AnIntroductioMetabolisKEY CONCEPTS

8.1 An organism's metabolism transforms matterand energy, subject to the Jaws ofthermodynamics

8.2 The free-energy change of a reaction tells uswhether or not the reaction occursspontaneously

8.3 AlP powers cellular work by couplingexergonic reactions to endergonic reactions

8.4 Enzymes speed up metabolic reactions bylowering energy barriers

8.5 Regulation of enzyme activity helps controlmetabolism

The Energy of Life

The living cell is a chemical factory in miniature,where thousands of reactions occur within a micro-scopic space. Sugars can be converted to amino

adds that are linked together into proteins when needed,and proteins are dismantled into amino acids that can beconverted to sugars when food is digested. Small moleculesare assembled into polymers, which may be hydrolyzed lateras the needs of the cell change. In multicellular organisms,many cells export chemical products that are used in otherparts of the organism. The process known as cellular respi-ration drives the cellular economy by extracting the energystored in sugars and other fuels. Cells apply this energy toperform various types of work, such as the transport ofsolutes across the plasma membrane, which we discussed inChapter 7. In a more exotic example, cells of the fungus inFigure 8.1 convert the energy stored in certain organic mol-ecules to light. a process called bioluminescence. (The glowmay attract insects that benefit the fungus by dispersing its

142

... Figure 8.1 What causes the bioluminescence in thesefungi?

spores.) Bioluminescence and all other metabolic activitiescarried out by a cell are precisely coordinated and con-trolled. In its complexity, its efficiency, its integration, andits responsiveness to subtle changes, the cell is peerless as achemical factory. The concepts of metabolism that youlearn in this chapter will help you understand how matterand energy flow during life's processes and how that flow isregulated.

metabolismtransforms matter and energy,subject to the laws ofthermodynamics

The totality of an organism's chemical reactions is calledmetabolism (from the Greek metabole, change). Metabolismis an emergent property of life that arises from interactionsbetween molecules within the orderly environment ofthe cell.

Organization of the Chemistry of Lifeinto Metabolic PathwaysWe can picture a cell's metabolism as an elaborate rood mapof the thousands of chemical reactions that occur in a cell,arranged as intersecting metabolic pathways. A metabolicpathway begins with a specific molecule, which is then alteredin a series ofdefined steps, resulting in a certain product. Eachstep of the pathway is catalyzed by a specific enzyme:

that matter possesses because of its location or structure. Wa-ter behind a dam, for instance, possesses energy because of itsaltitude above sea leveL Molecules possess energy because ofthe arrangement of their atoms. Chemical energy is a termused by biologists to refer to the potential energy available forrelease in a chemical reaction. Recall that catabolic pathwaysrelease energy by breaking down complex molecules. Biolo-gists say that these complex molecules, such as glucose, arehigh in chemical energy. During acatabolic reaction, atoms arerearranged and energy is released, resulting in lower-energybreakdown products. This transformation also occurs, for ex-ample, in the engine of a car when the hydrocarbons of gaso-line react explosively with oxygen, releasing the energy thatpushes the pistons and producing exhaust. Although less ex-plosive, a similar reaction of food molecules with oxygen pro-vides chemical energy in biological systems, producing carbondioxide and water as waste products. It is the structures andbiochemical pathways of cells that enable them to releasechemical energy from food molecules, powering life processes.How is energyconverted from one form toanother? Consider

the divers in Figure 8.2. The young man climbing the steps tothediving platform is releasingchemical energy from the food heate for lunch and using some ofthat energy to perform the work

... Figure 8.2 Transformations between potential andkinetic energy.

Analogous to the red, yellow, and green stoplights that controlthe flow of automobile traffic, mechanisms that regulate en-zymes balance metabolic supply and demand, avertingdeficitsor surpluses of important cellular molecules.Metabolism as a whole manages the material and energy re-

sources of the cell. Some metabolic pathways release energy bybreaking down complex molecules to simpler compounds.These degradative processes are called catabolic pathways, orbreakdown pathways. Amajor pathway ofcatabolism is cellularrespiration, in which the sugar glucose and other organic fuelsare brokendown in the presence ofoxygen to carbon dioxideandwater. (Pathways can have more than one starting moleculeand/or product.) Energy that was stored in the organicmoleculesbecomes available to do the work of the cell, such as ciliarybeating or membrane transport. Anabolic pathways, in contrast,consume energy to build complicated molecules from simplerones; they are sometimes called biosynthetic pathways. An ex-ample of anabolism is the synthesis of a protein from aminoacids. Catabolic and anabolic pathways are the and

avenues ofthe metabolic map. Energy released from thedownhill reactions ofcatabolic pathways can be stored and thenused to drive the uphill reactions ofanabolic pathways.In this chapter, we will focus on mechanisms common to

metabolic pathways. Because energy is fundamental to allmetabolic processes, a basic knowledge ofenergy is necessaryto understand how the living cell works. Although we will usesome nonliving examples to study energy, the conceptsdemonstrated by these examples also apply to bioenergetics,the study of how energy flows through living organisms.

forms of EnergyEnergy is the capacity to cause change. In everyday life, energyis important because some forms of energy can be used to dowork-that is, to move matter against opposing forces, such asgravity and friction. Put another way, energy is the ability to re-arrange a collection of matter. For example, you expend energyto turn the pages of this book, and your cells expend energy intransporting certain substances across membranes. Energy ex-ists in various forms, and the work of life depends on the abilityof cells to transform energy from one form into another.Energy can be associated with the relative motion of ob-

jects; this energy is called kinetic energy. Moving objects canperform work by imparting motion to other matter: A poolplayer uses the motion of the cue stick to push the cue ball,which in turn moves the other balls; water gushing through adam turns turbines; and the contraction ofleg muscles pushesbicycle pedals. Heat, or thermal energy, is kinetic energy as-sociated with the random movement of atoms or molecules.Light is also a type ofenergy that can be harnessed to performwork, such as powering photosynthesis in green plants.An object not presentlymoving may still possess energy. En-

ergy that is not kinetic is called potential energy; it is energy

A diver has more potentialenergy on the platformthan in the water.

Climbing up converts the kineticenergy of muscle movementto potential energy.

Diving convertspotential energy tokinetic energy.

Adiver has less potentialenergy in the waterthan on the platform.

ClIAPTER EIGHT An Introduction to Metabolism 143

ofclimbing. The kinetic energy ofmuscle movement is thus be-ing transformed into potential energy due to his increasingheight above the water. The young man diving is converting hispotential energy to kinetic energy, which is then transferred tothe water as he enters it. A small amount of energy is lost as heatdue to friction.Now let's go back one step and consider the original source

of the organic food molecules that provided the necessarychemical energy for the diver to climb the steps. This chemi-cal energy was itself derived from light energy by plants dur-ing photosynthesis. Organisms are energy transformers.

The Laws of Energy TransformationThe study of the energy transformations that occur in a collec-tion of matter is called thermodynamics. Scientists use theword system to denote the matter under study; they refer tothe rest ofthe universe-everything outside the system-as thesurroundings. An isolo.ted system, such as that approximated byliquid in a thermos bottle, is unable to exchange either energyor matter with its surroundings. In an open system, energy andmatter can be transferred between the system and its sur-roundings. Organisms are open systems. They absorb energy-for instance, light energy or chemical energy in the form oforganic molecules-and release heat and metabolic wasteproducts, such as carbon dioxide, to the surroundings. Twolaws of thermodynamics govern energy transformations in or·ganisms and all other collections ofmatter.

The First Law ofThermodynamics

According to the first law ofthermodynamics, the energy ofthe universe is constant. Energy can be transferred and trans-formed, but it cannot be created or destroyed. TIle first law is

also known as the principleofconservation ofenergy. The elec-tric company does not make energy, but merely converts it toa form that is convenient for us to use. By converting sunlightto chemical energy, a plant acts as an energy transformer, notan energy producer.The cheetah in Figure 8.3a will convert the chemical en·

ergy of the organic molecules in its food to kinetic and otherforms of energy as it carries out biological processes. Whathappens to this energy after it has performed work? The sec-ond law helps to answer this question.

The Second Law ofThermodynamics

If energy cannot be destroyed, why can't organisms simply re-cycle their energy over and over again? It turns out that duringevery energy transfer or transformation, some energy becomesunusable energy, unavailable to do work. In most energy trans-formations, more usable forms ofenergy are at least partly con-verted to heat, which is the energy associated with the randommotion of atoms or molecules. Only a small fraction of thechemical energy from the food in Figure 8.3a is transformedinto the motion of the cheetah shown in Figure 8.3b; most islost as heat, which dissipates rapidly through the surroundings.In the process of carrying out chemical reactions that per-

form various kinds of work, living cells unavoidably convertother forms of energy to heat. A system can put heat to workonly when there is a temperature difference that results in theheat flowing from a warmer location to a cooler one. If tem-perature is uniform, as it is in a living cell, then the only use forheat energy generated during a chemical reaction is to warm abody of matter, such as the organism. (This can make a roomcrowded with people uncomfortably warm, as each person iscarrying out a multitude of chemical reactions!)

(b) Second law of thermodynamics: Every energy transfer or transformation increasesthe disorder (entropy) of the universe. For example, disorder is added to the cheetah'ssurroundings in the form of heat and the small molecules that are the by-productsof metabolism.

(a) First law of thermodynamics: Energy canbe transferred or transformed but neithercreated nor destroyed. For eKample, thechemical (potential) energy in food will beconverted to the kinetic energy of thecheetah's movement in (b).

... Figure 8.3 The two laws of thermodynamics.

144 UNIT TWO The Cell

CONCEPT CHECK

Alogical consequence of the loss of usable energy during en-ergy transfer or transformation is that each such event makesthe universe more disordered. Scientists use a quantity calledentropy as a measure of disorder, or randomness. The morerandomly arranged a collection of matter is, the greater its en-tropy. We can now state the second law of thermodynamicsas follows: Every energy transfer or transfonnation increases theentropy of the universe. Although order can increase locally,there is an unstoppable trend toward randomization of the uni-verse as awhole.In many cases, increased entropy is evident in the physical

disintegration ofa system's organized structure. For example,you can observe increasing entropy in the gradual decay ofanunmaintained building. Much of the increasing entropy of theuniverse is less apparent, however, because it appears as in-creasing amounts ofheat and less ordered forms ofmatter. Asthe cheetah in Figure 8.3b converts chemical energy to kineticenergy, it is also increasing the disorder of its surroundings byproducing heat and the small molecules, such as the CO2 it ex-hales, that are the breakdown products of food.The concept of entropy helps us understand why certain

processes occur. It turns out that for a process to occur on itsown, without outside help (an input ofenergy), it must increasethe entropy of the universe. Let's first agree to use the wordspontaneous for a process that can occur without an input ofenergy. Note that as we're using it here, the word spontaneousdoes not imply that such a process would occur quickly. Somespontaneous processes may be virtually instantaneous, such asan explosion, while others may be much slower, such as therusting ofan old car over time. Aprocess that cannot occur onits own is said to be nonspontaneousi it will happen only if en-ergy isadded to the system.We know from experience that cer-tain events occur spontaneously and others do not Forinstance, we know that water flows downhill spontaneously,but moves uphill only with an input ofenergy, such as when amachine pumps the water against gravity. In fact, another wayto state the second law is: For a process to occur spontaneously,it must increase the entropy ofthe universe.

Biological Order and Disorder

Living systems increase the entropy of their surroundings, aspredicted by thermodynamic law. It is true that cells create or·dered structures from less organized starting materials. For ex-ample, amino acids are ordered into the specific sequences ofpolypeptide chains. At the organismal level, Figure 8,4 showsthe extremelysymmetrical anatomyofaplant's root, formed bybiological processes from simpler starting materials. However,an organism also takes in organized forms ofmatter and energyfrom the surroundings and replaces them with less orderedforms. For example, an animal obtains starch, proteins, andother complex molecules from the food it eats. As catabolicpathways break these molecules down, the animal releases car-

... Figure 8.4 Order as a characteristic of life. Order is evidentin the detailed anatomy of this root tissue from a buttercup plant (LM.cross section), As open systems. organisms can increase their order aslong as the order of their surroundings decreases,

bon dioxide and water-small molecules that possess lesschemical energy than the food did. The depletion ofchemicalenergy is accounted for by heat generated during metabolism.On a larger scale, energy flows into an ecosystem in the form oflight and exits in the form ofheat (see Figure 1.5).During the early history ofHfe, complex organisms evolved

from simpler ancestors. For example, we can trace the ances-try of the plant kingdom from much simpler organisms calledgreen algae to more complex flowering plants. However, thisincrease in organization over time in no way violates the sec-ond law. The entropy ofa particular system, such as an organ-ism, may actually decrease as long as the total entropy of theuniverse-the system plus its surroundings-increases. Thus,organisms are islands of low entropy in an increasingly ran-dom universe. The evolution of biological order is perfectlyconsistent with the laws of thermodynamics.

8.11. How does the second law of thermodynamics help ex-plain the diffusion ofa substance across a membrane?

2. Describe the forms ofenergy found in an apple as itgrows on a tree, then falls and is digested by someonewho eats it

3. _',ImUIA If you place a teaspoon of sugar in thebottom of a glass ofwater. it will dissolve completelyover time. Left longer, eventually the water will disap-pear and the sugar crystals will reappear. Explainthese observations in terms ofentropy.For suggested answers. see AppendiX A

ClIAPTER EIGHT An Introduction to Metabolism 145

changeof a reaction tells us whetheror not the reaction occursspontaneously

The laws of thermodynamics that we've just discussed applyto the universe as a whole. As biologists, we want to under·stand the chemical reactions ofhfe-for example, which reac-tions occur spontaneously and which ones require some inputofenergy from outside. But how can we know this without as-sessing the energy and entropy changes in the entire universefor each separate reaction?

Free-Energy Change, tJ.GRecall that the universe is really equivalent to "the system"plus "the surroundings." In 1878, J. Willard Gibbs, a professorat Yale, defined avery useful function called the Gibbs free en·ergy ofa system (without considering its surroundings), sym·bolized by the letter G. We'll refer to the Gibbs free energysimply as free energy. Free energy is the portion ofa system'senergy that can perform work when temperature and pres-sure are uniform throughout the system, as in a living cell.Let's consider how we determine the free-energy change thatoccurs when a system changes-for example, during a chem-ical reaction.The change in free energy, liG, can be calculated for a

chemical reaction with the following formula:

This formula uses only properties of the system (the reac-tion) itself: liH symbolizes the change in the system's enthalpy(in biological systems, equivalent to total energy); LlS is thechange in the system's entropy; and T is the absolute temper-ature in Kelvin (K) units (K = ·C +273; see Appendix C).Once we know the value ofLlG for a process, we can use it

to predict whether the process will be spontaneous (that is,whether itwill occurwithout an input ofenergy from outside).More than a century of experiments has shown that onlyprocesses with a negative LlG are spontaneous. For a processto occur spontaneously, therefore, the system must either giveup enthalpy (H must decrease), give up order (TS must in-crease), or both: When the changes inHand TS are tallied, LlGmust have a negative value (LlG <0) for a process to be spon-taneous. This means that every spontaneous process de-creases the system's free energy. Processes that have a positiveor zero LlG are never spontaneous.This information is immensely interesting to biologists, for

it gives us the power to predict which kinds ofchange can hap·pen without help. Such spontaneous changes can be har·

1% UNIT TWO The Cell

nessed to perform work. This principle is very important inthe study of metabolism, where a major goal is to determinewhich reactions can supply energy for cellular work.

Free Energy, Stability, and EquilibriumAs we saw in the previous section, when a process occursspontaneously in a system, we can be sure that LlG is negative.Another way to think ofLlG is to realize that it represents thedifference between the free energy of the final state and thefree energy of the initial state:

LlG = Gtin•1sl.te - Giniti.1 sl.te

Thus, LlG can be negative only when the process involves aloss of free energy during the change from initial state to finalstate. Because it has less free energy, the system in its final stateis less likely to change and is therefore more stable than it waspreviously.We can think of free energy as a measure of a system's in-

stability-its tendency to change to a more stable state. Un-stable systems (higher G) tend to change in such a way thatthey become more stable (lower G). For example, a diver ontop of a platform is less stable (more likely to fan) than whenfloating in the water, a drop of concentrated dye is less stable(more likely to disperse) than when the dye is spread randomlythrough the liquid, and a sugar molecule is less stable (morelikely to break down) than the simpler molecules into which itcan be split (Figure 8.5). Unless something prevents it, eachof these systems will move toward greater stability: The diverfalls, the solution becomes uniformly colored, and the sugarmolecule is broken down.Another term that describes a state ofmaximum stability is

equilibrium,which you learned about in Chapter 2 in connec-tion with chemical reactions. There is an important relation-ship between free energy and equilibrium, including chemicalequilibrium. Recall that most chemical reactions are reversibleand proceed to a point at which the forward and backward re-actions occur at the same rate. The reaction is then said to beat chemical equilibrium, and there is no further net change inthe relative concentration of products and reactants.As a reaction proceeds toward equilibrium, the free energy

ofthe mixture ofreactants and products decreases. Free energyincreases when a reaction is somehow pushed away from equi-librium, perhaps by removing some of the products (and thuschanging their concentration relative to that of the reactants).For a system at equilibrium, G is at its lowest possible value inthat system. We can think of the equilibrium state as a free-energy valley. Any change from the equilibrium position willhave a positive LlG and will not be spontaneous. For this rea-son, systems never spontaneously move away from equilib-rium. Because a system at equilibrium cannot spontaneouslychange, it can do no work. A process is spontaneous and canperform work only when it is moving toward equilibrium.

!I

""

!""""""" (I"

• More lree energy (higher G)• Less stable• Greater work capacity

In a spontaneous change• The free energy of the systemdecreases (aG < 0)

• The system becomes morestable

• The released free energy canbe harnessed to do work

• Less lree energy (lower G)• More stable• Less work capacity

(a) Gravitational motion. Objectsspontaneously from a

higher altitude to a lower one.

(b) Diffusion. Molecules in adropof dye diffuse until they arerandomly dispersed.

(c) Chemical reaction. In acell. asugar molecule is broken downinto simpler molecules.

(a) Exergonic reaction: energy released

•

•

----ly-Amount ofenergyrequired(aG>O)

___JL

Energy

------------------------ll---·Amount ofenergyreleased(aG<O)

__Jt

Products

(b) Endergonic reaction; energy required

1

1Free Energy and MetabolismWe can now apply the free-energy concept more specificallyto the chemistry oflife's processes.

C6H J20 6 +6O2 ----> 6 CO2 + 6H20J:\.G = -686 kcallmol (-2,870 kl/mol)

fxergonic and fndergonic Reactions in MetabolismBased on their free-energy changes, chemical reactions can beclassified as either exergonic (Uenergy outward") or ender-gonic ("energy inward'). An exergonic reaction proceedswith a net release of free energy (Figure 8.6a). Because thechemical mixture loses free energy (G decreases), dG is nega-tive for an exergonic reaction. Using dG as a standard forspontaneity, exergonic reactions are those that occur sponta-neously. (Remember, the word spontaneous does not implythat a reaction will occur instantaneously or even rapidly.)The magnitude ofdG for an exergonic reaction represents themaximum amount of work the reaction can perform.· Thegreater the decrease in free energy, the greater the amount ofwork that can be done.We can use the overall reaction for cellular respiration as an

example:

.. Figure 8.5 The relationship of free energy to stability,work capacity, and spontaneous change. Unstable systems(top diagrams) are rich In lree energy. G. They a tendency tochange spontaneously to amore stable state (bottom), and it ispossible to harness this "downhill" change to perform work.

• The word """'im/lm qualifies statement, because some of the free energyreleased as heat and cannot do work. Therefure. aG represents a theuret-

ical upper limit of available energy.... Figure 8.6 Free energy changes (!l.G) in exergonic andendergonic reactions.

CIiAPTER EIGIiT An Introduction to Metabolism 147

For each mole (180 g) of glucose broken down by respira-tion under what are called conditions" (1 M ofeachreactant and product, 25°C, pH 7), 686 kcal (2,870 kJ) ofenergyare made available for work. Because energy must be con-served, the chemical products of respiration store 686 kcallessfree energy per mole than the reactants. The products are, ina sense, the spent exhaust ofa process that tapped the free en·ergy stored in the sugar molecules.An endergonic reaction is one that absorbs free energy

from its surroundings (figure 8.Gb). Because this kind of re-action essentially stores free energy in molecules (G increases),toG is positive. Such reactions are nonspontaneous, and themagnitude of toG is the quantity of energy required to drivethe reaction. If a chemical process is exergonic (downhill), re-leasing energy in one direction, then the reverse process mustbe endergonic (uphill), using energy. A reversible process can-notbe downhill in both directions. If toG = -686 kcal/mol forrespiration, which converts sugar and oxygen to carbon diox·ide and water, then the reverse process-the conversion ofcarbon dioxide and water to sugar and oxygen-must bestrongly endergonic, with toG = +686 kcal/moL Such a reac-tion would never happen by itself.How, then, do plants make the sugar that organisms use for

energy? They get the required energy-686 kcal to make amole of sugar-from the environment by capturing light andconverting its energy to chemical energy. Next, in a long seriesofexergonic steps, they gradually spend that chemical energyto assemble sugar molecules.

Equilibrium and Metabolism

Reactions in an isolated system eventually reach equilibriumand can then do no work, as illustrated by the isolated hydro·electric system in Figure 8.7a. The chemical reactions ofme·tabolism are reversible, and they, too, would reach equi·librium if they occurred in the isolation ofa test tube. Becausesystems at equilibrium are at a minimum ofG and can do nowork, a cell that has reached metabolic equilibrium is dead!The fact that metabolism as a whole is never at equilibrium isone of the defining features of life.Like most systems, a living ceU is not in equilibrium. The con-

stant flow ofmaterials in and out of the cell keeps the metabolicpathways from ever reaching equilibrium, and the cell continuesto do work throughout its life. This principle is illustrated by theopen (and more realistic) hydroelectric system in figure 8.7b.However, unlike this simple single·step system, a catabolic path·way in a cell releases free energy in a series ofreactions. An exam-ple is ceUular respiration, illustrated by analogy in figure 8.7c.Some of the reversible reactions of respiration are constantly

in one direction-that is, they are kept out ofequilibrium.The key to maintaining this lack ofequilibrium is that the productofa reaction does not accumulate, but instead becomes a reactantin the next step; finally, waste products are expelled from the celL

148 UNIT TWO The Cell

(a) An isolated hydroelectric system. Water flowing downhill turns aturbine that drives agenerator providing electricity to a light bulb.but only until the system reaches equilibrium.

(b) An open hydroelectricsystem. Flowing waterkeeps driving the gen·erator because intake andoutflow of water keep thesystem from reachingequilibrium.

(c) A multistep open hydroelectric system. Cellular respirationis analogous to this system: Glucose is broken down in a seriesof exergonic reactions that power the work of the cell. Theproduct of each reaction becomes the reactant for the next,so no reaction reaches equilibrium.

.. Figure 8.7 Equilibrium and work in isolated and opensystems.

The overall sequence of reactions is kept going by the huge free-energydifference betv.'een glucoseand oxygen at the topoftheen·ergy "hill" and carbon dioxide and water at the end. Aslong as our cells have a steady supply ofglucose or other fuels andoxygen and are able to expel waste products to the surroWldings,their metabolic path\\'aYS never reach equilibriwn and can con·tinue to do the work ofllle.We see once again how important it is to think oforganisms

as open systems. Sunlight provides a daily source of free en-ergy for an ecosystem's plants and other photosynthetic or-ganisms. Animals and other nonphotosynthetic organisms inan ecosystem must have a source of free energy in the form ofthe organic products of photosynthesis. Now that we have ap-plied the free-energy concept to metabolism, we are ready tosee how a cell actually performs the work of life.

1. Cellular respiration uses glucose and oxygen, whichhave high levels of free energy, and releases CO2 andwater, which have low levels of free energy. Is respira-tion spontaneous or not? Is it exergonic or ender-gonic? \Vhat happens to the energy released fromglucose?

2. Akey process in metabolism is the transportofhy-drogen ions (H+) across a membrane to create a con-centration gradient. Other processes can result in anequal concentration of hydrogen ions on each side.Which arrangement ofhydrogen ions allows the H+to perform work in this system?

3. _','!:tUIA At nighttime celebrations, revelers cansometimes be seen wearing giow-in-the-dark neck-laces. The necklaces start glowing once they are "acti_vated;' which usually involves snapping the necklacein a way that allows two chemicals to react and emitlight in the form of"chemiluminescence:' Is thechemical reaction exergonic or endergonic? Explainyour answer.For suggested answers, see Appendix A.

CONCEPT CHECK 8.2 nine and a chain of three phosphate groups bonded to it(Figure 8.8). In addition to its role in energy coupling, ATP isalso one of the nucleoside triphosphates used to make RNA(see Figure 5.27).The bonds bem-een the phosphate groups ofATP can be bro·

ken by hydrolysis. \Vhen the terminal phosphate bond is broken,a molecule of inorganic phosphate (HOP032-, abbreviated ®ithroughout this book) leaves the ATP, which becomes adenosinediphosphate, or ADP (Figure 8.9). The reaction is exergonic andreleases 7.3 kcal of energy per mole ofATP hydrolyzed:

ATP+ Hp-. ADP +®;6G = -7.3 kcal/mol (-30.5 kUmol)

This is the free-energy change measured under standard con-ditions. In the cell, conditions do not conform to standardconditions, primarily because reactant and product concen·trations differ from I M. For example, when ATP hydrolysisoccurs under cellular conditions, the actual6G is about - 13kcallmol, 78% greater than the energy released by ATP hy-drolysis under standard conditions.Because their hydrolysis releases energy, the phosphate bonds

of ATP are sometimes referred to as high-energy phosphatebonds, but the term is misleading. The phosphate bonds ofATP

... Figure 8.8 The structure of adenosine triphosphate (ATP).In the cell. most hydroxyl groups of phosphates are ionized (-0-).

Inorganic phosphate Adenosine diphosphate (ADP)

.. Figure 8.9 The hydrolysis of ATP. The reaction of ATP andwater yields inorganic phosphate (®,) and ADP and releases energy.

NI

",CHN

Energy•

Adenosine triphosphate (ATP)

+®,

Adenine NHI '

l-cY'(000II II II \ /C

N

0·0·0· HHPhosphate groups

H H RiboseOH OH

A cell does three main kinds ofwork:

The Structure and Hydrolysis of AlPATP (adenosine triphosphate) was introduced in Chapter 4when we discussed the phosphate group as a functional group.ATP contains the sugar ribose, with the nitrogenous base ade-

... Chemical work, the pushing ofendergonic reactions, whichwould not occur spontaneously, such as the synthesis ofpolymers from monomers (chemical work will be discussedfurther here and will come up again in Chapters 9 and 10)

.. Transport work, the pumping of substances across mem-branes against the direction ofspontaneous movement (seeChapter 7)

... Mechanical work, such as the beatingofcilia (see Olapter6),the contraction ofmuscle cells, and the movement ofchro-mosomes during cellular reproduction

Akey feature in the waycells manage their energy resourcesto do this work is energy coupling, the use of an exergonicprocess to drive an endergonic one. ATP is responsible for me-diating most energy coupling in cells, and in most cases it actsas the immediate source ofenergy that powers cellular work.

work bycoupling exergonic reactionsto endergonic reactions

CIiAPTER EIGIiT An Introduction to Metabolism 149

The Regeneration of ATP

(a) Endergonie reaction. Amino acid conversion by itself is ender-gonic (DoG is positive). so it is not spontaneous.

to bind the cytoskeleton, resulting in movement ofthe proteinalong the cytoskeletal track.

DoG", +3,4 kcaVmol

DoG '" -73 kcaVmolNet AG '" -3,9 kcaVmol

, - ,G. ,34 k",VmolAmmonia GlutamineGlutamic

acid

Glu+NH3 - Glu-NH2ATP -ADP+®;

An organism at work uses ATP continuously, but ATP is a re-newable resource that can be regenerated by theadditionofphos-phate to ADP (Figure 8.12). The free energy required tophosphorylate ADP comes from exergonic breakdown reactions(catabolism) in the cell. This shuttlingofinorganic phosphateandenergy is called the ATP cycle, and it couples the cen's energy-yielding (exergonic) processes to the energy·consuming (ender-gonic) ones. The ATP cycle moves at an astonishing pace. Forexample, aworking muscle cell recycles its entire pool ofATP inless than aminute. That turnover represents 10million moleculesof ATP consumed and regenerated per second per cell. If ATPcould not be regenerated by the phosphorylation of ADP, hu-mans would use up nearly their body weight in ATP each day.

(e) Overall free-energy change. Adding the AG (under standardconditions) for the amino acid conversion to the DoG for ATPhydrolysis gives the free-energy change for the overall reaction.Because the overall process is exergonic (AG is negative), itoccurs spontaneously.

(b) Coupled with AlP hydrolysis, an exergonic reaction. In thecell. glutamine synthesiS occurs in two steps, coupled by aphosphorylated intermediate. OATP phosphorylates glutamicacid, making the amino acid less stable. f)Ammonia displacesthe phosphate group, forming glutamine

... Figure 8.10 How ATP drives chemical work: Energycoupling using AlP hydrolysis. In this example, the exergonic processof ATP hydroly;is is used to drive an endergoruc process-the cellularsynth€SlS of the amino aCId glutamine from glutamIC acid and ammonia

are not unusually strong bonds, as may imply;rather, the reactants (ATP and water) themselves have high en-ergyrelativetotheenergyoftheproducts (ADPand ®J There-lease of energy during the hydrolysis of ATP comes from thechemical change to a state of lower free energy, not from thephosphate bonds themselves.ATP is useful to the cell because the energy it releases on

losing a phosphate group is somewhat greater than the energymost other molecules could deliver. But why does this hydrol-ysis release so much energy? If we reexamine the ATP mol-ecule in Figure 8.8, we can see that all three phosphate groupsare negatively charged. These like charges are crowded to-gether, and their mutual repulsion contributes to the instabil-ity of this region of the ATP molecule. The triphosphate tail ofATP is the chemical equivalent ofa compressed spring.

\'(fhen ATP is hydrolyzed in a test tube, the release of free en-ergy merely heats the surrounding water. In an organism, thissame generation of heat can sometimes be beneficial. For in-stance, the process of shivering uses ATP hydrolysis duringmuscle contraction to generate heat and warm the body. Inmost cases in the cell, however, the generation of heat alonewould be an inefficient (and potentially dangerous) use of avaluable energy resource. Instead, the cell's proteins harnessthe energy released during ATP hydrolysis in several ways toperform the three types ofcellular work-chemical, transport,and mechanical.For example, with the help of specific enzymes, the cell is

able to use the energy released by ATP hydrolysis directly todrive chemical reactions that, by themselves, are endergonic.If the 6.G ofan endergonic reaction is less than the amount ofenergy released by ATP hydrolysis, then the two reactions canbe coupled so that, overall, the coupled reactions are exergonic(Figure 8.10). This usually involves the transfer of a phos-phate group from ATP to some other molecule, such as the re-actant. The recipient of the phosphate group is then said to bephosphorylated. The key to coupling exergonic and ender-gonic reactions is the formation of this phosphorylated inter-mediate, which is more reactive (less stable) than the originalunphosphorylated molecule.Transport and mechanical work in the cell are also nearly

always powered by the hydrolysis ofATP. In these cases, ATPhydrolysis leads to a change in a protein's shape and often itsability to bind another molecule. Sometimes this occurs via aphosphorylated intermediate, as seen for the transport pro-tein in Figure 8.11a.ln most instances ofmechanical work in-volving motor proteins "walking" along cytoskeletal elements(Figure 8.11 b), a cycle occurs in which ATP is first boundnoncovalently to the motor protein. Next, ATP is hydrolyzed,releasing ADP and ®;; another ATP molecule can then bind.At each stage, the motor protein changes its shape and ability

How ATP PerformsWork

150 UNIT TWO The Cell

Motor protein Protein moved(b) Mechanical work: ATP binds noncovalentlyto motor proteins. then is hydrolyzed

8.JCONCEPT CHECK

Thus, the ATP cycle is a turnstile through which energypasses during its transfer from catabolic to anabolic pathways.In fact, the chemical potential energy temporarily stored inATP drives most cellular work.

I. In most cases, how does ATP transfer energy fromexergonic to endergonic reactions in the cell?

2. -W:r.s1ljM \Vhich of the following combinations hasmore free energy: glutamic acid + ammonia +ATp, orglutamine +ADP +®j? Explain your answer.For suggested answers. see Appendix A

Up metabolicreactions by lowering energybarriers

ADP•

Cytoskeletal track

o

r Solole Solole(a) Transport work: ATP phosphorylatestransport proteins

L:", .......

Membrane protein

.. Figure 8.12 The ATP cycle. Energy reteased by breakdownreactions (catabolism) in the cell is used to phosphorylate ADp, regeneratingAlP: Chemical potential energy stored in ATP drives most cellular work.

.. Figure 8.11 How ATP drives transport and mechanicalwork. ATP hydrolysis causes changes In the shapes and bindingaffinities of proteins. This can occur either (a) diredly. byphosphorylation. as shown for membrane proteins involved in adivetransport of solutes. or (b) indirectly. via noncovalent binding of ATPand its hydrolytic products. as is the case for motor proteins that movevesicles (and organelles) along cyloskeletal "tracks" in the cell.

H OH OH HSucrose (C12H22011)

TIle laws of thermodynamics tell us what will and will not hap-pen under given conditions but say nothing about the rate ofthese processes. A spontaneous chemical reaction occurs with-out any requirement for outside energy, but it may occur soslowly that it is imperceptible. For example, even though the hy-drolysis of sucrose (table sugar) to glucose and fructose is exer-gonic, occurring spontaneously with a release of free energy(AG = -7 kcallmol), asolution ofsucrose dissolved in sterile wa-ter will sit for years at room temperature with no appreciable hy-drolysis. However, if we add a small amount of the enzymesucrase to the solution, then all the sucrose may be hydrolyzedwithin seconds (Figure 8.13). How does the enzyme do this?

Energy for cellularwork (endergonic.energy-consumingprocesses)

AlP hydrolysis toADP +®,yields energy

ADP + (D,

Energy fromcatabolism (exergonic.energy-releasingprocesses)

AlP synthesis fromADP + (D, requires energy

Because both directions of a reversible process cannot godownhill, the regeneration ofATP from ADP and ®; is neces-sarilyendergonic:

ADP + @lj---> ATP + H20AG = +7.3 kcal/mol (+30.5 kl/mol) (standard conditions)

Because ATP formation from ADP and ®; is not sponta-neous, free energy must be spent to make it occur. Catabolic(exergonic) pathways, especially cellular respiration, providethe energy for the endergonic process of making ATP. Plantsalso use light energy to produce ATP.

H OHGlucose (C;;H 120 6)

.. Figure 8.13 Example of an enzyme.catalyzed reaction:hydrolysis of sucrose by sucrase.

CIiAPTER EIGIiT An Introduction to Metabolism 151

Progress of the readlon _

.. Figure 8.14 Energy profile of an exergonic reaction. The"molecules" are hypothetical. with A. S, C. and Drepresentingportions of the molecules. Thermodynamically, this is an exergonicreaction. with a negative and the readion occurs spontaneously.However. the activation energy (EA) provides a barrier thatthe rate of the reaction."];""",,. Graph the progress of an endergonic reaction in whichEF and GH form products EG and FH. assuming that the reactantsmust pass through a transition state.

After bonds havebroken, new bondsform, releasing energyto the surroundings.

The reactants AB and CD must absorbenough energy from the surroundingsto reach the unstable transition state,where bonds can break.

Transition state

the reactant molecules, so they collide more often and moreforcefully. Also, thermal agitation of the atoms within the mol-ecules makes the bonds more likely to break. As the atoms settleinto their new, more stable bonding arrangements, energy is re-leased to the surroundings. Ifthe reaction is exergonic, EA will berepaid with interest, as the formation of new bonds releasesmore energy than was invested in the breaking ofold bonds.The reaction shown in Figure 8.14 is exergonic and occurs

spontaneously. However, the activation energy provides a bar-rier that determines the rate of the reaction. The reactants mustabsorb enough energy to reach the top of the activation energybarrier before the reaction can occur. For some reactions, EA ismodest enough that even at room temperature there is sufficientthermal energy for many ofthe reactants to reach the transitionstate in a short time. In most cases, however, EA is so high andthe transition state is reached so rarely that the reaction willhardly proceed at all. In these cases, the reaction will occur at anoticeable rate only ifthe reactants are heated. For example, thereaction ofgasolineand oxygen isexergonic and will occurspon-taneously, but energy is required for the molecules to reach thetransition state and react. Only when the spark plugs fire in anautomobile engine can there be the explosive release of energythat pushes the pistons. \Vithout a spark, a mixture ofgasoline

AS + CD---+ AC + SD

The Activation Energy Barrier

An enzyme is a macromolecule that acts as a catalyst, achemical agent that speeds up a reaction without being con-sumed by the reaction. In this chapter, we are focusing on en-zymes that are proteins. (RNA enzymes, also called ribozymes,are discussed in Chapters 17 and 25.) In the absence of regula-tion by enzymes, chemical traffic through the pathways ofme-tabolism would become terribly congested because manychemical reactions would take such a long time. In the next twosections, wewHi see what impedesaspontaneous reaction fromoccurring faster and how an enzyme changes the situation.

Every chemical reaction between molecules involves bothbond breaking and bond forming. For example, the hydrolysisof sucrose involves breaking the bond between glucose andfructose and one of the bonds of a water molecule and thenforming two new bonds, as shown in Figure 8.13. Changingone molecule into another generally involves contorting thestarting molecule into a highly unstable state before the reac-tion can proceed. This contortion can be compared to thebending ofa metal key ring when you pry it open to add a newkey. The key ring is highly unstable in its opened form but re-turns to astable state once the key is threaded all the way ontothe ring. To reach the contorted state where bonds canchange, reactant molecules must absorb energy from theirsurroundings. When the new bonds ofthe product moleculesform, energy is released as heat, and the molecules return tostable shapes with lower energy than the contorted state.The initial investmentofenergy for starting a reaction-the

energy required to contort the reactant molecules so thebonds can break-is known as the free energy ofactivation, oractivation energy, abbreviated EA in this book. We can thinkof activation energy as the amount of energy needed to pushthe reactants over an energy barrier, or hill, so that the "down-hill" part ofthe reaction can begin. Figure 8.14 graphs the en-ergy changes for a hypothetical exergonic reaction that swapsportions of two reactant molecules:

The energizing, or activation, of the reactants is representedby the uphill portion of the graph, in which the free-energycontent of the reactant molecules is increasing. At the sum-mit, the reactants are in an unstable condition known as thetransition state: They are activated, and their bonds can bebroken. The subsequent bond-forming phase of the reactioncorresponds to the downhill part of the curve, which showsthe loss of free energy by the molecules.Activation energy isoften supplied in the form ofheat that the

reactant molecules absorb from the surroundings. The bonds ofthe reactants break only when the molecules have absorbedenough energy to become unstable-to enter the transitionstate. The absorption of thermal energy increases the speed of

152 UNIT TWO The Cell

Sucrase +Glucose +Fructose

Enzyme +Product(s)

Sucrase-sucrose-H20complex

(b) When the substrate enters the active site, itinduces a change in the shape of the proteinThiS change allows more weak bonds toform, causing the active site to enfold thesubstrate and hold it in place,

Enzyme +Substrate(s)

Sucrase +Sucrose +H,O

Enzyme-substratecomplex

For example, the enzyme sucrase (most enzyme names endin -ase) catalyzes the hydrolysis of the disaccharide sucrose intoits twomonosaccharides, glucoseand fructose (see Figure 8.13):

Substrate Specificity of Enzymes

The reaction catalyzed by each enzyme is very specific; an en-zyme can recognize its specific substrate even among closelyrelated compounds, such as isomers. For instance, sucrase willact only on sucrose and will not bind to other disaccharides,such as maltose. What accounts for this molecular recogni-tion? Recall that most enzymes are proteins, and proteins aremacromolecules with unique three-dimensional configura-tions. The specificity of an enzyme results from its shape,which is a consequence of its amino acid sequence.Only a restricted region of the enzyme molecule actually

binds to the substrate. This region, called the activc sitc, is typi-callya pocketorgroove on the surface ofthe protein wherecatal-ysis occurs (Figure 8.16a). Usually, the active site is formed by

The reactant an enzyme acts on is referred to as the en-zyme's substrate. The enzyme binds to its substrate (or sub-strates, when there are two or more reactants), forming anenzyme-substrate complex. While enzyme and substrateare joined, the catalytic action ofthe enzyme converts the sub-strate to the product (or products) of the reaction. The over-all process can be summarized as follows:

lEA withenzymeis lower

6G is unaffectedby enzyme

(3) In this computer graphic model, the activesite of this enzyme (hexokinase, shown inblue) forms a groove on its surface Itssubstrate is glucose (red).

... Figure 8.16 Induced fit between an enzyme and its substrate.

Products

Progress of the reaction -----+-

Course ofreactionwith enzyme

(ourse ofreactionwithoutenzyme

1

How Enzymes lower the EA Barrier

hydrocarbons and oxygen will not react because the EA barrieris too high.

Proteins, DNA, and other complex molerules ofthe cell are rich infree energy and have the potential to decompose spontaneously;that is, the laws of thermodynamics favor their breakdown. Thesemolecules persist onlybecause at temperatures typical for cells, fewmolecules canmake it over the humpofactivation energy. Hoo'ever,the barriers for selected reactions must occasionally be surmountedfor cells to carry out the processes needed for life. Heat speeds a re-action byallowing reactants to attain the transition state more often,but this solution woukl be inappropriate for biological systems. FIrst,high temperature denatures proteins and kills ceUs. Second, heatwould speed up all reactions, not justthose that are needed, Organisms there-fore use an alternative: catalysis.An enzyme catalyzes a reaction by

lowering the EA barrier (Figure 8.15), en-abling the reactant molecules to absorbenough energy to reach the transitionstate even at moderate temperatures. Anenzyme cannotchange the AG for a reac-tion; it catmot make an endergonic reac-tion exergonic. Enzymes can only hastenreactions that would occur eventuallyanyway, but this function makes it possi-ble for the cell to have adynamic metabo-lism, routingchemicalssmoothly throughthe cell's metabolic pathways. And be-causeenzymes are very specific for the re-actions they catalyze, they determinewhich chemical processeswill begoingonin the cell at any particular time.

... Figure 8.15 The effect of an enzyme on activationenergy. Without affecting the free-energy change (boG) for a reaction,an enzyme speeds the reaction by reducing its activation energy (EA).

CIiAPTER EIGIiT An Introduction to Metabolism 153

only a few ofthe enzyme's amino acids, with the rest of the pro-tein molecule providing a framework that determines the con-figuration of the active site. The specificity of an enzyme isattributed to a compatible fit between the shape of its active siteand the shape of the substrate. The active site, however, is not arigid receptacle for the substrate. As the substrate enters the ac·tive site, interactions bety,'een its chemical groups and those onthe Rgroups {sidechains} ofthe amino acids that form theactivesite of the protein cause the enzyme to change its shape slightlyso that tile active site fits even more snugly around the substrate(Figure 8.16b). This induced fit is like a clasping handshake. In-duced fit brings chemical groups of the active site into positionsthat enhance their ability to catalyze the chemical reaction.

Catalysis in the Enzyme's Active SiteIn most enzymatic reactions, the substrate is held in the activesite by so-called weak interactions, such as hydrogen bondsand ionic bonds. Rgroups of a few of the amino acids thatmake up the active site catalyze the conversion ofsubstrate toproduct, and the product departs from the active site. The en·zyme is then free to take another substrate molecule into itsactive site. The entire cycle happens so fast that a single en-zyme molecule typically acts on about a thousand substrate

molecules per second. Some enzymes are much faster. En-zymes, like other catalysts, emerge from the reaction in theiroriginal form. Therefore, very small amounts of enzyme canhave a huge metabolic impact by functioning over and overagain in catalytic cycles. Figure 8.17 shows a catalytic cycleinvolving two substrates and two products.Most metabolic reactions are reversible, and an enzyme can

catalyze either the forward or the reverse reaction, dependingon which direction has a negative This in turn dependsmainlyon the relative concentrations ofreactants and products.The net effect is always in the direction ofequilibrium.Enzymes use a variety ofmechanisms that lower activation

energyand speed up a reaction (see Figure 8.17, step f)). First,in reactions involving two or more reactants, the active siteprovides a template on which the substrates can come to-gether in the proper orientation for a reaction to occur be-tween them. Second, as the active site ofan enzyme clutchesthe bound substrates, the enzyme may stretch the substratemolecules toward their transition·state form, stressing andbending critical chemical bonds that must be broken duringthe reaction. Because EA is proportional to the difficulty ofbreaking the bonds, distorting the substrate helps it approachthe transition state and thus reduces the amount offree energythat must be absorbed to achieve that state.

Enzyme-substratecomplex

... Figure 8.17 Theactive site andcatalytic cycle of anenzyme. An enzymecan convert one or morereactant molecules toone or more productmolecules. The enzymeshown here converts twosubstrate molecules totwo product molecules.

154 UNIT TWO The Cell

oSubstrates enter active site; enzymechanges shape such that its active siteenfolds the substrates (induced fit).

Enzyme

"Products are I -----"'''''d. I ,

Products

f)Substrates are heldin active site by weakinteractions, such ashydrogen bonds andionic bonds.

f)Active site can lower EAand speed up a reaction by• acting as a template forsubstrate orientation,• stressing the substratesand stabilizing thetransition state,• providing a favorablemicroenvironment, ami/or• participating directly in thecatalytic reaction.

oSubstrates areconverted toproducts.

The thermal agitation of the enzyme molecule disrupts thehydrogen bonds, ionic bonds, and other weak interactions thatstabilize the active shape of the enzyme, and the protein mol-ecule eventually denatures. Each enzyme has an optimal tem-perature at which its reaction rate is greatest. Withoutdenaturing the enzyme, this temperature allows the greatestnumber of molecular collisions and the fastest conversion ofthe reactants to product molecules. Most human enzymeshave optimal temperatures of about 35-40"C (dose to humanbody temperature). The thermophilic bacteria that live in hotsprings contain enzymeswith optimal temperatures of70'C orhigher (figure 8.18a).Just as each enzyme has an optimal temperature, it also has

a pH at which it is most active. The optimal pH values for mostenzymes fall in the range of pH 6-8, but there are exceptions.For example, pepsin, a digestive enzyme in the human stom-ach, works best at pH 2. Such an acidic environment denaturesmost enzymes, but pepsin is adapted to maintain its func-tional three-dimensional structure in the acidic environmentof the stomach. In contrast, trypsin, a digestive enzyme resid-ing in the alkaline environment of the human intestine, has anoptimal pH of 8 and would be denatured in the stomach(figure 8.18b).

Third, the active site may also provide a microenvironmentthat is more conducive to a particular type of reaction than thesolution itself would be without the enzyme. For example, ifthe active site has amino acids with acidic Rgroups, the activesite may be a pocket of low pH in an otherwise neutral cell. Insuch cases, an acidic amino acid may facilitate H+ transfer tothe substrate as a key step in catalyzing the reaction.A fourth mechanism of catalysis is the direct participation

of the active site in the chemical reaction. Sometimes thisprocess even involves briefcovalent bonding between the sub-strate and an Rgroup of an amino acid of the enzyme. Subse-quent steps of the reaction restore the R groups to theiroriginal states, so that the active site is the same after the reac-tion as it was before.The rate at which a particular amount of enzyme converts

substrate to product is partly a function of the initial concen-tration of the substrate: The more substrate molecules thatare available, the more frequently they access the active sitesofthe enzyme molecules. However, there is a limit to how fastthe reaction can be pushed by adding more substrate to afixed concentration of enzyme. At some point, the concen-tration of substrate will be high enough that all enzyme mol-ecules have their active sites engaged. As soon as the productexits an active site, another substrate molecule enters. At thissubstrate concentration, the enzyme is said to be saturated,and the rate of the reaction is determined by the speed atwhich the active site converts substrate to product. When anenzyme population is saturated, the only way to increase therate of product formation is to add more enzyme. Cells some-times increase the rate of a reaction by producing more en-zyme molecules.

Effects of Local Conditions on Enzyme Activity

t Optimal temperature fortypical human enzyme

Optimal temperature forenzyme of thermophilic

(heat-tolerant)bacteria

a 20 40 60 80 100Temperature (0C) _

(a) Optimal temperature for two enzymes

... Figure 8.18 Environmental factors affecting enzymeactivity. Each enzyme has an optimal (a) temperature and (b) pHthat favor the most active shape of the protein molecule.••I.p.\\I"1 Given that a mature /y5osome has an internal pH ofaround 4.5, draw a curve in (b) showing what you would predict for a/y5osomal enzyme, labeling its optimal pH.

023456pH_

(b) Optimal pH for two enzymes

9 1087

Optimal pH for pepsin(stomach enzyme)t

Effects ofTemperature and pHRecall from Chapter 5 that the three-dimensional structuresof proteins are sensitive to their environment. As a conse-quence, each enzyme works better under some conditionsthan under others, because these optimal conditions favor themost active shape for the enzyme molecule.Temperature and pH are environmental factors important

in the activity ofan enzyme. Up to a point, the rate ofan enzy-matic reaction increases with increasing temperature, partlybecause substrates collide with active sites more frequentlywhen the molecules move rapidly. Above that temperature,however, the speed of the enzymatic reaction drops sharply.

The activity of an enzyme-how efficiently the enzymefunctions-is affected by general environmental factors,such as temperature and pH. It can also be affected bychemicals that specifically influence that enzyme. In fact,researchers have learned much about enzyme function byemploying such chemicals.

CIiAPTER EIGIiT An Introduction to Metabolism 155

... Figure 8.19 Inhibition of enzyme activity.

cell often regulate enzyme activity by acting as inhibitors. Suchregulation-selective inhibition-is essential to the control ofcellular metabolism, as we discuss next.

Enzyme

Substrate

8.4

Noncompetitive inhibitor

(c) Noncompetitive inhibition

CONCEPT CHECK

A substrate canbind normally to theL----l

active site of anenzyme.

(b) Competitive inhibition

(a) Normal binding

A competitiveinhibitor mimics thesubstrate. competingfor the active site.

I. Many spontaneous reactions occur very slowly. \'(fhydon't all spontaneous reactions occur instantly?

2. Why do enzymes act only on very specific substrates?3. M'm'·i1iA Malonate is an inhibitor of the enzymesuccinate dehydrogenase. How would you determinewhether malonate is a competitive or noncompetitiveinhibitor?

For suggested answers. see Appendix A,

A noncompetitiveinhibitor binds to the

enzyme away from theactive site, altering theshape of the enzymeso that even if the

substrate can bind, theactive site functions

less effectively.

Cofactors

Certain chemicals selectively inhibit the action of specific en-zymes, and we have learned a lot about enzyme function bystudying the effects of these molecules. If the inhibitor at-taches to the enzyme by covalent bonds, inhibition is usuallyirreversible.Many enzyme inhibitors, however, bind to the enzyme by

weak interactions, in which case inhibition is reversible. Somereversible inhibitors resemble the normal substrate moleculeand compete for admission into the active site (figure 8.19aand b). These mimics, called competitive inhibitors, reducethe productivity of enzymes by blocking substrates from en-tering active sites. This kind of inhibition can be overcome byincreasing the concentration ofsubstrate so that as active sitesbecome available, more substrate molecules than inhibitormolecules are around to gain entry to the sites.In contrast, noncompetitive inhibitors do not directly

compete with the substrate to bind to the enzyme at the activesite (figure 8.19c).lnstead, they impede enzymatic reactionsby binding to another part of the enzyme. This interactioncauses the enzyme molecule to change its shape in such a waythat the active site becomes less effective at catalyzing the con-version of substrate to product.Toxins and poisons are often irreversible enzyme in-

hibitors. An example is sarin, a nerve gas that caused the deathof several people and injury to many others when it was re-leased by terrorists in the Tokyo subway in 1995. This smallmolecule binds covalently to the Rgroup on the amino acidserine, which is found in the active site ofacetylcholinesterase,an enzyme important in the nervous system. Other examplesinclude the pesticides DDT and parathion, inhibitors of keyenzymes in the nervous system. Finally, many antibiotics areinhibitors of specific enzymes in bacteria. For instance, peni-cillin blocks the active site of an enzyme that many bacteriause to make their cell walls.Citing enzyme inhibitors that are metabolic poisons may

give the impression that enzyme inhibition is generally abnor-mal and harmful. In fact, molecules naturally present in the

Many enzymes require nonprotein helpers for catalytic activ-ity. These adjuncts, called cofactors, may be bound tightly tothe enzyme as permanent residents, or they may bind looselyand reversibly along with the substrate. The cofactors of someenzymes are inorganic, such as the metal atoms zinc, iron, andcopper in ionic form. If the cofactor is an organic molecule, itis more specifically called a coenzyme. Most vitamins are im-portant in nutrition because they act as coenzymes or raw ma-terials from which coenzymes are made. Cofactors function invarious ways, but in all cases where they are used, they per-form a crucial function in catalysis. You'll encounter examplesof cofactors later in the book.

Enzyme Inhibitors

156 UNIT TWO The Cell

... Figure 8.20 Allosteric regulation of enzyme activity.

Stabilized inactiveform

Stabilized activeform

Allosteric activatorstabilizes active form.

•

Allosteric inhibitorstabilizes inactive form.

Stabilized active form

Substrate

..Inhibitor

Activator

Active site(one of four)

/

Active form

Inactive form

Allosteric enyzmewith four subunits

Non-----functional Inactive formactivesite(a) Allosteric activators and inhibitors. In the cell. activators andinhibitors diSSOCiate when at low concentrations. The enzymecan then oscillate agam.

Binding of one substrate molecule toactive site of one subunit locks allsubunits in active form.

(b) Cooperativity: another type of allosteric activation. Theinactive form shown on the left oscillates back and forthwith the active form when the active form is not stabilized bysubstrate.

cumulates and activates the enzymes that speed up catabolism,producing more ATP. If the supply of ATP exceeds demand,then catabolism slows down as ATP molecules accumulate andbind these same enzymes, inhibiting them. (You'll see specificexamples of this type of regulation when you learn about cellu-lar respiration in the next chapter.) ATP, ADP, and other related

In many cases, the molecules that naturally regulate enzyme ac-tivity in a cell behave something like reversible noncompetitiveinhibitors (see Figure 8.19<:): These regulatory molecules changean enzyme's shape and the functioning of its active site by bind-ing to a site elsewhere on the molecule, via noncovalent interac-tions. Allosteric regulation is the term used to describe anycase in which a protein's function at one site is affected by thebinding ofa regulatory molecule to a separate site. It may resultin either inhibition or stimulation ofan enzyme's activity.

Allosteric Regulation of Enzymes

Allosteric Activation and Inhibition

enzyme activityhelps control metabolism

Chemical chaos would result if all of a cell's metabolic path-ways were operating simultaneously. Intrinsic to the processof life is a cell's ability to tightly regulate its metabolic path-ways by controlling when and where its various enzymes areactive. It does this either by switching on and offthe genes thatencode specific enzymes (as we will discuss in Unit Three) Of,as we discuss here, by regulating the activity of enzymes oncethey are made.

Most enzymes known to be allosterically regulated are con-structed from two or more subunits, each composed of apolypeptide chain and having its own active site (figure 8.20).Each subunit has its own active site. The entire complex oscil-lates between two different shapes, one catalytically active andthe other inactive (figure a.20a). In the simplest case of al-losteric regulation, an activating or inhibiting regulatory mol-ecule binds to a regulatory site (sometimes called an allostericsite), often located where subunits join. The binding ofan act-ivator to a regulatory site stabilizes the shape that has func-tional active sites, whereas the binding ofan inhibitor stabilizesthe inactive form of the enzyme. The subunits ofan allostericenzyme fit together in such a way that a shape change in onesubunit is transmitted to all others. Through this interaction ofsubunits, a single activator or inhibitor molecule that binds toone regulatory site will affect the active sites of all subunits.Fluctuating concentrations ofregulators can cause a sophis-

ticated pattern of response in the activity ofcellular enzymes.The products of ATP hydrolysis (ADP and ®i)' for example,playa complex role in balancing the flow of traffic between an-abolic and catabolic pathways by their effects on key enzymes.ATP binds to several catabolic enzymes allosterically, loweringtheir affinity for substrate and thus inhibiting their activity.ADP, however, functions as an activator of the same enzymes.This is logical because a major function ofcatabolism is to re-generate ATP. IfATP production lags behind its use, ADP ac-

CIiAPTER EIGIiT An Introduction to Metabolism 157

Inactive form

5HActive form canbind substrate

•

Inhibitor

7

Substrate

Allostericallyinhibited form

Active form

It5H

Known active form

$Hbinding site.. Allosteric

Known Inactive form inhibitor Hypothesis: allostericinhibitor locks enzymein inactive form

To test this model, X-ray diffraction analysis was used to determinethe structure of caspase 1when bound to one of the inhibitorsand to compare it with the active and inactive structures,

CONCLUSION The inhibitory compound that was studied ap-parently locks the enzyme in its inactive form, as expected for atrue allosteric regulator, The data therefore support the existenceof an allosteric inhibitory site on caspase 1. which can be used tocontrol enzymatic activity,

SOURCE J M. Scheer EI al. Acommon alloslenc SOlE andmechanism in caspasl's, PNAS 103:7595-7600 (2006).

EXPERIMENT

Are there allosteric inhibitors of caspaseenzymes?

RESULTS Fourteen compounds were Identified that couldbind to the proposed allosteric site (red) of casP<lse 1and block en-zymatic activity. The enzyme's shape when one such inhibitor wasbound resembled the inactive caspase 1more than the active form,Caspase 1

In an effort to identify allosteric inhibitors ofcaspases. Justin Scheer and co·workers screened close to 8,000compounds for their ability to bind to a possible allosteric bindingsite in caspase 1and inhibit the enzyme's activity, Each compoundwas designed to form a disulfide bond with acysteine near thesite in order to stabilize the low-affinity interaction that ispected of an allosteric inhibitor. As the caspases are known to ex-ist in both active and inactive forms, the researchers hypothesizedthat this linkage might lock the enzyme in the inactive form.

• FI 8.21

Caspase 1

-·,1l:f.ilIA As a control, the researchers broke the disulfide link-age between one of the inhibitors and the caspase. Assumingthat the experimental solution contains no other inhibitors, howwould you expect the resulting caspase 1activity to be affected?

Identification ofAllosteric Regulators

Although allosteric regulation is probably quite widespread,relatively few ofthe many known metabolic enzymes have beenshown to be regulated in this way. Allosteric regulatory mole-cules are hard to characterize, in part because they tend to bindthe enzyme at low affinity and are thus hard to isolate. Recently,however, pharmaceutical companies have turned their atten-tion to allosteric regulators. These molecules are attractivedrug candidates for enzyme regulation because they exhibithigher specificity for particular enzymes than do inhibitors thatbind to the active site. (An active site may be similar to the ac-tive site in another, related enzyme, whereas allosteric regula-tory sites appear to be quite distinct between enzymes.)Figure 8.21 describes a search for allosteric regulators, car-

ried outas acollaboration between researchers at the Universityof California at San Francisco and a company called SunesisPharmaceuticals. The study was designed to find allosteric in-hibitors ofcaspases, protein-digesting enzymes that play an ac-tive role in inflammation and cell death. (You'll learn moreabout caspasesand cell death in Chapter 11.) By specifically reg-ulating these enzymes, we may be able to better manage inap-propriate inflammatory responses, such as those commonlyseen in vascular and neurodegenerative diseases.

molecules also affect key enzymes in anabolic pathways. In thisway, allosteric enzymes control the rates of key reactions inboth sorts of metabolic pathways.In another kind ofallosteric activation, asubstratemolecule

binding to one active site may stimulate the catalytic powers ofa multisubunit enzyme by affecting the other active sites(Figure 8.20b). If an enzyme has two or more subunits, a sub-strate molecule causing induced fit in one subunit can triggerthe same favorable shape change in all the other subunits of theenzyme. Called cooperativity, this mechanism amplifies theresponse of enzymes to substrates: One substrate moleculeprimes an enzyme to accept additional substrate moleculesmore readily.The vertebrate oxygen transport protein hemoglobin is a

classic example ofcooperativity. Although hemoglobin is notanenzyme, the study ofhow cooperative bindingworks in this pro-tein haselucidated the principle ofcooperativity. Hemoglobin ismade up of four subunits, each ofwhich has an oxygen-bindingsite (see Figure 5.21). The binding of an oxygen molecule toeach binding site increases the affinity for oxygen of the re-maining binding sites. Thus, in oxygen-deprived tissues, he-moglobin will be less likely to bind oxygen and will release itwhere it is needed. Where oxygen is at higher levels, such as inthe lungs or gills, the protein will have a greater affinity foroxygen as more binding sites are filled. An example of an en-zyme that exhibits cooperativity is the first enzyme in thepathway for pyrimidine biosynthesis in bacteria (this enzymeis called aspartyl transcarbamoylase).

158 UNIT TWO The Cell

.. Figure 8.22 Feedback inhibition in isoleucine synthesis.

8.5CONCEPT CHECK

1. How can an activator and an inhibitor have differenteffects on an allosterically regulated enzyme?

2. -WaUIA Imagine you are a pharmacological re-searcher who wants to design a drug that inhibits aparticular enzyme. Upon reading the scientific litera-ture, you find that the enzyme's active site is similar tothat of several other enzymes. What might be the bestapproach to developing your inhibitor drug?For suggested answers. see Appendix A.

The matrix containsenzymes in solution thatare involved in one stageof cellular respiration.

.. Figure 8.23 Organelles and structural order inmetabolism. Organelles such as these mitochondria (TEM) containenzymes that carry out specific functions. in this case cellular respiration.

Enzymes for anotherstage of cellularrespiration are

embedded in the innermembrane of the cristae.

into a multienzyme complex. The arrangement facilitates thesequence of reactions, with the product from the first enzymebecoming the substrate for an adjacent enzyme in the com-plex, and so on, until the end product is released. Some en-zymes and enzyme complexes have fixed locations within thecell and act as structural components of particular mem-branes. Others are in solution within specific membrane-enclosed eukaryotic organelles, each with its own internalchemical environment. For example, in eukaryotic cells, theenzymes for cellular respiration reside in specific locationswithin mitochondria (figure 8.23).In this chapter, you have learned that metabolism, the in-

tersecting set of chemical pathways characteristic of life, is achoreographed interplay ofthousands ofdifferent kinds ofcel-lular molecules. In the next chapter, we explore cellular respi-ration, the major catabolic pathway that breaks down organicmolecules, releasing energy for the crucial processes of life.

Enzyme 1(threonmedeaminase)

ThreonineIn active site

Initial substrate(threonine)

End product(isoleucine)

Intermediate A!'",m, ,Intermediate B!'",m, 3Intermediate (!'",m, 4Intermediate D!'",m, 5

Active site ofenzyme 1 nolonger bindsthreonine;pathway isswitched off

j Actlve siteavailable

Feedbackinhibition

Isoleucineused up bycell

Isoleucinebmds toallostericsite

Specific localization of Enzymes Within the CellThe cell is not just a bag of chemicals with thousands of dif-ferent kinds ofenzymes and substrates in a random mix. Thecell is compartmentalized, and cellular structures help bringorder to metabolic pathways. In some cases, a team of en-zymes for several steps ofa metabolic pathway are assembled

Feedback Inhibition

When ATP allosterically inhibits an enzyme in an ATP-generating pathway, as we discussed earlier, the result is feed-back inhibition, a common method of metabolic control. Infeedback inhibition, a metabolic pathway is switched off bythe inhibitory binding of its end product to an enzyme that actsearly in the pathway. figure 8.22 shows an example ofthis con-trol mechanism operating on an anabolic pathway. Some cellsuse this five-step pathway to synthesize the amino acidisoleucine from threonine, another amino acid. As isoleucineaccumulates, itslowsdown its ownsynthesis by allosterically in-hibiting the enzyme for the first step of the pathway. Feedbackinhibition thereby prevents the cell from wasting chemical re-sources by making more isoleucine than is necessary.

ClIAPTER EIGHT An Introduction to Metabolism 159

_,m,lt··

M' lilll'_ 8.4

lEA withillower

il.G il unaffectedby

Products

•

ofreactionwithout

••

•!III ofreaction

u. with enzyme

,\eIMty of ATPA'tivity Readions and ATP

.. How AlP Perfonns Work ATP hydrolysis drives endergonic re-actions by phosphorylation, the transfer ofa phosphate group tospecific reactants, making them more reactive. ATP hydrolysis(sometimes with protein phosphorylation) alsocauses changes inthe shape and binding affinities oftmnsport and motor proteins.

.. The Regeneralion of ATP Catabolic pathways drive the re-generation of ATP from ADP and phosphate.

Enzymes speed up metabolic reactions by loweringenergy barriers (pp. 151-156).. The Activation Energy Barrier In a chemical reaction, theenergy necessary to break the bonds of the reactants is the ac-tivation energy, EA'

.. How Enzymes lower the EA Barrier

..C a ter/.1

SUMMARY OF KEY CONCEPTS

-w·If·• Go to the Study Area at www.miilsteringbio.(omfor6ioFlix3-D Animations, MP3 Tutors, Videos. Practice Tests, an eBook, and more.

MPJ Tuwr flaskA'tivity Energy Transformations

_i,liiiil_ 8.1

_'.IIIiI'_ 8.2

An organism's metabolism transforms matter and energy,subject to the laws of thermodynamics (pp. 142-145).. Organization of the Chemistry of life into MetabolicPathways Metabolism is the collection of chemical reactionsthat occur in an organism. Aided by enzymes, it follows inter-secting pathways. which may be catabolic (breaking downmolecules, releasing energy) or anabolic (building molecules,consuming energy).

.. Forms of Energy Energy is the capacity to cause change:some forms of energy do work by moving matter. Kinetic en-ergy is associated with motion. Potential energy is related tothe location or structure of matter and includes chemical en-ergy possessed by a molecule due to its structure.

... The laws of Energy Transformation The first law, conser-vation of energy, states that energy cannot be created or de-stroyed, only transferred or transformed. The second lawstates that spontaneous changes, those requiring no outside in-put of energy, increase the entropy (disorder) of the universe.

The free.energy change of a reaction tells us whether ornot the reaction occurs spontaneously (pp. 146-149).. Free-Energy Change, !l.G A living system's free energy isenergy that can do work under cellular conditions. Thechange in free energy (!l.G) during a biological process is re-lated directly to enthalpy change (tlH) and to the change inentropy (6S): 6G = 6H - TOS.

.. Free Stability, and Equilibrium Organisms live at theexpense o(free energy. During a spontaneous change, freeenergy decreases and the stability of a system increases. At max-imum stability, the system is at equilibrium and can do no work.

... Free Energy and Metabolism In an exergonic (sponta-neous) chemical reaction, the products have less free energythan the reactants (-6G). Endergonic (nonspontaneous) re-actions require an input of energy (+ !loG). The addition ofstarting materials and the removal of end products preventmetabolism from reaching equilibrium.

M'.IIIiI'_ 8.3AlP powers cellular work by coupling exergonicreactions to endergonic reactions (pp. 149-151).. The Slruclure and Hydrolysis of AlP ATP is the cell's en-ergy shuttle. Hydrolysis at its terminal phosphate group pro-duces ADP and phosphate and releases free energy.

Progress of the

.. Substrate Specificity of Enzymes Each type of enzyme has aunique active site that combines specifically with its substrate,the reactant molecule on which it acts. The enzyme changesshape slightly when it binds the substrate (induced fit).

.. Catalysis in the Enzyme's Active Site The active site canlower an EA barrier by orienting substrates correctly, strainingtheir bonds, providing a favorable microenvironment, andeven covalently bonding with the substrate.

.. Effects of local Conditions on Enzyme Activity Each enzymehas an optimal temperature and pH. Inhibitors reduce enzymefunction. A competitive inhibitor binds to the active site. while anoncompetitive inhibitor binds to a different site on the enzyme.

A'tivity HowHow Is the Rate of Enzyme

Biology Labs On-Line EnzymeLab

_111111'_ 8.5Regulation of enzyme activity helps controlmetabolism (pp. 157-159).. Allosleric Regulation of Enzymes Many enzymes are al-losterically regulated: Regulatory molecules, either activators

160 UNIT TWO The Cell

or inhibitors, bind to specific regulatory sites, affecting theshape and function of the enzyme. In cooperativity, binding ofone substrate molecule can stimulate binding or activity atother active sites. In feedback inhibition, the end product of ametabolic pathwdy allosterically inhibits the enzyme for a pre-vious step in the pathway.

... Specific Localization of EnzymesWithin the Cell Someenzymes are grouped into complexes, some are incorporatedinto membranes, and some are contained inside organelles,increasing the efficiency of metabolic processes.

TESTING YOUR KNOWLEDGE

SELF·QUIZ

I. Choose the pair ofterms that correctly completes this sentence:Catabolism is to anabolism as is to 'a. exergonic; spontaneous d. work; energyb. exergonic; endergonic e. entropy; enthalpyc. free energy; entropy

2. Most cells cannot harness heat to perform work becausea. heat is not a form of energy.b. cells do not have much heat; they are relatively cool.c. temperature is usually uniform throughout a cell.d. heat can never be used to do work.e. heat must remain constant during work.

3. Which of the following metabolic processes can occur withouta net influx of energy from some other process?a. AOP + ---> ATP +H20b. C6Hl20 6 +602 ---> 6 CO2 + 6 H20c. 6 CO2 + 6 H20---> C6Hl20 6 +602d. amino acids ---> proteine. glucose + fructose ---> sucrose

4. If an enzyme in solution is saturated with substrate, the mosteffective way to obtain a faster yield of products is toa. add more of the enzyme.b. heat the solution to 9O'C.c. add more substrate.d. add an allosteric inhibitor.e. add a noncompetitive inhibitor.

5. If an enzyme is added to a solution where its substrate andproduct are in equilibrium, what would occur?a. Additional product would be formed.b. Additional substrate would be formed.c. The reaction would change from endergonic toexergonic.

d. The free energy of the system would change.e. Nothing; the reaction would stay at equilibrium.

6. Some bacteria are metabolically active in hot springs becausea. they are able to maintain a lower internal temperature.b. high temperatures make catalysis unnecessary.c. their enzymes have high optimal temperatures.d. their enzymes are completely insensitive to temperature.e. they use molecules other than proteins or RNAs as theirmain catalysts.

7. ,.p.W"1 Using a series of arrows, draw the branchedmetabolic reaction pathway described by the following state-ments, and then answer the question at the end. Use red ar·rows and minus signs to indicate inhibition.l. can form either M or N.M can form O.o can form either P or R.P can form Q.Rcan form S.o inhibits the reaction of l. to form M.Q inhibits the reaction of 0 to form P.S inhibits the reaction of 0 to form R.

Which reaction would prevail if bothQ and 5 were presentin the cell in high concentrations?a.l.--->M d.O--->Pb. M--->O e. R--->Sc. l. ---> N

For Self·Quiz answers, see Appendix A.

-$1401'·. Visit the Study Area at www.masteringbio.com lor aPractice Test

EVOLUTION CONNECTION

8. A recent reviv-dl ofthe antievolutionary "intelligent design"argument holds that biochemical pathways are too complex tohave evolved, because all intermediate steps in agiven pathwaymust be present to produce the final product. Critique this ar-gument. How could you use the diversity of metabolic pathwaysthat produce the same or similar products to support your case?

SCIENTIFIC INQUIRY

9. "]itiW"1 A researcher has develope<! an assay to me-dsure theactivity ofan important enzyme present in liver cells beinggrownin culture. She adds the enzyme's substrate to a dish of cells andthen measures the appearance of reaction products. The resultsare graphed as the amount of product on the y-axis versus timeon the x-axis. The ll'SC"Jrcher notes four sectionsofthegraph For ashort perioo oftime, no products appear (section A). Then (section B)the reaction rate is quite high (the slope ofthe line issteep). Next,the reaction gradually slows down (section q. Finally, the graphline becomes flat (section 0). Draw and label the graph, and pro-pose a model to explain the molecular events occurring at eachstage of this reaction profile.

SCIENCE. TECHNOLOGY. AND SOCIETY

10. The EPA is evaluating the safety of the most commonly usedorganophosphate insecticides (organic compounds containingphosphate groups). Organophosphates typically interfere withnerve transmission by inhibiting the enzymes that degradetransmitter molecules diffusing from one neuron to another.Noxious insects are not uniquely susceptible; humans and othervertebrates can be affected as well. Thus, the use of organophos-phate pesticides creates some health risks. As a consumer, whatlevel of risk are you willing to accept in exchange for an abun-dant and affordable food supply?

CIiAPTER EIGllr An Introduction to Metabolism 161