the reactions of the electron- matters, which is by no means complete, has enhanced our transport chain) across the inner mitochondrial understanding of how metabolic pathways are regulated, membrane drives the enzymatic synthesis of ATP from how cells respond to stimuli, and how organisms grow and

ADP and Pi (Section 22-3). Likewise, nerve impulses, change with time. JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 587

Section 16-6. Thermodynamics of Life 587

A. Living Systems Cannot Be at Equilibrium machine such as a computer. Both require a throughput of free energy to be active. However, the function of the ma- Classical or equilibrium thermodynamics (Chapter 3) chine is based on a static structure, so that the machine can applies largely to reversible processes in closed systems. be repeatedly switched on and off. Life, in contrast, is based The fate of any isolated system, as we discussed in Section on a self-destructing but self-renewing process, which once 3-4A, is that it must inevitably reach equilibrium. For ex- interrupted, cannot be reinitiated. ample, if its reactants are in excess, the forward reaction will proceed faster than the reverse reaction until equilibrium is attained (G 0). In contrast, open systems may B. Nonequilibrium Thermodynamics and remain in a nonequilibrium state as long as they are able to the Steady State acquire free energy from their surroundings in the form of In a nonequilibrium process, something (such as matter, reactants, heat, or work. While classical thermodynamics electrical charge, or heat) must flow, that is, change its spa- provides invaluable information concerning open systems tial distribution. In classical mechanics, the acceleration of by indicating whether a given process can occur sponta- mass occurs in response to force. Similarly, flow in a ther- neously, further thermodynamic analysis of open systems modynamic system occurs in response to a thermodynamic requires the application of the more recently elucidated force (driving force), which results from the system’s principles of nonequilibrium or irreversible thermodynamics. nonequilibrium state. For example, the flow of matter in In contrast to classical thermodynamics, this theory explic- diffusion is motivated by the thermodynamic force of a con- itly takes time into account. centration gradient; the migration of electrical charge (elec- Living organisms are open systems and therefore can tric current) occurs in response to a gradient in an electric never be at equilibrium. As indicated above, they continu- field (a voltage difference); the transport of heat results ously ingest high-enthalpy, low-entropy nutrients, which from a temperature gradient; and a chemical reaction they convert to low-enthalpy, high-entropy waste products. results from a difference in chemical potential. Such flows The free energy resulting from this process is used to do are said to be conjugate to their thermodynamic force. work and to produce the high degree of organization char- A thermodynamic force may also promote a nonconju- acteristic of life. If this process is interrupted, the organism gate flow under the proper conditions. For example, a gra- ultimately reaches equilibrium, which for living things is dient in the concentration of matter can give rise to an elec- synonymous with death. For example, one theory of aging tric current (a concentration cell), heat (such as occurs on holds that senescence results from the random but in- mixing H O and HCl), or a chemical reaction (the mito- evitable accumulation in cells of genetic defects that inter- 2 chondrial production of ATP through the dissipation of a fere with and ultimately disrupt the proper functioning of proton gradient). Similarly, a gradient in electrical poten- living processes. [The theory does not, however, explain tial can motivate a flow of matter (electrophoresis), heat how single-celled organisms or the germ cells of multicellu- (resistive heating), or a chemical reaction (the charging of lar organisms (sperm and ova), which are in effect immor- a battery). When a thermodynamic force stimulates a non- tal, are able to escape this so-called error catastrophe.] conjugate flow, the process is called energy transduction. Living systems must maintain a nonequilibrium state for several reasons: a. Living Things Maintain the Steady State 1. Only a nonequilibrium process can perform useful Living systems are, for the most part, characterized by work. being in a steady state. By this it is meant that all flows in the system are constant, so that the system does not change with 2. The intricate regulatory functions characteristic of time. Some environmental steady-state processes are life require a nonequilibrium state because a process at schematically illustrated in Fig. 16-31. Ilya Prigogine, a equilibrium cannot be controlled (similarly, a ship that is pioneer in the development of irreversible thermodynamics, dead in the water will not respond to its rudder). has shown that a steady-state system produces the maximum 3. The complex cellular and molecular systems that amount of useful work for a given energy expenditure under conduct biological processes can be maintained only in the the prevailing conditions. The steady state of an open system nonequilibrium state. Living systems are inherently unsta- is therefore its state of maximum thermodynamic efficiency. ble because they are degraded by the very biochemical re- Furthermore, in analogy with Le Châtelier’s principle (Sec- actions to which they give rise. Their regeneration, which tion 3-4A), slight perturbations from the steady state give must occur almost simultaneously with their degradation, rise to changes in flows that counteract these perturbations requires the continuous influx of free energy. For example, so as to return the system to the steady state. The steady state the ATP-generating consumption of glucose (Section 17-2), of an open system is therefore analogous to the equilibrium as has been previously mentioned, occurs with the initial state of an isolated system; both are stable states. consumption of ATP through its reactions with glucose to In the following chapters we shall see that many biological form glucose-6-phosphate and with fructose-6-phosphate regulatory mechanisms function to maintain a steady state. to form fructose-1,6-bisphosphate. Consequently, if metab- For example, the flow of reaction intermediates through a olism is suspended long enough to exhaust the available metabolic pathway is often inhibited by an excess of final prod- ATP supply, glucose metabolism cannot be resumed. Life uct and stimulated by an excess of starting material through therefore differs in a fundamental way from a complex the allosteric regulation of its key enzymes (Section 13-4). JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 588

588 Chapter 16. Introduction to Metabolism

(a) (b) Radiant energy Radiant energy from the sun from the sun Heat loss Rain

Heat loss CO2 Photosynthesis + H O Water 2 vapor

River flowing under steady Sea state conditions Breakdown of (gravity) carbohydrates

Figure 16-31 Two examples of open systems in a steady state. similarly maintained by the sun. Plants harness the sun’s radiant

(a) A constant flow of water in the river occurs under the energy to synthesize carbohydrates from CO2 and H2O. The influence of the force of gravity.The water level in the reservoir eventual metabolism of the carbohydrates by the plants or by the is maintained by rain, the major source of which is the animals that eat them results in the release of their stored free

evaporation of seawater. Hence the entire cycle is ultimately energy and the return of the CO2 and H2O to the environment to powered by the sun. (b) The steady state of the biosphere is complete the cycle.

Living things have apparently evolved so as to take maximum that are also possible. As an example, let us consider the re- thermodynamic advantage of their environments. actions of ATP, glucose, and water. Two thermodynamically favorable reactions that ATP can undergo are phosphoryl transfer to form ADP and glucose-6-phosphate, and hydroly- C. Thermodynamics of Metabolic Control sis to form ADP and Pi (Fig. 16-23a).The free energy profiles a. Enzymes Selectively Catalyze Required Reactions of these reactions are diagrammed in Fig. 16-32.ATP hydrol- Biological reactions are highly specific; only reactions that ysis is thermodynamically favored over the phosphoryl lie on metabolic pathways take place at significant rates de- transfer to glucose. However, their relative rates are deter- spite the many other thermodynamically favorable reactions mined by their free energies of activation to their transition

= (ATP•Glucose)uncatalyzed + H2O

= Δ G3 = (ATP•Glucose)enzymatic + H2O

= ΔG 1 = Δ G2 G ATP + H O 2 ΔG , ΔG + glucose 2 3

Δ G1 (ATP•Glucose)enzymatic g ADP + H2O + lucose-6-P

(ADP•Glucose-6-P)enzymatic g ADP + Pi + lucose Reaction coordinate Figure 16-32 Reaction coordinate diagrams. These are (1) the the hydrolysis of ATP is a more exergonic reaction than the reaction of ATP and water (purple curve), and the reaction of phosphorylation of glucose ( G1 is more negative than G2), the ATP and glucose (2) in the presence (orange curve) and (3) in latter reaction is predominant in the presence of a suitable ¢ ‡ ¢ ‡ the absence (yellow curve) of an appropriate enzyme. Although enzyme because it is kinetically favored ( G2 G1 ). JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 589

Chapter Summary 589

‡ ¿ states ( G values; Section 14-1Cb) and the relative concen- has an equilibrium constant of Keq 300 but under physio- trations of glucose and water.The larger G‡, the slower the logical conditions in rat heart muscle has the mass action ra- reaction. In the absence of enzymes, G‡ for the phosphoryl- tio [fructose-1,6-bisphosphate][ADP]/[fructose-6-phos- transfer reaction is greater than that for hydrolysis, so the phate][ATP] 0.03, which corresponds to G 25.7 hydrolysis reaction predominates (although neither reaction kJ ؒ mol1 (Eq. [3.15]). This situation arises from a buildup occurs at a biologically significant rate). of reactants because there is insufficient phosphofructoki- The free energy barriers of both of the nonenzymatic re- nase activity to equilibrate the reaction. Changes in sub- actions are far higher than that of the enzyme-catalyzed strate concentrations therefore have relatively little effect phosphoryl transfer to glucose. Hence enzymatic forma- on the rate of the phosphofructokinase reaction; the en- tion of glucose-6-phosphate is kinetically favored over the zyme is close to saturation. Only changes in the activity of nonenzymatic hydrolysis of ATP. It is the role of an enzyme, the enzyme, through allosteric interactions, for example, can in this case hexokinase, to selectively reduce the free energy significantly alter this rate.An enzyme such as phosphofruc- of activation of a chemically coupled reaction so that it ap- tokinase is therefore analogous to a dam on a river. Sub- proaches equilibrium faster than the more thermodynami- strate flux (rate of flow) is controlled by varying its activity cally favored uncoupled reaction. (allosterically or by other means), much as a dam controls the flow of a river below the dam by varying the opening of b. Many Enzymatic Reactions Are Near Equilibrium its floodgates (when the water levels on the two sides of the Although metabolism as a whole is a nonequilibrium dam are different, that is, when they are not at equilibrium). process, many of its component reactions function close to Understanding of how reactant flux in a metabolic path- equilibrium. The reaction of ATP and creatine to form way is controlled requires knowledge of which reactions phosphocreatine (Section 16-4Cd) is an example of such a are functioning near equilibrium and which are far from it. reaction. The ratio [creatine]/[phosphocreatine] depends Most enzymes in a metabolic pathway operate near equi- on [ATP] because creatine kinase, the enzyme catalyzing librium and therefore have net rates that are sensitive only this reaction, has sufficient activity to equilibrate the reac- to their substrate concentrations. However, as we shall see tion rapidly.The net rate of such an equilibrium reaction is in the following chapters (particularly in Section 17-4), cer- effectively controlled by varying the concentrations of its tain enzymes, which are strategically located in a metabolic reactants and/or products. pathway, operate far from equilibrium. These enzymes, which are targets for metabolic regulation by allosteric inter- c. Pathway Throughput Is Regulated by Controlling actions and other mechanisms, are responsible for the main- Enzymes Operating Far from Equilibrium tenance of a stable steady-state flux of metabolites through Other biological reactions function far from equilibrium. the pathway. This situation, as we have seen, maximizes the For example, the phosphofructokinase reaction (Fig. 16-28) pathway’s thermodynamic efficiency.

CHAPTER SUMMARY

1 Metabolic Pathways Metabolic pathways are series of carbon–carbon bonds. Most of these reactions involve het- consecutive enzymatically catalyzed reactions that produce erolytic bond cleavage or formation occurring through the addi- specific products for use by an organism. The free energy re- tion of nucleophiles to electrophilic carbon atoms. Group-trans- leased by degradation (catabolism) is, through the intermedi- fer reactions therefore involve transfer of an electrophilic group acy of ATP and NADPH, used to drive the endergonic from one nucleophile to another.The main electrophilic groups processes of biosynthesis (anabolism). Carbohydrates, lipids, transferred are acyl groups, phosphoryl groups, and glycosyl and proteins are all converted to the common intermediate groups. The most common nucleophiles are amino, hydroxyl, im-

acetyl-CoA, whose acetyl group is then converted to CO2 and idazole, and sulfhydryl groups. Electrophiles participating in H2O through the action of the citric acid cycle and oxidative metabolic reactions are protons, metal ions, carbonyl carbon phosphorylation.A relatively few metabolites serve as starting atoms, and cationic imines. Oxidation–reduction reactions materials for a host of biosynthetic products. Metabolic path- involve loss or gain of electrons. Oxidation at carbon usually ways have five principal characteristics: (1) Metabolic path- involves C¬H bond cleavage, with the ultimate loss by C of ways are irreversible; (2) if two metabolites are interconvertible, the two bonding electrons through their transfer to an elec- the synthetic route from the first to the second must differ tron acceptor such as NAD. The terminal electron acceptor

from the route from the second to the first; (3) every meta- in aerobes is O2. Elimination reactions are those in which a bolic pathway has an exergonic first committed step; (4) all C“C double bond is created from two saturated carbon cen-

metabolic pathways are regulated, usually at the first commit- ters with the loss of H2O, NH3, ROH, or RNH2. Dehydration ted step; and (5) metabolic pathways in eukaryotes occur in reactions are the most common eliminations. Isomerizations specific subcellular compartments. involve shifts of double bonds within molecules. Rearrange- 2 Organic Reaction Mechanisms Almost all metabolic ments are biochemically uncommon reactions in which in- reactions fall into four categories: (1) group-transfer reactions; tramolecular C¬C bonds are broken and reformed to produce (2) oxidation–reduction reactions; (3) eliminations, isomeriza- new carbon skeletons. Reactions that make and break C¬C tions, and rearrangements; and (4) reactions that make or break bonds form the basis of both degradative and biosynthetic JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 590

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metabolism. In the synthetic direction, these reactions involve dergo pyrophosphate cleavage to yield PPi, whose subsequent addition of a nucleophilic carbanion to an electrophilic carbon hydrolysis adds further thermodynamic impetus to the reaction. atom. The most common electrophilic carbon atom is the car- ATP is present in too short a supply to act as an energy reser- bonyl carbon, whereas carbanions are usually generated by re- voir. This function, in vertebrate nerve and muscle cells, is car- moval of a proton from a carbon atom adjacent to a carbonyl ried out by phosphocreatine, which under low-ATP conditions group or by decarboxylation of a -keto acid. readily transfers its phosphoryl group to ADP to form ATP. 3 Experimental Approaches to the Study of Metabolism 5 Oxidation–Reduction Reactions The half-reactions of Experimental approaches employed in elucidating metabolic redox reactions may be physically separated to form two elec- pathways include the use of metabolic inhibitors, growth stud- trochemical half-cells.The redox potential for the reduction of ies, and biochemical genetics. Metabolic inhibitors block path- A by B, ways at specific enzymatic steps. Identification of the resulting An B Δ A Bn intermediates indicates the course of the pathway. Mutations, ox red red ox which occur naturally in genetic diseases or can be induced by in which n electrons are transferred, is given by the Nernst mutagens, X-rays, or genetic engineering, may also result in the equation absence or inactivity of an enzyme. Modern genetic techniques n RT [Ared ][Box ] make it possible to express foreign genes in higher organisms ¢e ¢e° ln nf a n b (transgenic animals) or inactivate (knock out) a gene and study [Aox ][Bred] the effects of these changes on metabolism. When isotopic la- The redox potential of such a reaction is related to the reduc- e e bels are incorporated into metabolites and allowed to enter a tion potentials of its component half-reactions,A and B , by metabolic system, their paths may be traced from the distribu- ¢e e e tion of label in the intermediates. NMR is a noninvasive tech- A B e e n nique that may be used to detect and study metabolites in vivo. If A B , then Aox has a greater electron affinity than does n Studies on isolated organs, tissue slices, cells, and subcellular or- Box .The reduction potential scale is defined by arbitrarily set- ganelles have contributed enormously to our knowledge of the ting the reduction potential of the standard hydrogen half-cell localization of metabolic pathways. Systems biology endeavors to zero. Redox reactions are of great metabolic importance. to quantitatively describe the properties and dynamics of bio- For example, the oxidation of NADH yields 2.5 ATPs through logical networks as a whole through the integration of genomic, the mediation of the electron-transport chain. transcriptomic, proteomic, and metabolomic information. 6 Thermodynamics of Life Living organisms are open sys- 4 Thermodynamics of Phosphate Compounds Free en- tems and therefore cannot be at equilibrium.They must contin- ergy is supplied to endergonic metabolic processes by the ATP uously dissipate free energy in order to carry out their various produced via exergonic metabolic processes. ATP’s 30.5 kJ ؒ functions and preserve their highly ordered structures. The mol1 G°¿ of hydrolysis is intermediate between those of study of nonequilibrium thermodynamics has indicated that the “high-energy” metabolites such as phosphoenolpyruvate and steady state, which living processes maintain, is the state of max- “low-energy” metabolites such as glucose-6-phosphate. The imum efficiency under the constraints governing open systems. “high-energy” phosphoryl groups are enzymatically trans- Control mechanisms that regulate biological processes preserve ferred to ADP, and the resulting ATP, in a separate reaction, the steady state by regulating the activities of enzymes that are phosphorylates “low-energy” compounds. ATP may also un- strategically located in metabolic pathways.

REFERENCES

Metabolic Studies Freifelder, D., Biophysical Chemistry (2nd ed.), Chapters 5 and 6, Aebersold, R., Quantitative proteome analysis: Methods and ap- Freeman (1982). [A discussion of the principles of radioactive plications, J. Infect. Dis. 182 (supplement 2), S315–S320 (2003). counting and autoradiography.] Beadle, G.W., Biochemical genetics, Chem. Rev. 37, 15–96 (1945). Go,V.L.W.,Nguyen, C.T.H.,Harris, D.M., and Lee, W.-N.P.,Nutrient- [A classic review summarizing the “one gene–one enzyme” gene interaction: Metabolic genotype-phenotype relationship, hypothesis.] J. Nutr. 135, 2016s–3020s (2005). Campbell A.M. and Heyer L.J., Discovering Genomics, Pro- Hevesy,G., Historical sketch of the biological application of tracer teomics and Bioinformatics (2nd ed.), Pearson Benjamin Cum- elements, Cold Spring Harbor Symp. Quant. Biol. 13, 129–150 mings, New York (2007). [An interactive introduction to these (1948). subjects.] Jeffrey, F.M.H., Rajagopal, A., Malloy, C.R., and Sherry, A.D., Cerdan, S. and Seelig, J., NMR studies of metabolism, Annu. Rev. 13C-NMR:A simple yet comprehensive method for analysis of in- Biophys. Biophys. Chem. 19, 43–67 (1990). termediary metabolism, Trends Biochem. Sci. 16, 5–10 (1991). Choi, S. (Ed.), Introduction to Systems Biology, Humana Press Michal, G. (Ed.), Biochemical Pathways. An Atlas of Biochemistry (2007). and Molecular Biology, Wiley (1999). [An encyclopedic com- Cooper, T.G., The Tools of Biochemistry, Chapter 3, Wiley- pendium of metabolic pathways.] Interscience (1977). [A presentation of radiochemical tech- Shemin, D. and Rittenberg, D.,The biological utilization of glycine niques.] for the synthesis of the protoporphyrin of hemoglobin, J. Biol Duarte, N.C., Becker, S.A., Jamshidi, N., Thiele, I., Mo, M.L., Vo, Chem. 166, 621–625 (1946). T.D., Srivas, R., and Palsson, B. Ø., Global reconstruction of the Shulman, R.G. and Rothman, D.L., 13C NMR of intermediary me- human metabolic network based on genomic and bibliomic tabolism: Implications for systematic physiology, Annu. Rev. data, Proc. Natl. Acad. Sci. 104, 1777–1782 (2007). Physiol. 63, 15–48 (2001). JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 591

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Smolin, L.A. and Grosvenor, M.B, Nutrition: Science and Applica- Bioenergetics tions, Wiley (2008). [A good text for those interested in pursu- Alberty, R.A., Standard Gibbs free energy, enthalpy and entropy ing nutritional aspects of metabolism]. changes as a function of pH and pMg for reactions involving Suckling, K.E. and Suckling, C.J., Biological Chemistry, Cam- adenosine phosphates, J. Biol. Chem. 244, 3290–3302 (1969). bridge University Press (1980). [Presents the organic chemistry Alberty, R.A., Calculating apparent equilibrium constants of of biochemical reactions.] enzyme-catalyzed reactions at pH 7, Biochem. Ed. 28, 12–17 Walsh, C., Enzymatic Reaction Mechanisms, Chapter 1, Freeman (2000). (1979). [A discussion of the types of biochemical reactions.] Caplan, S.R., Nonequilibrium thermodynamics and its application Wang, N.-D., Finegold, M.J., Bradley, A., Ou, C.N., Abdelsayed, to bioenergetics, Curr. Top. Bioenerg. 4, 1–79 (1971). S.V., Wilde, M.D., Taylor, L.R., Wilson, D.R., and Darlington, Crabtree, B. and Taylor, D.J.,Thermodynamics and metabolism, in G.J., Impaired energy homeostasis in C/EBP knockout mice, Jones, M.N. (Ed.), Biochemical Thermodynamics, pp. 333–378, Science 269, 1108–1112 (1995). Elsevier (1979). Weckwerth, W. (Ed.), Metabolomics. Methods and Protocols, Dickerson, R.E., Molecular Thermodynamics, Chapter 7, Humana Press (2007). Benjamin (1969). [An interesting chapter on the thermody- Westheimer, F.H., Why nature chose phosphates, Science 235, namics of life.] 1173–1178 (1987). Henley, H.J.M., An introduction to nonequilibrium thermody- Xia, Y.,Yu, H., Jansen, R., Seringhaus, M., Baxter, S., Greenbaum, namics, J. Chem. Ed. 41, 647–655 (1964). D., Zhao, H., and Gerstein, M.,Analyzing cellular biochemistry Katchelsky,A. and Curran, P.F., Nonequilibrium Thermodynamics in terms of molecular networks, Annu. Rev. Biochem. 73, in Biophysics, Harvard University Press (1965). 1051–1087 (2004). Morowitz, H.J., Foundations of Bioenergetics, Academic Press Zhu, H., Bilgin, M., and Snyder, M., Proteomics, Annu. Rev. (1978). Biochem. 72, 783–812 (2003).

PROBLEMS

1. Glycolysis (glucose breakdown) has the overall stoichiome- In a nucleophilic substitution reaction, would two cycles of try: pseudorotation, so as to place the leaving group (X) in an apical position and the attacking group (Y) in an equatorial position, Glucose 2ADP 2P 2NAD ¡ i lead to retention or inversion of configuration on the departure of 2 pyruvate 2ATP 2NADH 4H 2H2O the leaving group? whereas that of gluconeogenesis (glucose synthesis) is 3. One Curie (Ci) of radioactivity is defined as 3.70 1010 dis- ¡ 2 pyruvate 6ATP 2NADH 4H 6H2O integrations per second, the number that occurs in 1 g of pure ؒ 14 226 Ra. A sample of CO2 has a specific radioactivity of 5 Ci glucose 6ADP 6Pi 2NAD mol1. What percentage of its C atoms are 14C? What is the overall stoichiometry of the glycolytic breakdown of 4. In the hydrolysis of ATP to ADP and P , the equilibrium 1 mol of glucose followed by its gluconeogenic synthesis? Explain i concentration of ATP is too small to be measured accurately. A why it is necessary that the pathways of these two processes be in- better way of determining K¿ , and hence G°¿ of this reaction, is dependently controlled and why they must differ by at least one eq ¿ reaction. to break it up into two steps whose values of G° can be accurately determined. This has been done using the following pair of 2. It has been postulated that a trigonal bipyramidal penta- reactions (the first being catalyzed by glutamine synthetase): covalent phosphorus intermediate can undergo a vibrational de- Δ formation process known as pseudorotation in which its apical (1) ATP glutamate NH4 ADP Pi glutamine H ligands exchange with two of its equatorial ligands via a tetragonal G°¿ 16.3 kJ ؒ mol1¢ pyramidal transition state: 1 Δ X O3 (2) Glutamate NH4 glutamine H2O H O2 X G°¿ 14.2 kJ ؒ mol1¢ O1 P O1 P 2 O3 Y Y O2 What is the G°¿ of ATP hydrolysis according to these data?

Trigonal bipyramid Trigonal bipyramid *5. Consider the reaction catalyzed by hexokinase: [X and Y apical] [O2 and O3 apical] ATP glucose Δ ADP glucose-6-phosphate A mixture containing 40 mM ATP and 20 mM glucose was incu- bated with hexokinase at pH 7 and 25°C. Calculate the equilibrium X O3 concentrations of the reactants and products (see Table 16-3). O2 X O1 P O1 P 6. In aerobic metabolism, glucose is completely oxidized in O3 Y the reaction Y O2 Δ Glucose 6 O2 6CO2 6H2O Tetragonal pyramidal with the coupled generation of 32 ATP molecules from 32 ADP transition state and 32 Pi.Assuming the G for the hydrolysis of ATP to ADP and JWCL281_c16_557-592.qxd 7/20/10 5:42 PM Page 592

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Ϫ ؒ Ϫ1 Ϫ Pi under intracellular conditions is 50 kJ mol and that for the when the initial concentrations of acetoacetate and NADH are Ϫ1 combustion of glucose is Ϫ2823.2 kJ ؒ mol , what is the efficiency 0.01 and 0.005M, respectively, and ␤-hydroxybutyrateϪ and NADϩ of the glucose oxidation reaction in terms of the free energy se- are initially absent. Assume the reaction takes place at 25°C and questered in the form of ATP? pH 7.

7. Typical intracellular concentrations of ATP,ADP, and Pi in 11. In anaerobic bacteria, the final metabolic electron accep- muscles are 5.0, 0.5, and 1.0 mM, respectively. At 25°C and pH 7: tor is some molecule other than O2. A major requirement for any (a) What is the free energy of hydrolysis of ATP at these concen- redox pair utilized as a metabolic free energy source is that it pro- trations? (b) Calculate the equilibrium concentration ratio of vides sufficient free energy to generate ATP from ADP and Pi.In- phosphocreatine to creatine in the creatine kinase reaction: dicate which of the following redox pairs are sufficiently exer- Creatine ϩ ATP Δ phosphocreatine ϩ ADP gonic to enable a properly equipped bacterium to utilize them as a major energy source. Assume that redox reactions forming ATP if ATP and ADP have the above concentrations. (c) What concen- require two electrons and that ¢e ϭ ¢e°¿ . tration ratio of ATP to ADP would be required under the forego- ϩ Ϫ ϩ ing conditions to yield an equilibrium concentration ratio of phos- (a) Ethanol NO3 (c) H2 S (b) FumarateϪ ϩ SO2Ϫ (d) Acetaldehyde ϩ acetaldehyde phocreatine to creatine of 1? Assuming the concentration of Pi 3 remained 1.0 mM, what would the free energy of hydrolysis of 12. Calculate ⌬G°¿ for the following pairs of half-reactions at ATP be under these latter conditions? pH 7 and 25°C.Write a balanced equation for the overall reaction *8. Assuming the intracellular concentrations of ATP, ADP, and indicate the direction in which it occurs spontaneously under standard conditions. and Pi, are those given in Problem 7: (a) Calculate the concentration of AMP at pH 7 and 25°C under the condition that the (a) (Hϩ 1H ) and (1 O ϩ 2Hϩ H O) adenylate kinase reaction: >2 2 2 2 > 2 (b) (PyruvateϪ ϩ 2Hϩ/lactateϪ) and (NADϩ ϩ Hϩ/NADH) 2ADP Δ ATP ϩ AMP *13. The chemiosmotic hypothesis (Section 22-3A) postulates is at equilibrium. (b) Calculate the equilibrium concentration of that ATP is generated in the two-electron reaction: AMP when the free energy of hydrolysis of ATP to ADP and Pi is ϩ ϩ ϩ Δ Ϫ1 ADP Pi 2H (low pH) Ϫ55 kJ ؒ mol . Assume [P ] and ([ATP] ϩ [ADP]) remain ϩ i ATP ϩ H O ϩ 2H (high pH) constant. 2 9. Using the data in Table 16-4, list the following substances in which is driven by a metabolically generated pH gradient in the order of their decreasing oxidizing power: (a) fumarateϪ, (b) cys- mitochondria. What is the magnitude of the pH gradient required ϩ 3ϩ tine, (c) O2, (d) NADP , (e) cytochrome c (Fe ), and (f) lipoic for net synthesis of ATP at 25°C and pH 7, if the steady-state acid. concentrations of ATP, ADP, and Pi are 0.01, 10, and 10 mM, respectively? 10. Calculate the equilibrium concentrations of reactants and products for the reaction: 14. Gastric juice is 0.15M HCl.The blood plasma, which is the source of this Hϩ and ClϪ, is 0.10M in ClϪ and has a pH of 7.4. Cal- AcetoacetateϪ ϩ NADH ϩ Hϩ Δ culate the free energy necessary to produce the HCl in 0.1 L of ␤-hydroxybutyrateϪ ϩ NADϩ gastric juice at 37°C.