Chapter 14

Metabolic functions in the cytosol

-glycolysis -pentose phosphate pathway -fatty acid biosynthesis -many reactions of gluconeogenesis

Which of the mechanisms for flux control are short term and which are long term?

1. Allosteric control 2. Covalent modification 3. Substrate cycles 4. Genetic control Mechanisms 1 to 3 can respond rapidly (within seconds or minutes) to external stimuli and are therefore classified as "short-term" control mechanisms. Mechanism 4 responds more slowly to changing conditions (within hours or days in higher organisms) and is therefore regarded as a "long-term" control mechanism.

What are 4 mechanisms that cells use to control flux through the rate-determining steps of metabolic pathways?

1. Allosteric control 2. Covalent modification 3. Substrate cycles 4. Genetic control

Three levels of a metabolic pathway

1. In terms of the sequence of reactions by which a specific nutrient is converted to end products, and the energetics of the conversions. 2. In terms of the mechanisms by which each intermediate is converted to its successor. Such an analysis requires the isolation and characterization of the specific enzymes that catalyze each reaction. 3. In terms of the control mechanisms that regulate the flow of metabolites through the pathway. These mechanisms include the interorgan relationships that adjust metabolic activity to the needs of the entire organism.

Most enzymes in a metabolic pathway operate near equilibrium and therefore have net rates that vary with their substrate concentrations. However, certain enzymes that operate far from equilibrium are strategically located in metabolic pathways. This has several important implications:

1. Metabolic pathways are irreversible. A highly exergonic reaction (one with ΔG«0) is irreversible; that is, it goes to completion. If such a reaction is part of a multistep pathway, it confers directionality on the pathway; that is, it makes the entire pathway irreversible. 2. Every metabolic pathway has a first committed step. Although most reactions in a metabolic pathway function close to equilibrium, there is generally an irreversible (exergonic) reaction early in the pathway that "commits" its product to continue down the pathway (likewise, water that has gone over a dam cannot spontaneously return). 3. Catabolic and anabolic pathways differ. If a metabolite is converted to another metabolite by an exergonic process, free energy must be supplied to convert the second metabolite back to the first. This energetically "up-hill" process requires a different pathway for at least one of the reaction steps. The existence of independent interconversion routes is an important property of metabolic pathways because it allows independent control of the two processes. If metabolite 2 is required by the cell, it is necessary to "turn off the pathway from 2 to 1 while "turning on" the pathway from 1 to 2. Such independent control would be impossible without different pathways.

Does a favorable free energy change for a reaction indicate how quickly the reaction occurs?

A favorable free energy change for a reaction does not indicate how quickly the reaction occurs. Despite their high group-transfer potentials, ATP and related phosphoryl compounds are kinetically stable and do not react at a significant rate unless acted upon by an appropriate enzyme.

A striking characteristic of degradative metabolism is that the pathways for the catabolism of a large number of diverse substances (carbohydrates, lipids, and proteins) converge on a few common intermediates, in many cases, a two-carbon acetyl unit linked to ______________ to form _________________.

A striking characteristic of degradative metabolism is that the pathways for the catabolism of a large number of diverse substances (carbohydrates, lipids, and proteins) converge on a few common intermediates, in many cases, a two-carbon acetyl unit linked to coenzyme A to form acetyl-coenzyme A (acetyl-CoA). These intermediates are then further metabolized in a central oxidative pathway.

Roles of ATP and NADP+ in metabolism

ATP and NADPH, generated through the degradation of complex metabolites such as carbohydrates, lipids, and proteins, are sources of free energy for biosynthetic and other reactions. In degradative pathways, the major nutrients, referred to as complex metabolites, are exergonically broken down into simpler products. The free energy released in the degradative process is conserved by the synthesis of ATP from ADP + Pi or by the reduction of a coenzyme such as NADP+ (Fig. 11-4) to NADPH. ATP and NADPH are the major free energy sources for biosynthetic reactions.

Is Acetyl-CoA a "high-energy" or "low-energy" compound? Explain.

Acetyl-CoA is a "high-energy" compound. The ΔG°' for the hydrolysis of its thioester bond is -31.5 kJ • mol-1 which makes this reaction slightly (1 kJ • mol -I) more exergonic than ATP hydrolysis. The hydrolysis of thioesters is more exergonic than that of ordinary esters because the thioester is less stabilized by resonance. This destabilization is a result of the large atomic radius of S, which reduces the electronic overlap between C and S compared to that between C and O. The formation of a thioester bond in a metabolic intermediate conserves a portion of the free energy of oxidation of a metabolic fuel. That free energy can then be used to drive an exergonic process. In the citric acid cycle, for example, cleavage of a thioester (succinyl-CoA) releases sufficient free energy to synthesize GTP from GDP and Pi (Section 17-3E).

Explain how inorganic pyrophosphatase catalyzes additional phosphoanhydride bond cleavage

Although many reactions involving ATP yield ADP and Pi (orthophosphate cleavage), others yield AMP and PPi (pyrophosphate cleavage). In these latter cases, the PPi is rapidly hydrolyzed to 2 Pi by inorganic pyrophosphatase (ΔG°' = -19.2 kJ • mol-1) so that the pyrophosphate cleavage of ATP ultimately consumes two "high-energy" phosphoanhydride bonds. The attachment of amino acids to tRNA molecules for protein synthesis is an example of this phenomenon. The two steps of the reaction are readily reversible because the free energies of hydrolysis of the bonds formed are comparable to that of ATP hydrolysis. The overall reaction is driven to completion by the irreversible hydrolysis of PPi. Nucleic acid biosynthesis from nucleoside triphosphates also releases PPi. The standard free energy changes of these reactions are around 0, so the subsequent hydrolysis of PPi is also essential for the synthesis of nucleic acids.

Isozymes and their relation to specialization of tissues and subcellular compartments

An intriguing manifestation of specialization of tissues and subcellular compartments is the existence of isozymes, enzymes that catalyze the same reaction but are encoded by different genes and have different kinetic or regulatory properties. For example, we have seen that mammals have three isozymes of glycogen phosphorylase, those expressed in muscle, brain, and liver (Section 12-3B). Similarly, vertebrates possess two homologs of the enzyme lactate dehydrogenase: the M type, which predominates in tissues subject to anaerobic conditions such as skeletal muscle and liver, and the H type, which predominates in aerobic tissues such as heart muscle. Lactate dehydrogenase catalyzes the interconversion of pyruvate, a product of glycolysis, and lactate (Section 15-3A). The M-type isozyme appears mainly to function in the reduction by NADH of pyruvate to lactate, whereas the H-type enzyme appears to be better adapted to catalyze the reverse reaction. The existence of isozymes allows for the testing of various illnesses. For example, heart attacks cause the death of heart muscle cells, which consequently rupture and release H-type LDH into the blood. A blood test indicating the presence of H-type LDH is therefore diagnostic of a heart attack.

Anabolism

Anabolism, or biosynthesis, in which biomolecules are synthesized from simpler components.

Are animals obligate aerobes or facultative anaerobes? Explain

Animals are obligate aerobic heterotrophs, whose nutrition depends on a balanced intake of the macronutrients proteins, carbohydrates, and lipids. These are broken down by the digestive system to their component amino acids, monosaccharides, fatty acids, and glycerol- the major nutrients involved in cellular metabolism- which are then transported by the circulatory system to the tissues. The metabolic utilization of the latter substances also requires the intake of O2 and water, as well as micronutrients composed of vitamins and minerals.

What is the affect of genetic defects on metabolic intermediates?

Archibald Garrod's realization, in the early 1900s, that human genetic diseases are the consequence of deficiencies in specific enzymes also contributed to the elucidation of metabolic pathways. For example, upon the ingestion of either phenylalanine or tyrosine, individuals with the largely harmless inherited condition known as alcaptonuria, but not normal subjects, excrete homogentisic acid in their urine Box 21-2). This is because the liver of alcaptonurics lacks an enzyme that catalyzes the breakdown of homogentisic acid (Fig. 14-17).

What is the nature of the "energy" in "high-energy" compounds?

Bonds whose hydrolysis proceeds with large negative values of ΔG°' (customarily less than -25 kJ • mol-1) are often referred to as "high-energy" bonds or "energy-rich" bonds and are frequently symbolized by the squiggle (~). Thus, ATP can be represented as AR—P~P~P, where A, R, and P symbolize adenyl, ribosyl, and phosphoryl groups, respectively. Yet the phosphoester bond joining the adenosyl group of ATP to its α-phosphoryl group appears to be not greatly different in electronic character from the "high-energy" bonds bridging its α- and β- and its β- and γ-phosphoryl groups. In fact, none of these bonds has any unusual properties, so the term "high-energy" bond is somewhat of a misnomer (in any case, it should not be confused with the term "bond energy," which is defined as the energy required to break, not hydrolyze, a covalent bond).

Autotrophs

Can synthesize all their cellular constituents from simple molecules such as H2O, CO2, NH3, and H2S. There are two possible free energy sources for this process.

Catabolism

Catabolism, or degradation, in which nutrients and cell constituents are broken down to salvage their components and/or to make energy available.

How are labeled metabolites traced?

Chemical labeling has the disadvantage that the chemical properties of labeled metabolites differ from those of normal metabolites. This problem is largely eliminated by labeling molecules with isotopes. The fate of an isotopically labeled atom in a metabolite can therefore be elucidated by following its progress through the metabolic pathway of interest. The advent of isotopic labeling and tracing techniques in the 1940s revolutionized the study of metabolism. Some of the most common radioactive isotopes (radionuclides) used in biochemistry are listed in Table 14-6, along with their half-lives and the type of radioactivity emitted by the spontaneously disintegrating atomic nuclei. Radioactive compounds can be detected by their ability to expose photographic film. Alternatively, β particles and γ rays can excite fluorescent compounds, and the emitted light can be measured.

Metabolic functions in the mitochondrion

Citric acid cycle, electron transport and oxidative phosphorylation, fatty acid oxidation, amino acid breakdown

What are the compounds whose phosphoryl group-transfer potentials are less than ATP?

Compounds such as glucose-6-phosphate and glycerol-3-phosphate, which are below ATP in Table 14-3, have no significantly different resonance stabilization or charge separation compared to their hydrolysis products. Their free energies of hydrolysis are therefore much less than those of the preceding "high-energy" compounds.

Supply-demand process of metabolic pathways

Control of most metabolic pathways involves several nonequilibrium steps. Hence, the flux of material through a pathway that supplies intermediates for use by an organism may depend on multiple effectors whose relative importance reflects the overall metabolic demands of the organism at a given time. Thus, a metabolic pathway is part of a supply-demand process.

Transcriptomics

Creating an accurate picture of gene expression is the goal of transcriptomics, the study of a cell's transcriptome. Identifying and quantifying all the transcripts from a single cell type reveals which genes are active. Cells transcribe thousands of genes at once, so this study requires the use of new techniques, including DNA microarray technology.

DNA microarrays or DNA chips

DNA microarrays or DNA chips are made by depositing numerous (up to several hundred thousand) different DNA segments of known gene sequences in a precise array on a solid support such as a coated glass surface. These DNAs are often PCR-amplified cDNA clones derived from mRNAs (PCR is discussed in Section 3-5C) or their robotically synthesized counterparts. The mRNAs extracted from cells, tissues, or other biological sources grown under differing conditions are then reverse-transcribed to cDNA, labeled with a fluorescent dye (a different color for each growth condition), and allowed to hybridize with the DNAs on the DNA microarray. After the unhybridized cDNA is washed away, the resulting fluorescence intensity and color at each site on the DNA microarray indicates how much cDNA (and therefore how much mRNA) has bound to a particular complementary DNA sequence for each growth condition. Figure 14-19 shows a DNA chip that indicates the changes in yeast gene expression when yeast grown on glucose have depleted their glucose supply.

Metabolic functions in the lysosome

Enzymatic digestion of cell components and ingested matter

Controlling flux via Genetic control

Enzyme concentrations, and hence enzyme activities, may be altered by protein synthesis in response to metabolic needs. The processes of transcribing a gene to messenger RNA and then translating the RNA to a polypeptide chain offer numerous points for regulation.

How do reduction potential differences determine spontaneity?

Equation 14-7 shows that the free energy change of a redox reaction can be determined by directly measuring its change in reduction potential with a voltmeter (Fig. 14-15). Such measurements make it possible to determine the order of spontaneous electron transfers among a set of electron carriers such as those of the electron-transport pathway that mediates oxidative phosphorylation in cells.

Overview of catabolism

Figure 14-3 outlines the breakdown of various foodstuffs to their monomeric units and then to acetyl-CoA. This is followed by the oxidation of the acetyl carbons to CO2 by the citric acid cycle (Chapter 17). When one substance is oxidized (loses electrons), another must be reduced (gain electrons; Box 14-1). The citric acid cycle thus produces the reduced coenzymes NADH and FADH2 (Section 14-3A), which then pass their electrons to O2 to produce H2O in the processes of electron transport and oxidative phosphorylation. Figure caption: Complex metabolites such as carbohydrates, proteins, and lipids are degraded first to their monomeric units, chiefly glucose, amino acids, fatty acids, and glycerol, and then to the common intermediate, acetyl-CoA. The acetyl group is oxidized to CO2 via the citric acid cycle with concomitant reduction of NAD+ and FAD to NADH and FADH2. Reoxidation of NADH and FADH2 by O2 during electron transport and oxidative phosphorylation yields H2O and ATP.

What sets the flux of a metabolic pathway? (Flux = rate of flow)

For the pathway as a whole, flux is set by the rate-determining step of the pathway. By definition, this step is the pathway's slowest step, which is often the first committed step of the pathway. In some pathways, flux control is distributed over several enzymes, all of which help determine the overall rate of flow of metabolites through the pathway. Because a rate-determining step is slow relative to other steps in the pathway, its product is removed by succeeding steps in the pathway before it can equilibrate with reactant. Thus, the rate-determining step functions far from equilibrium and has a large negative free energy change. In an analogous manner, a dam creates a difference in water levels between its upstream and downstream sides, and a large negative free energy change results from the hydrostatic pressure difference. The dam can release water to generate electricity, varying the water flow according to the need for electrical power.

Explain the connection between vitamin B2 and FAD

Humans cannot synthesize the flavin moiety of FAD but, rather, must obtain it from their diets, for example, in the form of riboflavin (vitamin B2; Fig. 14-13). Nevertheless, riboflavin deficiency is quite rare in humans, in part because of the light binding of flavin prosthetic groups to their apoenzymes. The symptoms of riboflavin deficiency, which are associated with general malnutrition or bizarre diets, include an inflamed tongue, lesions in the corner of the mouth, and dermatitis.

Controlling flux via Substrate cycles

If v(f) and v(r) represent the rates of two opposing nonequilibrium reactions that are catalyzed by different enzymes, v(f) and v(r) may be independently varied. For example, flux (v(f) - v(r)) can be increased not just by accelerating the forward reaction but by slowing the reverse reaction. The flux through such a substrate cycle, is more sensitive to the concentrations of allosteric effectors than is the flux through a single unopposed nonequilibrium reaction.

Metabolomics

In order to describe a cell's functional state (its phenotype) we need, in addition to the cell's genome, transcriptome, and proteome, a quantitative description of all of the metabolites it contains under a given set of conditions, its metabolome. However, a cell or tissue contains thousands of metabolites with vastly different properties, so that identifying and quantifying all these substances is a daunting task, requiring many different analytical tools. Consequently, this huge undertaking is often subdivided. For example, lipidomies is the subsection of metabolomics aimed at characterizing all lipids in a cell under a particular set of conditions, including how these lipids influence membrane structure, cell signaling, gene expression, cell-cell interactions, and so on. For example, a panel of 10 lipid metabolites in blood has been used to predict the development of cognitive impairment in Alzheimer's disease. Another application of metabolomics is the comparison of the different types of cancers. Although all cells contain the same "core" metabolites, their patterns of use yield different profiles that could be exploited to inhibit cancer growth.

What happens to phosphoanhydride bonds in the absence of an appropriate enzyme?

In the absence of an appropriate enzyme, phosphoanhydride bonds are stable; that is, they hydrolyze quite slowly, despite the large amount of free energy released by these reactions. This is because these hydrolysis reactions have unusually high free energies of activation (ΔG**: Section 11-2). Consequently, ATP hydrolysis is thermodynamically favored but kinetically disfavored. For example, consider the reaction of glucose with ATP that yields glucose-6-phosphate (Fig. 14-7a). ΔG** for the nonenzymatic transfer of a phosphoryl group from ATP to glucose is greater than that for ATP hydrolysis, so the hydrolysis reaction predominates (although neither reaction occurs at a biologically significant rate). However, in the presence of the appropriate enzyme, hexokinase (Section 15-2A), glucose-6-phosphate is formed far more rapidly than ATP is hydrolyzed. This is because the catalytic influence of the enzyme reduces the activation energy for phosphoryl group transfer from ATP to glucose to less than the activation energy for ATP hydrolysis. This example underscores the point that a thermodynamically favored reaction (ΔG < 0) may not occur at a significant rate in a living system in the absence of a specific enzyme that catalyzes the reaction (i.e., lowers ΔG** to increase the rate of product formation; Box 12-2).

Where is ATP ranked in the thermodynamic hierarchy of phosphoryl-transfer agents?

Just as ATP drives endergonic reactions through the exergonic process of phosphory1 group transfer and phosphoanhydride hydrolysis, ATP itself can be regenerated by coupling its formation to a more highly exergonic metabolic process. As Table 14-3 indicates, in the thermodynamic hierarchy of phosphoryl-transfer agents, ATP occupies the middle rank. ATP can therefore be formed from ADP by direct transfer of a phosphory1 group from a "high-energy" compound (e.g., phosphoenolpyruvate). Such a reaction is referred to as a substrate-level phosphorylation. Other mechanisms generate ATP indirectly, using the energy supplied by transmembrane proton concentration gradients. In oxidative metabolism, this process is called oxidative phosphorylation, whereas in photosynthesis, it is termed photophosphorylation. The flow of energy from "high-energy" phosphate compounds to ATP and from ATP to "low-energy" phosphate compounds is diagrammed in Fig. 14-9. These reactions are catalyzed by enzymes known as kinases, which transfer phosphoryl groups from ATP to other compounds or from phosphorylated compounds to ADP.

Metabolic functions in the smooth endoplasmic reticulum

Lipid and steroid biosynthesis

Are living systems in equilibrium or a steady state? Explain.

Living organisms are thermodynamically open systems that tend to maintain a steady state rather than reaching equilibrium (Section 1-3E). This is strikingly demonstrated by the observation that, over a 40-year time span, a normal human adult consumes literally tons of nutrients and imbibes more than 20,000 L of water but does so without major weight change. The flux of intermediates through a metabolic pathway in a steady state is more or less constant; that is, the rates of synthesis and breakdown of each pathway intermediate maintain it at a constant concentration. A steady state far from equilibrium is thermodynamically efficient, because only a nonequilibrium process (ΔG =/=0) can perform useful work. Indeed, living systems that have reached equilibrium are dead.

Nucleoside Triphosphates are freely inter converted

Many biosynthetic processes, such as the synthesis of proteins and nuclei acids, require nucleoside triphosphates other than ATP. For example, RNA synthesis requires the ribonucleotides CTP, GTP, and UTP, along with ATP, and DNA synthesis requires dCTP, dGTP, dTTP, and dATP (Section 3-1). All these nucleoside triphosphates (NTPs) are synthesized from ATP and the corresponding nucleoside diphosphate (NDP) in a reaction catalyzed by the nonspecific enzyme nucleoside diphosphate kinase: ATP + NDP <==> ADP + NTP The ΔG°' values for these reactions are nearly 0, as might be expected from the structural similarities among the NTPs. These reactions are driven by the depletion of the TPs through their exergonic utilization in subsequent reactions.

Pellagra

Many coenzymes (Section 11-1C) were discovered as growth factors for microorganisms or as substances that cure nutritional deficiency diseases in humans and/or animals. For example, the NAD+ component nicotinamide, or its carboxylic acid analog nicotinic acid (niacin; Fig. 14-1), relieves the ultimately fatal dietary deficiency disease in humans known as pellagra. Pellagra (Italian: pelle, skin + agra, sour), which is characterized by dermatitis, diarrhea, and dementia, was endemic in the rural southern United States in the early twentieth century. Most animals, including humans, can synthesize nicotinamide from the amino acid tryptophan. However, the corn (maize)-rich diet that was prevalent in the rural South contained little available nicotinamide or tryptophan from which to synthesize it. (Corn actually contains significant quantities of niacin but in a form that requires treatment with base before it can be intestinally absorbed. The Mexican Indians, who domesticated the corn plant but did not suffer from pellagra, customarily soak corn meal in lime water--dilute Ca(OH)2 solution--before using it to make their staple food, tortillas.) Dietary supplementation with nicotinamide or niacin has all but eliminated pellagra in the developed world.

Controlling flux via Allosteric control

Many enzymes are allosterically regulated by effectors that are often substrates, products, or coenzymes of the pathway but not necessarily of the enzyme in question. For example, in negative feedback regulation, the product of a pathway inhibits an earlier step in the pathway: Thus, as we have seen, CTP, a product of pyrimidine biosynthesis, inhibits ATCase, which catalyzes the rate-determining step in the pathway.

Controlling flux via covalent modification

Many enzymes that control pathway fluxes have specific sites that may be enzymatically phosphorylated and dephosphorylated or covalently modified in some other way. Such enzymatic modification processes, which are themselves subject to control by external signals such as hormones, greatly alter the activities of the modified enzymes.

Metabolic pathways

Metabolic pathways are series of connected enzymatic reactions that produce specific products. Their reactants, intermediates, and products are referred to as metabolites. There are around 4000 known metabolic reactions, each catalyzed by a distinct enzyme. The types of enzymes and metabolites in a given cell vary with the identity of the organism, the cell type, its nutritional status, and its developmental stage. Many metabolic pathways are branched and interconnected, so delineating a pathway from a network of thousands of reactions is somewhat arbitrary and is driven by tradition as much as by chemical logic.

Metabolism

Metabolism, the overall process through which living systems acquire and use free energy to carry out their various functions, is traditionally divided into two parts: 1. Catabolism, or degradation, in which nutrients and cell constituents are broken down to salvage their components and/or to make energy available. 2. Anabolism, or biosynthesis, in which biomolecules are synthesized from simpler components.

What is the role of phosphocreatine in ATP Formation?

Muscle and nerve cells, which have a high ATP turnover, rely on phosphoguanidines to regenerate ATP rapidly. In vertebrates, phosphocreatine is synthesized by the reversible phosphorylation of creatine by ATP catalyzed by creatine kinase: ATP + creatine <==> phosphocreatine + ADP ΔG° = +12.6 kJ • mol-1 Note that this reaction is endergonic under standard conditions; however, the intracellular concentrations of its reactants and products are such that it operates close to equilibrium (ΔG ~= 0). Accordingly, when the cell is in a resting state, so [ATP] is relatively high, the reaction proceeds with net synthesis of phosphocreatine, whereas at times of high metabolic activity, when [ATP] is low, the equilibrium shifts so as to yield net synthesis of ATP from phosphocreatine and ADP. Phosphocreatine thereby acts as an ATP "buffer" in cells that contain creatine kinase. A resting vertebrate skeletal muscle normally has sufficient phosphocreatine to supply its free energy needs for several minutes (but for only a few seconds at maximum exertion). In the muscles of some invertebrates, such as lobsters, phosphoarginine performs the same function. These phosphoguanidines are collectively named phosphagens.

Nutrition

Nutrition, the intake and utilization of food, affects health, development, and performance. Food supplies the energy that powers life processes and provides the raw materials to build and repair body tissues. The nutritional requirements of an organism reflect its source of metabolic energy.

Chemolithotrophs

Obtain their energy through the oxidation of inorganic compounds such as NH3, H2S, or even Fe2+

Obligate aerobes vs facultative anaerobes

Organisms can be further classified by the identity of the oxidizing agent for nutrient breakdown. Obligate aerobes (which include animals) must use O2, whereas anaerobes employ oxidizing agents such as sulfate or nitrate. Facultative anaerobes, such as E. coli, can grow in either the presence or the absence of O2. Obligate anaerobes, in contrast, are poisoned by the presence of O2. Their metabolisms are thought to resemble those of the earliest life-forms, which arose more than 3.5 billion years ago when the earth's atmosphere lacked O2. Most of our discussion of metabolism will focus on aerobic processes.

Adenylate kinase

Other kinases reversibly convert nucleoside monophosphates to their diphosphate forms at the expense of ATP. One of these phosphoryl group-transfer reactons is catalyzed by adenylate kinase: AMP + ATP <==> 2 ADP This enzyme is present in all tissues, where it functions to maintain equilibrium concentrations of the three nucleotides. When AMP accumulates, it is converted to ADP, which can be used to synthesize ATP through substrate-level phosphorylation, oxidative phosphorylation, or photophosphorylation. The reverse reaction helps restore cellular ATP because rapid consumption of ATP increases the level of ADP.

When metabolic reactions are not in equilibrium

Other metabolic reactions function far from equilibrium; that is, they are irreversible. This is because an enzyme catalyzing such a reaction has insufficient catalytic activity (the rate of the reaction it catalyzes is too slow) to allow the reaction to come to equilibrium under physiological conditions. Reactants therefore accumulate in large excess of their equilibrium amounts, making ΔG «0. Changes in substrate concentrations therefore have relatively little effect on the rate of an irreversible reaction; the enzyme is essentially saturated. Only changes in the activity of the enzyme—through allosteric interactions, for example—can significantly alter the rate. The enzyme is therefore analogous to a dam on a river: It controls the flow of substrate through the reaction by varying its activity, much as a dam controls the flow of a river by varying the opening of its floodgates.

Oxidation—reduction reactions

Oxidation-reduction reactions resemble other types of group-transfer reactions except that the "groups" transferred are electrons, which are passed from an electron donor (reductant or reducing agent) to an electron acceptor (oxidant or oxidizing agent). For example, in the reaction Fe3+ + Cu+ <==> Fe2+ + Cu2+ Cu+, the reductant, is oxidized to Cu2+ while Fe3+, the oxidant, is reduced to Fe2+.

Metabolic functions in the peroxisome (glyoxysome in plants)

Oxidative reactions catalyzed by amino acid oxidases and catalase; glyoxylate cycle reactions in plants

Photoautotrophs

Photoautotrophs obtain energy via photosynthesis, a process in which light energy powers the transfer of electrons from inorganic donors to CO2 to produce carbohydrates, (CH2O)n, which are later oxidized to release free energy.

Metabolic functions in the Golgi apparatus

Posttranslational processing of membrane and secretory proteins; formation of plasma membrane and secretory vesicles

Reduction of NAD+ to NADH

R represents the ribose-pyrophosphory|-adenosine portion of the coenzyme. Only the nicotinamide ring is affected by reduction, which is formally represented here as occurring by hydride transfer.

Do reactions that function near equilibrium respond rapidly or slowly to changes in substrate concentration?

Reactions that function near equilibrium respond rapidly to changes in substrate concentration. For example, upon a sudden increase in the concentration of reactant for a near-equilibrium reaction, the enzyme catalyzing it would increase the net reaction rate to rapidly achieve the new equilibrium level. Thus, a series of near-equilibrium reactions downstream from the rate-determining step all have the same flux. Likewise, the flux of water in a river is the same at all points downstream from a dam.

Half-reactions

Redox reactions can be divided into two half-reactions, such as Fe3+ + e- <==> Fe2+ (Reduction) Cu+ <==> Cu2+ + e- (Oxidation) whose sum is the whole reaction above. These particular half-reactions occur during the oxidation of cytochrome c oxidase in the mitochondrion (Section 18-2F). Note that for electrons to be transferred, both half-reactions must occur simultaneously. In fact, the electrons are the two half-reactions' common intermediate. A half-reaction consists of an electron donor and its conjugate electron acceptor; in the oxidative half-reaction shown above, Cu+ is the electron donor and Cu2+ is its conjugate electron acceptor. Together these constitute a redox couple or conjugate redox pair analogous to a conjugate acid-base pair (HA and A; Section 2-2B). An important difference between redox pairs and acid-base pairs, however, is that the two half-reactions of a redox reaction, each consisting of a conjugate redox pair, can be physically separated to form an electrochemical cell (Fig. 14-15). 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 electrodes. A salt bridge is necessary to complete the electrical circuit by providing a conduit for ions to migrate and thereby maintain electrical neutrality.

How are standard reduction potentials used to compare electron affinities?

Reduction potentials, like free energies, must be defined with respect to some arbitrary standard, in this case, the hydrogen half-reaction 2 H+ + 2 e- <==> H2(g) in which H+ is in equilibrium with H2(g) that is in contact with a Pt electrode. This half-cell is arbitrarily assigned a standard reduction potential E of 0 V (1 V = 1J • C-1) at pH 0, 25°C, and 1 atm. Under the biochemical convention, where the standard state is pH 7.0, the hydrogen half-reaction has a standard reduction potential E of -0.421 V. When ΔE is positive, ΔG is negative (Eq. 14-7), indicating a spontaneous process. In combining two half-reactions under standard conditions, the direction of spontaneity therefore involves the reduction of the redox couple with the more positive standard reduction potential. In other words, the more positive the standard reduction potential, the higher the affinity of the redox couple's oxidized form for electrons; that is, the greater the tendency for the redox couple's oxidized form to accept electrons and thus become reduced.

Why are the phosphoryl group-transfer reactions of ATP so exergonic?

Several factors appear to be responsible for the "high-energy" character of phosphoanhydride bonds such as those in ATP (Fig. 14-6): 1. The resonance stabilization of a phosphoanhydride bond is less than that of its hydrolysis products. This is because a phosphoanhydride's two strongly electron-withdrawing groups must compete for the lone pairs of electrons of its bridging oxygen atom, whereas this competition is absent in the hydrolysis products. In other words, the electronic requirements of the phosphoryl groups are less satisfied in a phosphoanhydride than in its hydrolysis products. 2. Of perhaps greater importance is the destabilizing effect of the electrostatic repulsions between the charged groups of a phosphoanhydride compared to those of its hydrolysis products. In the physiological pH range, ATP has three to four negative charges whose mutual electrostatic repulsion are partially relieved by ATP hydrolysis. 3. Another destabilizing influence, which is difficult to assess, is the smaller solvation energy of a phosphoanhydride compared to that of its hydrolysis products. Some estimates suggest that this factor provides the dominant thermodynamic driving force for the hydrolysis of phosphoanhydrides. Of course, the free energy change for any reaction, including phosphoryl group transfer from a "high-energy" compound, depends in part on the concentrations of the reactants and products (Eq. 14-1). Furthermore, because ATP and its hydrolysis products are ions, ΔG also depends on pH and ionic strength

The flux of metabolites

Since a metabolic pathway is a series of enzyme-catalyzed reactions, it is easiest to describe the flux of metabolites through the pathway by considering its reaction steps individually. The flux of metabolites, J, through each reaction step is the rate of the forward reaction, v(f) less that of the reverse reaction, v(r): J = v(f) - v(r) At equilibrium, by definition, there is no flux (J = 0), although v(f) and v(r) may be quite large. In reactions that are far from equilibrium, v(f) >> v(r), the flux is essentally equal to the rate of the forward reaction ( J ~= v(f)).

Metabolic functions in the rough endoplasmic reticulum

Synthesis of membrane-bound and secretory proteins

ATP has a high phosphoryl group-transfer potential because...

The "high-energy" intermediate adenosine triphosphate (ATP; Fig. 14-5) occurs in all known life-forms. ATP consists of an adenosine moiety (adenine + ribose) to which three phosphory| (—PO3^2-) groups are sequentially linked via a phosphoester bond followed by two phosphoanhydride bonds. The biological importance of ATP rests in the large free energy change that accompanies cleavage of its phosphoanhydride bonds. This occurs when either a phosphoryl group is transferred to another compound, leaving ADP, or a nucleotidyl (AMP) group is transferred, leaving pyrophosphate (P2O7^4- ; PPi). When the acceptor is water, the process is known as hydrolysis: ATP + H2O <==> ADP + Pi ATP + H2O <==> AMP + PPi Most biological group-transfer reactions involve acceptors other than water. However, knowing the free energy of hydrolysis of various phosphoryl compounds allows us to calculate the free energy of transfer of phosphoryl groups to other acceptors by determining the difference in free energy of hydrolysis of the phosphoryl donor and acceptor.

Conformational changes in E. Coli adenylate kinase on binding substrate

The X-ray structure of adenylate kinase, determined by Georg Schulz, reveals that, in the reaction catalyzed by the enzyme, two ~30-residue domains of the enzyme close over the substrates (Fig. 14-10), thereby tightly binding them and preventing water from entering the active site (which would lead to hydrolysis rather than phosphoryl group transfer). The movement of one of the domains depends on the presence of four invariant charged residues. Interactions between those groups and the bound substrates apparently trigger the rearrangements around the substrate-binding site (Fig. 14-10b). Once the adenylate kinase reaction is complete, the tightly bound products must be rapidly released to maintain the enzyme's catalytic efficiency. Yet since the reaction is energetically neutral (the net number of phosphoanhydride bonds is unchanged), another source of free energy is required for rapid product release. Comparison of the X-ray structures of unliganded adenylate kinase and adenylate kinase in complex with the bisubstrate model compound Ap5A (AMP and ATP connected by a fifth phosphate) show how the enzyme avoids the kinetic trap of tight-binding substrates and products: On binding substrate, a portion of the protein remote from the active site increases its chain mobility and thereby consumes some of the free energy of substrate binding. The region "resolidifies" when the binding site is opened and the products are released. This mechanism is thought to act as an "energetic counterweight" to help adenylate kinase maintain a high reaction rate.

Significance of biochemical half-reactions

The biochemical standard reduction potentials (E°') of some biochemically important half-reactions are listed in Table 14-4. The oxidized form of a redox couple with a large positive standard reduction potential has a high affinity for electrons and is a strong electron acceptor (oxidizing agent), whereas its conjugate reductant is a weak electron donor (reducing agent). For example, O2 is the strongest oxidizing agent in Table 14-4, whereas H2O, which tightly holds its electrons, is the table's weakest reducing agent. The converse is true of half-reactions with large negative standard reduction potentials. Since electrons spontaneously flow from low to high reduction potentials, they are transferred, under standard conditions, from the reduced products in any half-reaction in Table 14-4 to the oxidized reactants of any half-reaction above it cytochromes listed in Table 14-4 have significantly different reduction potentials. This indicates that the protein components of redox enzymes play active roles in electron-transfer reactions by modulating the reduction potentials of their bound redox-active centers. Electron-transfer reactions are of great biological importance. For example, in the mitochondrial electron-transport chain (Section 18-2), electrons are passed from NADH along a series of electron acceptors of increasing reduction potential (including ubiquinone and others listed in Table 14-4) to O2. ATP is generated from ADP and P; by coupling its synthesis to this free energy cascade. NADH thereby functions as an energy-rich electron-transfer coenzyme. In fact, the oxidation by O2 of one NADH to NAD+ supplies sufficient free energy to generate almost three ATPs. NAD+ is an electron acceptor in many exergonic metabolite oxidations. In serving as the electron donor in ATP synthesis, it fulfills its cyclic role as a free energy conduit in a manner analogous to ATP (Fig. 14-9).

"High-energy" intermediates

The complete oxidation of a metabolic fuel such as glucose C6H12O6 + 6O2 —> 6 CO2 + 6H2O releases considerable energy (ΔG°'= -2850 kJ • mol-1). The complete oxidation of palmitate, a typical fatty acid, C16H32O2 + 23 O2 —> 16 CO2 + 16H2O is even more exergonic (ΔG°' = -9781 kJ • mol). Oxidative metabolism proceeds in a stepwise fashion, so the released free energy can be recovered in a manageable form at each exergonic step of the overall process. These "packets" of energy are conserved by the synthesis of a few types of "high-energy" intermediates whose subsequent exergonic breakdown drives endergonic processes. These intermediates therefore form a sort of free energy "currency" through which free energy-producing reactions such as glucose oxidation or fatty acid oxidation "pay for" the free energy-consuming processes in biological systems (Box 14-2). The cell uses several forms of energy currency, including phosphorylated compounds such as the nucleotide ATP (the cell's primary energy currency), compounds that contain thioester bonds, and reduced coenzymes such as NADH. Each of these represents a source of free energy that the cell can use in various ways, including the synthesis of ATP.

What are the compounds whose phosphoryl group-transfer potentials are greater than ATP?

The compounds whose phosphoryl group-transfer potentials are greater than that of ATP have additional stabilizing effects. For example, the hydrolysis of acyl phosphates (mixed phosphoric-carboxylic anhydrides), such as acetyl phosphate and 1,3-bisphosphoglycerate, is driven by the same competing resonance and differential solvation effects that influence the hydrolysis of phosphoanhydrides (Fig. 14-6). Apparently, these effects are more pronounced for acyl phosphates than for phosphoanhydrides.

Reduction of FAD to FADH2

The conjugated ring system of FAD can accept one or two electrons to produce the stable radical (semiquinone) FADH• or the fully reduced (hydroquinone) FADH2 (Fig. 14-14). The change in the electronic state of the ring system on reduction is reflected in a color change from brilliant yellow (in FAD) to pale yellow (in FADH2). The metabolic functions of NAD and FAD demand that they undergo reversible reduction so that they can accept electrons, pass them on to other electron carriers, and thereby be regenerated to participate in additional cycles of oxidation and reduction.

Why can't we synthesize vitamins?

The distant ancestors of humans probably had the ability to synthesize the various vitamins, as do many modern plants and microorganisms. Yet since vitamins are normally available in the diets of animals, which all eat other organisms, or are synthesized by the bacteria that normally inhabit their digestive systems, it seems likely that the superfluous cellular machinery to synthesize them was lost through evolution. For example, vitamin C (ascorbic acid) is required in the diets of only humans, apes, and guinea pigs (Section 6-1C and Box 6-2) because, in what is apparently a recent evolutionary loss, they lack a key enzyme for ascorbic acid biosynthesis.

How does phosphoanhydride hydrolysis effect biochemical processes?

The free energy of the phosphoanhydride bonds of "high-energy" compounds such as ATP can be used to drive reactions even when the phosphoryl groups are not transferred to another organic compound. For example, ATP hydrolysis (i.e., phosphoryl group transfer directly to H2O) provides the free energy for the operation of molecular chaperones, muscle contraction, and transmembrane active transport. In these processes, proteins undergo conformational changes in response to binding ATP. The exergonic hydrolysis of ATP and release of ADP and Pi renders these changes irreversible and thereby drives the processes forward. GTP hydrolysis functions similarly to drive some of the reactions of signal transduction and protein synthesis.

Electrochemical cell

The half-cell undergoing oxidation (here Cu+ —> Cu2+ + e-) passes the liberated electrons through the wire to the half-cell undergoing reduction (here e- + Fe3+ —> Fe2+). Electroneutrality in the two half-cells is maintained by the transfer of ions through the electrolyte-containing salt bridge.

Why do phosphoguanidines have high phosphoryl group-transfer potentials?

The high phosphoryl group-transfer potentials of phosphoguanidines, such as phosphocreatine and phosphoarginine, largely result from the competing resonances in the guanidino group, which are even more pronounced than they are in the phosphate group of phosphoanhydrides: Consequently, phosphocreatine can transfer its phosphoryl group to ADP to form ATP.

Is it a useful reaction when there's hydrolysis of a "high-energy" compound while releasing considerable free energy? explain

The hydrolysis of a "high-energy" compound, while releasing considerable free energy, is not in itself a useful reaction. However, the exergonic reactions of "high-energy" compounds can be coupled to endergonic processes to drive them to completion. The thermodynamic explanation for the coupling of an exergonic and an endergonic process is based on the additivity of free energy. Consider the following two-step reaction pathway: (1) A + B <==> C + D ΔG1 (2) D + E <==> F + G ΔG2 If ΔG1 ≥ 0, Reaction 1 will not occur spontaneously. However, if ΔG2 is sufficiently exergonic so ΔG1 + ΔG2 <0, then although the equilibrium concentration of D in Reaction 1 will be relatively small, it will be larger than that in Reaction 2. As Reaction 2 converts D to products, Reaction 1 will operate in the forward direction to replenish the equilibrium concentration of D. The highly exergonic Reaction 2 therefore "drives" or "pulls" the endergonic Reaction 1, and the two reactions are said to be coupled through their common intermediate, D. That these coupled reactions proceed spontaneously can also be seen by summing Reactions 1 and 2 to yield the overall reaction where ΔG3 = ΔGI + ΔG2 <0. As long as the overall pathway is exergonic, it will operate in the forward direction. (1 + 2) A + B + E <==> C + F + G ΔG3

(Coupled reactions) the phosphorylation of glucose & the phosphorylation of ADP

The initial step in the metabolism of glucose is its conversion to glucose-6-phosphate. Yet the direct reaction of glucose and P1 is thermodynamically unfavorable (ΔG°' = + 13.8 kJ • mol-l; Fig. 14-70). In cells, however, this reaction is coupled to the exergonic cleavage of ATP (for ATP hydrolysis, ΔG°'= -30.5 kJ • mol-1), so the overall reaction is thermodynamically favorable (ΔG°' = +13.8 - 30.5 = -16.7 kJ • mol-1). ATP can be similarly regenerated (ΔG°' = +30.5 kJ • mol-1) by coupling its synthesis from ADP and Pi to the even more exergonic cleavage of phosphoenolpyruvate (ΔG°' = -61.9 kJ • mol-1; Fig. 14-7b and Section 15-2J). Note that the half-reactions shown in Fig. 14-7 do not actually occur as written in an enzyme active site. Hexokinase, the enzyme that catalyzes the formation of glucose-6-phosphate (Fig. 14-7a), does not catalyze ATP hydrolysis but instead catalyzes the transfer of a phosphoryl group from ATP directly to glucose. Likewise, pyruvate kinase, the enzyme that catalyzes the reaction shown in Fig. 14-7b, does not add a free phosphoryl group to ADP but transfers a phosphoryl group from phosphoenolpyruvate to ADP to form ATP.

Describe the thioester bond and its relationship with Coenzyme A

The thioester bond is involved in substrate-level phosphorylation, an ATP-generating process that is independent of—and presumably arose before—oxidative phosphorylation. The thioester bond appears in modern metabolic pathways as a reaction intermediate (involving a Cys residue in an enzyme active site) and in the form of acetyl-CoA (Fig. 14-11), the common product of carbohydrate, fatty acid, and amino acid catabolism. Coenzyme A (CASH or CoA) consists of a ß-mercaptoethylamine group bonded through an amide linkage to the vitamin pantothenic acid, which, in turn, is attached to a 3'-phosphoadenosine moiety Via a pyrophosphate bridge. The acetyl group of acetyl-CoA is bonded as a thioester to the sulfhydryl portion of the β-mercaptoethylamine group. CoA thereby functions as a carrier of acetyl and other acyl groups (the A of COA stands for "'acetylation"). Thioesters also take the form of acyl chains bonded to a residue that is linked to a Ser OH group in a protein (Section 20-4C) rather than to 3'-phospho-AMP, as in COA.

The _________ -soluble vitamins in the human diet are all coenzyme precursors

The water-soluble vitamins in the human diet are all coenzyme precursors. In contrast, the fat-soluble vitamins, with the exception of vitamin K (Section 9-1F), are not components of coenzymes, although they are also required in small amounts in the diets of many higher animals.

Describe the electron carriers NAD+ and FAD

Two of the most widely occurring electron carriers are the nucleotide coenzymes nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD). The nicotinamide portion of NAD+ (and its phosphorylated counterpart NADP+; Fig. 11-4) is the site of reversible reduction, which formally occurs as the transfer of a hydride ion (H; a proton with two electrons) as indicated in Fig. 14-12. The terminal electron acceptor in aerobic organisms, O2, can accept only unpaired electrons (because each of its two available lowest energy molecular orbitals is already occupied by one electron); that is, electrons must be transferred to O2 one at a time. Electrons that are removed from metabolites as pairs (e.g., with the two-electron reduction of NAD+) must be transferred to other carriers that can undergo both two-electron and one-electron redox reactions. FAD (Fig. 14-13) is such a coenzyme.

Proteomics

Unfortunately, the correlation between the amount of a particular mRNA and the amount of its protein product is imperfect. This is because the various mRNAs and their corresponding proteins are synthesized and degraded at different rates. Furthermore, many proteins are posttranslationally modified, sometimes in several different ways (e.g., by phosphorylation or glycosylation). Consequently, the number of unique proteins in a cell exceeds the number of unique mRNAs. A more reliable way than transcriptomics to assess gene expression is to examine a cell's proteome. This proteomics approach requires that the proteins first be separated, usually by two-dimensional (2D) gel electrophoresis (a technique that separates proteins by isoelectric point in one direction and by mass in the perpendicular direction; Section 5-2D). Individual proteins are then identified by using tandem mass spectrometry to obtain amino acid sequence information (Section 5-3D) and correlating it with protein sequence databases. Because many peptides are generated from a single protein, the technique enables the redundant and unambiguous identification of that protein from the database. In this way we can catalog all the proteins that are contained in a cell or tissue under a given set of conditions. Can we compare all the proteins synthesized by a cell under two different sets of conditions as is done for mRNA? The answer is yes, by using different isotopically labeled reagents that are either contained in the growth medium (e.g., deuterated amino acids) or that are reacted with the cell extract. The proteins are then purified and analyzed by tandem mass spectrometry. A hope for the future is that samples from diseased and normal subjects can be compared in this manner to find previously undetected protein markers that would allow early diagnosis of various diseases.

Vitamins and Minerals

Vitamins are organic molecules that an animal is unable to synthesize and must therefore obtain from its diet. Vitamins can be divided into two groups: water-soluble vitamins and fat-soluble vitamins. Table 14-2 lists the essential minerals and trace elements necessary for metabolism. They participate in metabolic processes in many ways. Mg2+, for example, is involved in nearly all reactions that involve ATP and other nucleotides, including the synthesis of DNA, RNA, and proteins. Zn2+ is a cofactor in a variety of enzymes, including carbonic anhydrase (Section 11-3C). Ca2+, in addition to being the major mineral component of bones and teeth, is a vital participant in signal transduction processes (Section 13-4).

With what levels can gene expression be explored?

We can assess the genome, transcriptome (the entire collection of RNA transcribed by a cell), proteome (the complete set of proteins synthesized by a cell in response to changing conditions), and metabolome (the cell's collection of metabolic intermediates) as well as their interrelationships (Fig. 14-18). The term bibliome (Greek: biblion, book) has even been coined to denote the systematic incorporation of preexisting information about reaction mechanisms and metabolic pathways.

When metabolic reactions are in equilibrium

When the reactants are present at values close to their equilibrium values, [C]eq [D]eq / [A]eq [B]eq ~= Keq, and ΔG ~= 0. This is the case for many metabolic reactions, which are said to be near-equilibrium reactions. Because their ΔG values are close to zero, they can be relatively easily reversed by changing the ratio of products to reactants. When the reactants are in excess of their equilibrum concentrations, the net reaction proceeds in the forward direction until the excess reactants have been converted to products and equilibrium is attained. Conversely, when products are in excess, the net reaction proceeds in the reverse direction to convert products to reactants until the equilibrium concentration ratio is again achieved. Enzymes that catalyze near-equilibrium reactions tend to act quickly to restore equilibrium concentrations, and the net rates of such reactions are effectively controlled by the relative concentrations of substrates and products.

Heterotrophs

obtain free energy through the oxidation of organic compounds (carbohydrates, lipids, and proteins) and hence ultimately depend on autotrophs for those substances.