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88 Chapter2: CellChemistryand Biosynthesis HOWCELLS OBTAIN ENERGY FROMFOOD The constant supply of energy that cells need to generate and maintain the biological order that keeps them alive comes from the chemical bond energy in food molecules, which thereby serve as fuel for cells. The proteins, lipids, and polysaccharidesthat make up most of the food we eat must be broken down into smaller molecules before our cells can use themeither as a source of energy or as building blocks for other molecules. EnzFnatic digestion breaks down the large polymeric molecules in food into their monomer subunits-proteins into amino acids, polysaccharides into sugars, and fats into fatty acids and glycerol. After d^igestion, the small orginic molecules derived from food enter the cltosol of cells, where their gradual oxidation begins. Sugars are particularly important fuel molecules, and they are oxidized in small controlled steps to carbon dioxide (coz) and water (Figure 2-69). In this section we trace the major steps in the breakdor.tm,or catabolism, of sugarsand show how they produce ATB NADH, and other activated carrier molecules in animal cells. A very similar pathway also operates in plants, fungi, and many bacteria. As we shall see, the oxidation of fatty acids is equally important for cells. other molecules, such as proteins, can also serve as energy sourceswhen they are funneled through appropriate enzymatic pathways. Glycolysis ls a CentralATP-Producing Pathway The major process for oxidizing sugars is the sequence of reactions known as verted into two molecules of pyruuate, each of which contains three carbon atoms. For each glucose molecule, two molecules of ATp are hydrolyzed to provide energy to drive the early steps, but four molecules of Arp are produced in the later steps.At the end of glycolysis,there is consequently a nef gain of two molecules of AIP for each glucose molecule broken down. The glycolltic pathway is outlined in Figure 2-zo and shown in more detail in Panel 2-8 (pp. I?}-IZL). Glycolysis involves a sequence of l0 separate reac_ tions, each producing a different sugar intermediate and each caialvzed bv a (A) stepwiseoxidation of sugar in cells I a q o E ( B ) d i r e c tb u r n i n go f s u g a r s m a l la c t i v a t i o ne n e r g i e s o v e r c o m ea t b o d y t e m p e r a t u r eo w i n g t o t h e presence of enzymes S U G A R+ O , q activated c ar n e r molecules store energy 1w allfree energy rs reteaSed as heat; none rs stored & C O ,+ H r O CO, + HrO Figure2-69 Schematicrepresentation of the controlled stepwiseoxidation of sugarin a cell,comparedwith ordinary burning,(A)In the cell,enzymescatalyze oxidationvia a seriesof smallsteosin whichfreeenergyis transferred in conveniently sizedpacketsto carrier molecules-mostoftenATPand NADH. At eachstep,an enzymecontrolsthe reactionby reducingthe activation energybarrierthat hasto be surmounted beforethe specificreactioncan occur. The total free energyreleasedis exactly the samein (A)and (B).But if the sugar wereinsteadoxidizedto CO2and H2Oin a singlestep,as in (B),it would release an amountof energymuch largerthan could be capturedfor usefulpurposes. 89 HOWCELLSOBTAINENERGY.FROM FOOD one molecule of glucose energy investment to be recoupeo later Figure2-70 An outlineof glycolysis' Eachofthe 10 stepsshownis <GGGC> catalyzedby a differentenzyme.Note sugar a six-carbon that step4 cleaves so that the sugars, into two three-carbon at everystageafter numberof molecules step6 begins this doubles.As indicated, the energygenerationphaseof of ATP two molecules glycolysis. Because in the early,energY arehydrolyzed resultsin the investmentphase,glycolysis of 2 ATPand 2 NADH net synthesis moleculesper moleculeof glucose(see alsoPanel2-B). fructose 1,6bisphosphate I I CHO CHOH CHOH I two moleculesof glyceraldehyde 3-phosphate i cH2o P I @ I a | I I I < two molecules of pyruvate srres CHO COO C:o I CHr srEPT ,',r, e srEP c l e a v a g eo f s i x - c rab o n sugarto two th ree-carbon s u g ar s I I t---* I I I I t I energy generation sTEP1O l- cooI I CH: different enzyrne. Like most enzymes, these have names ending in ase--such as isomerase and dehydro genase-,lo indicate the type of reaction they catalyze. Although no molecular oxygen is used in glycolysis,oxidation occurs, in that electrons are removed by NAD+ (producing NADH) from some of the carbons derived from the glucose molecule. The stepwise nature of the process releases the energy of oxidation in small packets,so that much of it can be stored in activated carrier molecules rather than all of it being released as heat (see Figure 2-69). Thus, some of the energy releasedby oxidation drives the direct slmthesis of ATP molecules from ADP and Pi, and some remains with the electrons in the high-energy electron carrier NADH. TWomolecules of NADH are formed per molecule of glucosein the course of glycolysis. In aerobic organisms (those that require molecular oxygen to live), these NADH molecules donate their electrons to the electron-transport chain described in Chapter 14, and the NAD+ formed from the NADH is used again for glycolysis (see step 6 in Panel 2-8, pp. 120-l2I). ProduceATPin the Absenceof Oxygen Fermentations For most animal and plant cells, glycolysis is only a prelude to the final stage of the breakdo'ornof food molecules. In these cells, the p],'ruvateformed by glycolysis is 90 Chapter2: CellChemistryand Biosynthesis rapidly transported into the mitochondria, where it is converted into co2 plus acetyl CoA, which is then completely oxidized to CO2and H2O. In contrast, for many anaerobic organisms-which do not utilize morecular oxygen and can grow and divide without it-glycolysis is the principal source of the cell'sArP This is also true for certain animal tissues,such as skeletalmuscle, that can continue to function when molecular oxygen is limiting. In these anaerobic conditions, the pyruvate and the NADH electrons stay in the cytosol. The pyruvate is converted into products excreted from the cell-for example, into ethanol and co2 in the yeastsused in brewing and breadmaking, or into lactate in muscle. In this process,the NADH gives up its electrons and is converted back into NAD+. This regeneration of NAD+ is required to maintain the reactions of glycolysis (Figure 2--7l). Anaerobic energy-yielding pathways like these are called fermentations. process.The piecing together of the complete glycolltic pathway in the 1930swas a major triumph of biochemistry, and it was quickly followed by the recognition of the central role of ArP in cell processes.Thus, most of the fundamentalioncepts discussed in this chapter have been understood for manv vears. (A) FERMENTATION LEADINGTO EXCRETION OF LACTATE grucose 2 ADF- .r -2l|{4q: \ E/ --E -E-; 2 x pyruvate oo\,, o. o\./ I I H- C-OH C C I I cH:r CH: 2 x lactate ( B ) F E R M E N T A T I OLN EADING T O E X C R E T I OONFA L C O H O L ANDCO. grucose - ^-' ........._.\ '6 t->I =- /.-' EDlr 2 x pyruvate o. o\// C I HC:O I CH: I CH: 2 x acetaldehyde H-, C l -OH CH: 2x COz 2 x ethanol Figure2-71 Two pathwaysfor the anaerobicbreakdownof pyruvate. (A)Whenthereis inadequate oxygen,for example,in a musclecellundergoing vigorouscontraction, the pyruvate producedby glycolysisis convertedto lactateas shown.Thisreactton regenerates the NADt consumedin step 6 of glycolysis,but the whole pathway yieldsmuch lessenergyoverallthan completeoxidation.(B)In some organisms that can grow anaerobically, suchasyeasts,pyruvateis convertedvia acetaldehyde into carbondioxideand ethanol.Again,this pathwayregenerates NAD+from NADH,as requiredto enable glycolysis to continue.Both(A)and (B) are exampfes of fermentations. HOWCELLS OBTAIN ENERGY FROMFOOD Glycolysis lllustrates HowEnzymes CoupleOxidationto Energy Storage Returning to the paddle-wheel analogy that we used to introduce coupled reactions (see Figure 2-56), we can now equate enzymes with the paddle wheel. Enzymes act to harvest useful energy from the oxidation of organic molecules by coupling an energetically unfavorable reaction with a favorable one. To demonstrate this coupling, we examine a step in glycolysis to see exactly how such coupled reactions occur. TWo central reactions in glycolysis (steps 6 and 7) convert the three-carbon sugar intermediate glyceraldehyde3-phosphate (an aldehyde) into 3-phosphoglycerate(a carboxylic acid; seePanel2-8, pp. 120-121).This entails the oxidation of an aldehyde group to a carboxylic acid group in a reaction that occurs in two steps.The overall reaction releasesenough free energy to convert a molecule of ADP to AIP and to transfer two electrons from the aldehyde to NAD* to form NADH, while still releasing enough heat to the environment to make the overall reaction energeticallyfavorable (AG for the overall reaction is -3.0 kcal/mole). Figure 2-72 otttlines the means by which this remarkable feat of energy harvesting is accomplished. The indicated chemical reactions are precisely guided by two enzymes to which the sugar intermediates are tightly bound. In fact, as detailed in Figure 2-72, the first enzyme (glyceraldehyde 3-phosphate dehydrogenase) forms a short-lived covalent bond to the aldehyde through a reactive -SH group on the enzyme, and catalyzes its oxidation by NAD+ in this attached state. The reactive enzyme-substrate bond is then displaced by an inorganic phosphate ion to produce a high-energy phosphate intermediate, which is released from the enzyme. This intermediate binds to the second enzyme (phosphoglycerate kinase), which catalyzesthe energetically favorable transfer of the high-energy phosphate just created to ADB forming AIP and completing the process of oxidizing an aldehyde to a carboxylic acid. We have shown this particular oxidation process in some detail because it provides a clear example of enzyme-mediated energy storage through coupled reactions (Figure 2-73). Steps 6 and 7 are the onlyreactions in glycolysis that create a high-energy phosphate linkage directly from inorganic phosphate. As such, they account for the net yield of two AIP molecules and two NADH molecules per molecule of glucose (seePanel 2-8, pp.l20-I2l). As we have just seen,AIP can be formed readily from ADP when a reaction intermediate is formed with a phosphate bond of higher-energy than the phosphate bond in AIP Phosphatebonds can be ordered in energy by comparing the standard free-energy change (AGl for the breakage of each bond by hydrolysis. Figure 2-74 compares the high-energy phosphoanhydride bonds in ATP with the energy of some other phosphate bonds, several of which are generated during glycolysis. in SpecialReservoirs StoreFoodMolecules Organisms All organisms need to maintain a high ATP/ADP ratio to maintain biological order in their cells. Yet animals have only periodic accessto food, and plants need to survive overnight without sunlight, when they are unable to produce sugar from photosynthesis. For this reason, both plants and animals convert sugars and fats to special forms for storage (Figure 2-75). To compensate for long periods of fasting, animals store fatty acids as fat droplets composed of water-insoluble triacylglycerols,largely in the cytoplasm of specialized fat cells, called adipocltes. For shorter-term storage, sugar is stored as glucose subunits in the large branched polysaccharide glycogen, which is present as small granules in the cltoplasm of many cells,including liver and muscle. The synthesis and degradation of glycogen are rapidly regulated according to need. \.A/hencells need more AIP than they can generate from the food molecules taken in from the bloodstream, they break down glycogen in a reaction that produces glucose 1-phosphate,which is rapidly converted to glucose 6-phosphate for glycolysis. 91 92 Chapter2: CellChemistryand Biosynthesis (A) HO \ /(_ / I H-C-OH glyceraldehyde 3-phosphate SH A covalent bond is formed between glyceraldehyde3-phosphate(the substrate)and the -5H group of a cysteineside chain of the enzyme glyceraldehyde3-phosphate d e h y d r o g e n a s ew,h i c h a l s ob i n d s noncovalentlyto NAD+. t-? I H -C-OH I H_C_OH I Oxidation of glyceraldehyde 3-phosphateoccurs,as two electronsplus a proton (a hydride ion, see Figure2-60) are transferredfrom glyceraldehyde 3-phosphateto the bound NAD+, forming NADH.Part of the energy releasedby the oxidation of the a l d e h y d ei s t h u s s t o r e di n N A D H , and part goes into convertingthe bond between the enzymeand its substrateglyceraldehyde 3-phosphateinto a high-energy thioester bond cH2oo (o c r F I H-C-OH HO- A m o l e c u l eo f i n o r g a n i cp h o s p h a t e displacesthe high-energybond to the enzymeto create 1,3-bisphosphoglycerate,which contains a high-energyacyl-anhydride bond. PIO ti rU- iAL J 1,3-bisphosphoglycerate C:O I H-C-OH l^ cH2o(L) @@o F 4 H r fr_A-r, C I H- C-OH l^ cH2o(L) (B) 3 - p h o s p h olgy c e r a t e The high-energybond to phosphate is transferredto ADP to form ATP. SUMMARYOF STEPS 6 AND 7 Much of the energy of oxidation has been stored in the activateo carriersATPand NADH. Figure2-72 Energystoragein steps6 and 7 of glycolysis.In thesestepsthe oxidationof an aldehydeto a carboxylic acidis coupledto the formationof ATP and NADH.(A)Step6 beginswith the formationof a covalentbond between the substrate(glyceraldehyde 3-phosphate) and an -5H groupexposed on the surfaceof the enzyme (glyceraldehyde 3-phosphate dehydrogenase). Theenzymethen catalyzestransferof hydrogen(asa hydrideion-a protonplustwo electrons) from the bound glyceraldehyde 3-phosphate to a moleculeof NAD+.Partof the energy released in this oxidationis usedto form a moleculeof NADHand part is usedto convertthe originallinkagebetweenthe enzymeand its substrate to a highenergythioesterbond (shownin red.). A moleculeof inorganicphosphatethen displaces this high-energy bond on the enzyme,creatinga high-energy sugarphosphatebond instead(red).At this point the enzymehas not only stored energyin NADH,but alsocoupledthe energetically favorableoxidationof an aldehydeto the energetically unfavorable formationof a high-energy phosphate bond.Thesecondreactionhasbeen drivenby the first,therebyactinglikethe "paddle-wheel" couplerin Figure2-56. In reactionstep7, the high-energy just made, sugar-phosphate intermediate 1,3-bisphosphoglycerate, bindsto a secondenzyme,phosphoglycerate kinase.The reactivephosphateis transferredto ADP,forming a moleculeof ATPand leavinga freecarboxylic acid groupon the oxidizedsugar. (B)Summaryof the overallchemical changeproducedby reactions6 and 7. 93 HOWCELLSOBTAINENERGY FROMFOOD o ME o c o a o o Figure 2-73 Schematicview of the coupledreactionsthat form NADHand ATPin steps 6 and 7 of glycolysis.The C-H bond oxidationenergydrivesthe formationof both NADHand a highenergyphosphatebond.The breakageof bond then drivesATP the high-energy formation. \,/ C I \,, C I C-H bond oxidation energy + STEP6 7 STEP t o t a l e n e r g yc h a n g ef o r s t e p6 f o l l o w e d b y s t e p 7 i s a f a v o r a b l e- 3 k c a l / m o l e o-o \// CO enol phosphate bond rll H'' C : C - O - . P Hro -O- / ,/lo- p h o s p h o e n oply r u v a t e isee ianel Z-8,'pp. 120-121) - 14'6 (-61 e) oo tltl c-c-o y'P o- anhydride bondto carbon o Hro/ phosphate bond in creaTtne phosphate anhydride bondto phosphate (phosphoanhydride bond) a._a)- ooo ililtl i-"-i-o7i-"o- o- Hzo c r e a t i n eo h o s p h a t e ( a c t i v a t e dc a r r i e rt h a t storesenergy in muscle) (-43.0) for example, ATPwhen hydrolyzed tOADP -7.3 (-306) -10.3 /o- HO phosphoester bond for example, 1,3-bisphosphoglycerate (seePanel2-8) lll , -i-"vP-o- for example, g l u c o s e6 - p h o s p h a t e (seePanel2-8) ",/o- Hzo type of phosphatebond specificexamplesshowing the standardfree-energychange (AG') for hydrolysisof phosphatebond Figwe 2-74 Phosphatebonds have different energies.Examplesof differenttypes of phosphatebondswith their sitesof hydrolysisare shown in the moleculesdepictedon the left.Thoseitarting with a gray catbonatom show only part of a molecule.Examplesof molecules (kilojoules transfer in parentheses)'The in kilocalories changefor hydrolysis containingsuchbondsaregivenon the right,with the free-energy of a phosfhate group from one moleculeto anotheris energeticallyfavorableif the standardfree-energychange(AG')for hydrolysisof the Thus,a phosphategroup of the phosphatebond in the second. phosphatebond of the firstmoleculeis more negativethan that for hydrolysis to ADPto form ATPThe hydrolysisreactioncan be viewedas the transferof the phosphate is readilytransferredfrom 1,3-bisphosphoglycerate group to water. 94 Chapter2: CellChemistyand Biosynthesis 9rycogen g r a n u l e isn the cytoplasm o f a l i v e rc e l l b r a n c hp o i n t g l u c o s es u b u n i t s 1pm ql a 1,4-glycosidic bond in backbone ,,- Figure2-75 The storageof sugarsand fats in animaland plant cells.(A)The structures of starchand glycogen, the storageform of sugarsin plantsand animals,respectively. Botharestorage polymersof the sugarglucoseand differ only in the frequencyof branchpoints (theregioninyellowisshownenlarged below).Therearemanymore branchesin glycogenthan in starch.(B)An electron micrographshowsglycogengranulesin the cytoplasmof a livercell.(C)A thin sectionof a singlechloroplast from a plantcell,showingthe starchgranules and lipid(fatdroplets)that have accumulated asa resultof the biosyntheses occurringthere.(D)Fat droplets(stainedred)beginningto accumulate in developingfat cellsof an animal.(8,courtesyof RobertFletterick and DanielS.Friend;C,courtesyof K.Plaskitt; D,courtesyof RonaldM. Evans and PeterTotonoz.) o 1 , 6 - 9 l y c o s i dbi co n d at branch point / o-cH2 OH l;------., Quantitatively, fat is far more important than glycogen as an energy store for animals, presumably becauseit provides for more efficient storage.The oxidation of a gram of fat releasesabout twice as much energy as the oxidation of a gram of glycogen. Moreover, glycogen differs from fat in binding a great deal of water, producing a sixfold difference in the actual mass of glycogen required to store the same amount of energy as fat. An averageadult human stores enough glycogen for only about a day of normal activities but enough fat to last for nearly a month. If our main fuel reservoir had to be carried as glycogen instead of fat, body weight would increase by an averageof about 60 pounds. Although plants produce NADPH and Arp by photosynthesis,this important process occurs in a specialized organelle, called a chloroplast, which is isolated from the rest of the plant cell by a membrane that is impermeable to both types of activated carrier molecules. Moreover, the plant contains many other cellssuch as those in the roots-that lack chloroplasts and therefore cannot produce their or,rmsugars.Therefore, for most of its ATP production, the plant relies on an 50u. HOWCELLSOBTAINENERGY FROMFOOD .95 Figure 2-76 How the ATPneeded for most plant cell metabolismis made.In plants,the chloroplasts and mitochondria to supplycellswith collaborate metabolitesand ATP.(Fordetails,see Chapter14.) light chloroplast metabolites export of sugars from its chloroplasts to the mitochondria that are located in all cells of the plant. Most of the AIP needed by the plant is synthesized in these mitochondria and exported from them to the rest of the plant cell, using exactly the same pathways for the oxidative breakdor,rrnof sugars as in nonphotosynthetic organisms (Figure 2-76). During periods of excessphotosynthetic capacity during the day, chloroplasts convert some of the sugars that they make into fats and into starch, a polgner of glucose analogous to the glycogen of animals. The fats in plants are triacylglycerols, just like the fats in animals, and differ only in the types of fatty acids that predominate. Fat and starch are both stored in the chloroplast as reservoirs to be mobilized as an energy source during periods of darkness (see Figure 2-75C). The embryos inside plant seedsmust live on stored sources of energy for a prolonged period, until they germinate to produce leaves that can harvest the energy in sunlight. For this reason plant seeds often contain especially large amounts of fats and starch-which makes them a malor food source for animals, including ourselves (Figare 2-7 7), MostAnimalCellsDeriveTheirEnergyfrom FattyAcidsBetween Meals After a meal, most of the energy that an animal needs is derived from sugars derived from food. Excesssugars,if any, are used to replenish depleted glycogen stores,or to synthesizefats as a food store. But soon the fat stored in adipose tissue is called into play, and by the morning after an overnight fast, fatty acid oxidation generatesmost of the ATP we need. Low glucose levels in the blood trigger the breakdown of fats for energy production. As illustrated in Figure 2-78, the triacylglycerols stored in fat droplets in adipocl'tes are hydrolyzed to produce fatty acids and glycerol, and the fatty acids released are transferred to cells in the body through the bloodstream. \.\hile animals readily convert sugars to fats, they cannot convert fatty acids to sugars.Instead, the fatty acids are oxidized directly. Figure2-77 SomePlant seedsthat serveas important foods for humans. Corn,nuts,and Peasall containrich storesof starchand fat that providethe youngplantembryoin the seedwith energyand buildingblocksfor (Courtesy of the JohnInnes biosynthesis. Foundation.) 96 Chapter 2:CellChemistry and Biosynthesis stored fat bloodstream glycerol MUSCLE CELL fatty acids o x i d a t i o ni n mitochondria Figure 2-78 How stored fats are mobilized for energy production in animals.Low glucoselevelsin the blood triggerthe hydrolysisof the triacylglycerolmoleculesin fat droplets to free fatty acidsand glycerol,as illustrated.Thesefatty acidsenter the bloodstream,wherethey bind to the abundantblood protein,serumalbumin. Specialfatty acidtransportersin the plasmamembraneof cellsthat oxidize fatty acids,suchas musclecells,then pass thesefatty acidsinto the cytosol,from whichthey aremovedinto mitochondria for energyproduction(seeFigure2-80). ) Sugarsand FatsAre Both Degradedto AcetylCoAin Mitochondria The fatty acids imported from the bloodstream are moved into mitochondria, where all of their oxidation takes place ). Each molecule of fatty acid (as the activated molecule /a tty acyl coA) is broken down completely by a cycle of reactions that trims two carbons at a time from its carboxyl end, generating one molecule of acetyl coA for each turn of the cycle. A molecule of NADH and a molecule of FADH2 are also produced in this proces Sugars and fats are the major energy sources for most nonorganisms, including humans. However, most of the useful ene 8 t r i m e r so f lipoamide reductasetransacetylase +6 dimersof dihydrolipoyl dehydrogenase + 1 2 d i m e r so f pyruvatedecarboxylase o ,// cH.c si*ht{iiii acetyl coA (B) Figure 2-79 The oxidation of pyruvate to acetylCoA and COz.(A)The structure of the pyruvatedehydrogenase complex, whichcontains60 polypeptidechains. Thisis an exampleof a large multienzymecomplexin which reaction intermediatesare passeddirectlyfrom one enzymeto another.In eucaryotic cellsit is locatedin the mitochondrion. (B)The reactionscarriedout by the pyruvatedehydrogenase complex.The complexconvertspyruvateto acetylcoA in the mitochondrial matrix;NADHis also producedin this reaction.A, B,and C are the three enzymespyruvate decarboxylase,Iipoam ide reductasetronsacetylose,and dihydrolipoyI dehydrogenase,respectively.These enzymesareillustrated in (A);their activities arelinkedas shown. 97 HOWCELL5OBTAINENERGY FROMFOOD S u g a r sa n d polysaccharides Fats+fatty acids CYTOSOL Figure2-80 Pathwaysfor the production of acetyl CoAfrom sugarsand fats.The mitochondrionin lt is eucaryotic cellsis the placewhereacetylCoAis producedfrom both typesof majorfood molecules. occurand wheremostof its ATPis made. thereforethe olacewheremostof the cell'soxidationreactions in detailin Chapter14. arediscussed Thestructureand functionof mitochondria extracted from the oxidation of both types of foodstuffs remains stored in the acetyl CoA molecules that are produced by the two t)?es of reactions just described. The citric acid cycle of reactions, in which the acetyl group in acetyl CoA is oxidized to CO2and H2O,is therefore central to the energy metabolism of aerobic organisms. In eucaryotesthese reactions all take place in mitochondria. We should therefore not be surprised to discover that the mitochondrion is the place where most of the ATP is produced in animal cells. In contrast, aerobic bacteria carry out all of their reactions in a single compartment, the cytosol, and it is here that the citric acid cycle takes place in these cells. TheCitricAcidCycleGenerates NADHby OxidizingAcetylGroups to COz In the nineteenth century, biologists noticed that in the absence of air (anaerobic conditions) cells produce lactic acid (for example, in muscle) or ethanol (for example, in yeast), while in its presence (aerobic conditions) they consume 02 and produce CO2and H2O.Efforts to define the pathways of aerobic metabolism Figure2-81 The oxidation of fatty acids to acetyl CoA.(A)Electronmicrographof a lipid droplet in the cytoplasm(top),and the structureof fats (bottom).Fatsare The glycerolportion,to triacylglycerols. whichthreefattyacidsarelinked throughesterbonds,is shownherein areinsolublein waterand form blue.Fats fat largelipiddropletsin the specialized in whichthey are cells(calledadipocytes) stored.(B)The fatty acid oxidationcycle. The cycleis catalyzedby a seriesof four Each enzymesin the mitochondrion. turn of the cycleshortensthe fattyacid chain by two carbons(shownin red)and generatesone moleculeof acetylCoA and one moleculeeachof NADHand The structureof FADHzis FADHz. presentedin Figure2-838.(4, courtesy of Daniel5. Friend.) (B) fatty acyl CoA Rr,-CH2-CH2- CH2-C hydrocarbontail fatty acyl CoA shortenedby .:fl,- CH2 - C two carDons // o \s-coA / C H'?\ S -C 1pm o C- acetylCoA hydrirclrrbohtail o tlC e s t e rb o n d 6it Hzo o C- @ //o -c i$tct-tr-cu: cH 's hydrocarbon tail \? R-CH2-C-CH2-C. ,,o OHH r$,-CHr- Ct l- C HH hydrocarbontail C 2 o .5{6A 98 Chapter2:CellChemistryand Biosynthesis eventually focused on the oxidation ofpyruvate and led in 1937to the discovery of the citric acid cycle, also knoum as the tricarboxylic acid cycle or the Krebs cycle.Thecitric acid cycle accounts for about two-thirds of the total oxidation of carbon compounds in most cells, and its major end products are CO2and highenergy electrons in the form of NADH. The CO2 is released as a waste product, while the high-energy electrons from NADH are passed to a membrane-bound electron-transport chain (discussedin Chapter 14), eventually combining with 02 to produce H2O. Although the citric acid cycle itself does not use 02, it requires 02 in order to proceed because there is no other efficient way for the NADH to get rid of its electrons and thus regeneratethe NAD+ that is needed to keep the cycle going. The citric acid cycle takes place inside mitochondria in eucaryotic cells. It results in the complete oxidation of the carbon atoms of the acetyl groups in acetyl CoA, converting them into CO2. But the acetyl group is not oxidized directly. Instead, this group is transferred from acetyl CoA to a larger, four-carbon molecule, oxaloacetate,to form the six-carbon tricarboxylic acid, citric acid, for which the subsequent cycle of reactions is named. The citric acid molecule is then gradually oxidized, allowing the energy of this oxidation to be harnessedto produce energy-rich activated carrier molecules. The chain of eight reactions forms a cycle because at the end the oxaloacetate is regenerated and enters a new turn of the cycle, as shown in outline in Figure 2-82. we have thus far discussed only one of the three types of activated carrier molecules that are produced by the citric acid cycle, the NAD+-NADH pair (see Figure 2-60). In addition to three molecules of NADH, each turn of the cycle also produces one molecule of FADH2 (reduced flavin adenine dinucleotide) from FAD and one molecule of the ribonucleotide GTP (guanosine triphosphate) from GDP The structures of these two activated carrier molecules are illustrated in Figure 2-83. GTP is a close relative of ATB and the transfer of its terminal phosphate group to ADP produces one ATP molecule in each cycle. Like NADH, FADHz is a carrier of high-energy electrons and hydrogen. As we discussshortly, the energy that is stored in the readily transferred high-energy electrons of NADH and FADH2will be utilized subsequently for Arp production through the process of oxidatiue phosphorylation, the only step in the oxidative catabolism of foodstuffs that directly requires gaseousoxygen (oz) from the atmosphere. Panel 2-9 (pp. 122-123)presents the complete citric acid cycle.Water, rather than molecular oxygen, supplies the extra oxygen atoms required to make co2 from the acetyl groups entering the citric acid cycle.As illustrated in the panel, o -t - s-coA Hrc acetylCoA 2C t oxd toacelale 4C +H* / I 4C STEP1 *{ srEP2 -)l u4lllil-,,/ ./ V srEP8 5C \ srEP 3 r*E!E-f.- co, I 5C N E TR E S U L O T N ET U R NO FT H EC Y C L E P R O D U C ETSH R E EN A D H ,O N EG T BA N D O N EF A D H 2A, N D R E L E A S E TS W O M O L E C U L EOSF C O I Figure2-82 Simpleoverviewof the citric acid cycle.<TAGT>The reactionof acetylcoA with oxaloacetatestartsthe cycleby producingcitrate(citricacid).In eachturn of the cycle,two molecules of CO2are producedas wasteproducts,plus threemolecules of NADH,one molecule of GTP, and one moleculeof FADH2. The numberof carbonatomsin each intermediateis shown in a yellowbox. Fordetails,see Panel2-9 (pp. 122-123). guanine o- o ll c 2H- ???\'.,-i 2e OH ill - N - - c- t - r n c llrtll HrC-9 t-r-t--N-C\O H3C-C OH OH tl ,rN '' c,. ,rC'-N.' n CH, I' H-C -OH I H-C-OH I H-C-OH (B) three moleculesof water are split in each cycle,and the orygen atoms of some of them are ultimately used to make CO2. In addition to pyruvate and fatty acids, some amino acids pass from the cytosolinto mitochondria, where they are alsoconvertedinto acetylCoAor one of the other intermediatesof the citric acid cycle.Thus,in the eucaryoticcell,the mitochondrion is the center toward which all energy-yieldingprocesseslead, whether they begin with sugars,fats,or proteins. Both the citric acid cycle and glycolysisalso function as starting points for important biosynthetic reactionsby producing vital carbon-containing intermediates,such as oxaloacetateand a-ketoglutarate.Someof these substances produced by catabolism are transferredback from the mitochondrion to the cytosol,where they servein anabolicreactionsasprecursorsfor the synthesisof many essentialmolecules,such as amino acids (Figure244). H2C-O- -O- Figure2-83 The structuresof GTPand FADHz.(A)GTPand GDPare close relativesof ATPand ADP,respectively. (B)FADH2is a carrierof hydrogensand high-energyelectrons,like NADHand NADPH.lt is shown here in its oxidized form (FAD)with the hydrogen-canying atoms h ighlightedin yellow. nucleotides glucose6-phosp nur. / amrno sugars + fructose 6-phosphate ' glycolipids glycoproteins + /\ I + + + serine d i h y d r o x y a c e t o n+e PhosPhate 3-phosphoglycerate lipids a m i n oa c i d s pyrimidines phosphoenolpyruvate alanine .*- .- orrrlura" andthe citric Figure2-84Glycolysis acidcycleprovidethe precursors manyimportant neededto synthesize Theaminoacids, biologicalmolecules. andother lipids,sugars, nucleotides, hereasproducts-in molecules-shown for the many turnserveasthe precursors the cell.Eachb/ack macromoleculesof arrowinthisdiagramdenotesa single thered reaction; enzyme-catalyzed with arrowsgenerallyrepresentpathways to produce manystepsthatarerequired products. the indicated 100 Chapter2:CellChemistryand Biosynthesis ElectronTransportDrivesthe Synthesis of the Majorityof the ATP in MostCells Most chemical energy is released in the last step in the degradation of a food molecule. In this final process the electron carriers NADH and FADH2 transfer the electrons that they have gained when oxidizing other molecules to the electron-transport chain, which is embedded in the inner membrane of the mitochondrion (seeFigure 14-10).As the electrons pass along this long chain of specialized electron acceptor and donor molecules, they fall to successivelylower energy states.The energy that the electrons release in this process pumps H+ ions (protons) across the membrane-from the inner mitochondrial compartment to the outside-generating a gradient of H+ ions (Figure 2-85). This gradient servesas a source of energy,being tapped like a battery to drive a variety of energy-requiring reactions.The most prominent of these reactions is the generation of ATP by the phosphorylation of ADP At the end of this series of electron transfers, the electrons are passed to molecules of oxygen gas (Oz) that have diffused into the mitochondrion, which simultaneously combine with protons (H*) from the surrounding solution to produce water molecules. The electrons have now reached their lowest energy Ievel, and therefore all the available energy has been extracted from the oxidized food molecule. This process, termed oxidative phosphorylation (Figure 2-86), also occurs in the plasma membrane of bacteria. As one of the most remarkable achievements of cell evolution, it is a central topic of Chapter 14. In total, the complete oxidation of a molecule of glucose to H2O and CO2is used by the cell to produce about 30 molecules of ATP In contrast, only 2 molecules of ATP are produced per molecule of glucose by glycolysis alone. AminoAcidsand Nucleotides ArePartof the NitrogenCycle So far we have concentrated mainly on carbohydrate metabolism and have not yet considered the metabolism of nitrogen or sulfur. These two elements are important constituents of biological macromolecules. Nitrogen and sulfur atoms pass from compound to compound and between organisms and their environment in a seriesof reversible cycles. Although molecular nitrogen is abundant in the Earth's atmosphere, nitrogen is chemically unreactive as a gas.Only a few living speciesare able to incorporate it into organic molecules, a process called nitrogen fixation. Nitrogen fixation occurs in certain microorganisms and by some geophysical processes, such as lightning discharge.It is essentialto the biosphere as a whole, for without it life could not exist on this planet. Only a small fraction of the nitrogenous compounds in today's organisms, however, is due to fresh products of nitrogen fixation from the atmosphere. Most organic nitrogen has been in circulation for pyruvatefrom gl y c o l y s i s I Coz I N A D Hf r o m glycolysis I Oz I pyruvate :*Hl * P II t acetyl CoA ctTRtc ACID CYCLE CoA MITOCHONDRION 2eI -- u^rDAilvE PHOSPHORYLATION t Hzo Figure2-85 The generationof an H+gradientacrossa membraneby electron-transportreactions. A high-energy electron(derived, for example,from the oxidationof a metabolite) is passedsequentially by carriers A, B,and C to a lowerenergy state.In this diagramcarrierB is arranged in the membranein sucha way that it takesup H+from one sideand releases it to the otherasthe electronpasses. The resultis an H+gradient.As discussed in Chapter14,this gradientis an important form of energythat is harnessed by other membraneoroteinsto drivethe formationof ATP. Figure2-86 Thefinal stagesof oxidation of food molecules.Molecules of NADH (FADHz and FADH2 is not shown)are producedby the citricacidcycle.These activatedcarriers donatehigh-energy electrons that areeventuallyusedto reduceoxygengasto water. A majorportionof the energyreleased duringthe transferof theseelectrons alongan electron-transfer chainin the mitochondrial innermembrane(or in the plasmamembraneof bacteria) is harnessed to drivethe synthesis of ATPhencethe nameoxidative (discussed phosphorylation in Chapter14). 101 HOW CELLSOBTAINENERGY FROMFOOD some time, passing from one living organism to another. Thus present-day nitrogen-fixing reactions can be said to perform a "topping-up" function for the total nitrogen supply. Vertebrates receive virtually all of their nitrogen from their dietary intake of proteins and nucleic acids. In the body these macromolecules are broken down to amino acids and the components of nucleotides, and the nitrogen they contain is used to produce new proteins and nucleic acids-or utilized to make other molecules. About half of the 20 amino acids found in proteins are essential amino acids for vertebrates (Figure 2-87), which means that they cannot be synthesizedfrom other ingredients of the diet. The others can be so synthesized, using a variety of raw materials, including intermediates of the citric acid cycle as described previously.The essentialamino acids are made by plants and other organisms, usually by long and energeticallyexpensivepathways that have been lost in the course of vertebrate evolution. Roshanl(eab 02l-66950639 The nucleotides needed to make RNA and DNA can be synthesized using specializedbiosynthetic pathways. All of the nitrogens in the purine and pyrimidine bases (as well as some of the carbons) are derived from the plentiful amino acids glutamine, aspartic acid, and glycine, whereas the ribose and deoxyribose sugars are derived from glucose. There are no "essential nucleotides" that must be provided in the diet. Amino acids not used in biosynthesis can be oxidized to generatemetabolic energy.Most of their carbon and hydrogen atoms eventually form COz or HzO, whereas their nitrogen atoms are shuttled through various forms and eventually appear as urea, which is excreted.Each amino acid is processeddifferently, and a whole constellation of enzymatic reactions exists for their catabolism. Sulfur is abundant on Earth in its most oxidized form, sulfate (SOaz-).To convert it to forms useful for life, sulfate must be reduced to sulfide (S2-),the oxidation state of sulfur required for the synthesis of essential biological molecules. These molecules include the amino acids methionine and cysteine,coenzymeA (seeFigure 2-62), and the iron-sulfur centers essentialfor electron transport (see Figure 14-23). The process begins in bacteria, fungi, and plants, where a special group of enzymes use ATP and reducing power to create a sulfate assimilation pathway. Humans and other animals cannot reduce sulfate and must therefore acquire the sulfur they need for their metabolism in the food that they eat. Metabolismls Organized and Regulated One gets a sense of the intricacy of a cell as a chemical machine from the relation of glycolysis and the citric acid cycle to the other metabolic pathways sketched out in Figure 2-88. This type of chart, which was used earlier in this chapter to introduce metabolism, represents only some of the enzymatic pathways in a cell. It is obvious that our discussion of cell metabolism has dealt with only a tiny fraction of cellular chemistry. All these reactions occur in a cell that is less than 0.1 mm in diameter, and each requires a different enzyme. As is clear from Figure 2-88, the same molecule can often be part of many different pathways. Pyruvate,for example, is a substrate for half a dozen or more different enzymes,each of which modifies it chemically in a different way. One enzyme converts pyruvate to acetyl CoA, another to oxaloacetate;a third enzyrne changespyruvate to the amino acid alanine, a fourth to lactate, and so on. All of these different pathways compete for the same pyruvate molecule, and similar competitions for thousands of other small molecules go on at the same time. The situation is further complicated in a multicellular organism. Different cell tlpes will in general require somewhat different sets of enzymes. And different tissues make distinct contributions to the chemistry of the organism as a whole. In addition to differences in specialized products such as hormones or antibodies, there are significant differences in the "common" metabolic pathways among various types of cells in the same organism. Although virtually all cells contain the enzymes of glycolysis, the citric acid cycle, lipid synthesis and breakdown, and amino acid metabolism, the levels of THEESSENTIAL AMINOACIDS Figure?-87The nine essentialamino by acids.Thesecannotbe synthesized humancellsand so mustbe suppliedin the diet. 102 Chapter2:CellChemistryand Biosynthesis Figure2-88 Glycolysisand the citric acid cycleare at the center of metabolism.Some500 metabolic reactions of a typicalcellareshown with the reactions schematically of glycolysis and the citricacidcyclein red. Otherreactions eitherleadinto these two centralpathways-delivering small molecules to be catabolized with productionof energy-or they lead outwardand therebysupplycarbon compoundsfor the purposeof biosynthesis. these processes required in different tissues are not the same. For example, nerve cells, which are probably the most fastidious cells in the body, maintain almost no reservesof glycogen or fatty acids and rely almost entirely on a constant supply of glucose from the bloodstream. In contrast, liver cells supply glucose to actively contracting muscle cells and recycle the lactic acid produced by muscle cells back into glucose.All types of cells have their distinctive metabolic traits, and they cooperate extensivelyin the normal state, as well as in response to stressand starvation. One might think that the whole system would need to be so finely balanced that any minor upset, such as a temporary change in dietary intake, would be disastrous. In fact, the metabolic balance of a cell is amazingly stable.\.A/henever the balance is perturbed, the cell reacts so as to restore the initial state. The cell can adapt and continue to function during starvation or disease.Mutations of many kinds can damage or even eliminate particular reaction pathways, and yet-provided that certain minimum requirements are met-the cell survives.It does so because an elaborate network of control mechanismsregulates and coordinates the rates of all of its reactions.These controls rest, ultimately, on the remarkable abilities of proteins to change their shape and their chemistry in response to changesin their immediate environment. The principles that underlie how large molecules such as proteins are built and the chemistry behind their regulation will be our next concern. END-OF-CHAPTER PROBLEMS 103 Sum mar y Glucoseand otherfood moleculesare broken down by controlled stepwiseoxidation to prouide chemical energy in the form of ATP and NADH. Thereare three main setsof reactions that act in series-the products of each being the starting material for the next:glycolysis(which occursin the cytosol),the citric acid cycle(in the mitochondrial matrix), and oxidatiue phosphorylation (on the inner mitochondrial membrane).The intermediate products of glycolysk and the citric acid cycleare usedboth as sourcesof metabolic energyand to produce many of the small moleculesusedas the raw materials for biosynthesis.Cellsstore sugar moleculesas glycogenin animals and starch in plants; both plants and animals also usefats extensiuelyas a food store.Thesestorage materials in turn serueas a major sourceof food for humans, along with the proteins that comprisethe majority of the dry massof most of the cellsin thefoods we eet. PROBLEMS TableQ2-1 Radioactiveisotopesand someof their properties(Problem2-12). Whichstatementsare true?Explainwhy or why not. 2-1 Of the original radioactivityin a sample,only about 1/ 1000will remain after 10 half-lives. 2-2 A 10-BM solution of HCI has a pH of B. 2-3 Most of the interactions between macromolecules could be mediatedjust aswell by covalentbonds as by noncovalentbonds. 2-4 Animals and plants use oxidation to extract energy from food molecules. 2-5 If an oxidation occurs in a reaction, it must be accompaniedby a reduction. 2-6 Linking the energetically unfavorable reaction A -+ B to a second,favorablereaction B -+ C will shift the equilibrium constantfor the first reaction. 2-7 The criterion for whether a reaction proceedsspontaneouslyis AG not AGo,becauseAG takesinto accountthe concentrationsof the substratesand products. 2-8 Becauseglycolysis is only a prelude to the oxidation of glucosein mitochondria, which yields l5-fold more AIB glycolysisis not really important for human cells. 2-9 The oxygen consumed during the oxidation of glucosein animal cellsis returned as COzto the atmosphere. Discussthe following problems. 2- 10 The organicchemistryof living cellsis said to be special for two teasons:it occurs in an aqueous environment and it accomplishessome very complex reactions.But do you suppose it is really all that much different from the organic chemistry carried out in the top laboratoriesin the world? \A/tryor why not? 2-11 The molecular weight of ethanol (CHgCHzOH)is 46 and its density is 0.789g/cm3. A. \A4ratis the molarity of ethanol in beer that is 5% ethanol by volume? [Alcohol content of beer varies from about 4Vo(lite beer) to B% (stout beer).1 B. The legal limit for a driver's blood alcohol content varies,but 80 mg of ethanol per 100 mL of blood (usually 14c 36 35s 32P B particle B particle B particle B particle 5730years 12.3years 87.4days 14.3days 0.062 29 1490 9120 referredto as a blood alcohollevel of 0.08)is t)?ical. \ /hat is the molarity of ethanol in a person at this legal limit? t. How many l2-oz (355-mL)bottles of 5% beer could a 70-kgpersondrink and remain under the legallimit? A 70-kg person contains about 40 liters of water. Ignore the metabolism of ethanol, and assumethat the water content of the person remains constant. D. Ethanol is metabolizedat a constant rate of about 120 mg per hour per kg body weight, regardlessof its concentration. If a 70-kg person were at twice the legal limit (160 mg/f 00 mL), how long would it take for their blood alcohol level to fall below the legal limit? 2-12 Specificactivity refers to the amount of radioactivity per unit amount of substance,usually in biology expressedon a molar basis,for example,as Ci/mmol. [One curie (Ci) corresponds to 2.22 x 1012disintegrations per minute (dpm;.1 As apparent in Table Q2-1, which lists properties of four isotopes commonly used in biology, there is an inverserelationship between maximum specific activity and half-life. Do you suppose this is just a coincidence or is there an underlying reason? Explain your answer. 2-13 By a convenientcoincidencethe ion product ofwater, K- = lH+l[OH-],is a nice round number: 1.0x 10-14M2. A. \AIhyis a solution at pH 7.0 said to be neutral? B. \A/tratis the H+ concentrationand pH of a I mM solution of NaOH? C. If the pH of a solution is 5.0,what is the concentration of OH- ions? 2-14 Suggesta rank order for the pKvalues (from lowestto highest)for the carboxylgroup on the aspartateside chain 104 Chapter2:CellChemistryand Biosynthesis in the following environments in a protein. Explain your ranking. 1. An aspartateside chain on the surfaceof a protein with no other ionizable groupsnearby. 2. An aspartateside chain buried in a hydrophobic pocket on the surlaceof a protein. 3. An aspartateside chain in a hydrophobic pocket adjacent to a glutamateside chain. 4. An aspartateside chain in a hydrophobic pocket adjacent to a lysine side chain. 2-15 A histidine side chain is knol,rrnto play an important role in the cataly.ticmechanismof an enz).ryne; however,it is not clear whether histidine is required in its protonated (charged)or unprotonated (uncharged)state.To answerthis question you measureenzyrneactivity over a range of pH, with the resultssho\^Trin Figure Q2-1. \Ahich form of histidine is required for enz)ryneactivity? FigureQ2-1 Enzyme activityasa functionof pH(Problem 2-15). c f E o E o ,r_ ,_a, , ! C- O FigureQ2-2 Threemolecules that illustrate the sevenmostcommonfunctionalgroupsin biology(Problem2-17).1,3-Bisphosphoglycerate and pyruvateareintermediates in glycolysis and cysteineis an aminoacid. HO-CH 1 , 3 - b i s p h o s p h o g l y c e r a t e pyruvare SH cyslerne Calculatethe instantaneousvelocity of a water molecule (molecularmass= 1Bdaltons),a glucosemolecule (molecular mass = lB0 daltons),and a myoglobin molecule (molecular mass = 15,000daltons) at 37"C. Just for fun, convert thesenumbers into kilometers/hour.Beforeyou do any calculations,try to guesswhether the moleculesare moving at a slow crawl (<1km/hr), an easywalk (5 km/hr), or a recordsettingsprint (40km/hr). 2-19 Polymerization of tubulin subunits into microtubules occurs with an increasein the orderlinessof the subunits (Figure Q2-3). Yet tubulin polymerization occurs with an increasein entropy (decreasein order). How can that be? o 5 7 2-16 During an all-out sprint, musclesmetabolizeglucose anaerobically,producing a high concentrationoflactic acid, which lowers the pH of the blood and of the cytosol and contributes to the fatigue sprinters experiencewell before their fuel reservesare exhausted.The main blood buffer againstpH changesis the bicarbonate/CO2system. PKr= PK2= 2.3 38 CO2+CO2 +: +H* H2CO3 (gas) (dissolved) = PtrG lo" + HCO3-JsH* POLYMERIZATION .+ FigureQ2-3Polymerization of tubulinsubunits intoa microtubule (Problem 2-19).The fatesof onesubunit(shoded) anditsassociated (smallspheres) watermolecules areshown. + CO32- To improve their performance,would you advisesprinters to hold their breath or to breatherapidly for a minute immediately before the race?Explain your answer. 2-17 The three molecules in Figure Q2-2 contain the seven most common reactive groups in biology. Most moleculesin the cell are built from thesefunctional groups. Indicate and name the functional groups in these molecules. 2-18 "Diffusion" sounds slow-and over everyday distancesit is-but on the scaleof a cell it is very fast.The average instantaneousvelocity of a particle in solution, that is, the velocity betweencollisions,rs y = (kTlm)h where k= 1.38x 10-16g cmzlKsecz,T = temperaturein K (37'Cis 310 K), m = massin g/molecule. 2-2O A 70-kg adult human (154lb) could meet his or her entire energyneedsfor one day by eating3 moles of glucose (5a0g). (We don't recommend this.) Each molecule of glucose generates30 AIP when it is oxidizedto CO2.The concentration of AIP is maintained in cellsat about 2 mM, and a 70-kg adult has about 25 liters of intracellularfluid. Given that the ATPconcentrationremains constantin cells,calculate how many times per day,on average,eachAIP molecule in the body is hydrolyzedand reslmthesized. 2-21 Assuming that there are 5 x 1013cells in the human body and that AIP is turning over at a rate of 10eAIP per minute in each cell, how many watts is the human body consuming?(A watt is a joule per second,and there are 4.18 joules/calorie.) Assume that hydrolysis of AIP yields 12 kcal/mole. 2-22 Does a SnickersrMcandy bar (65 g, 325 kcal) provide enough energyto climb from Zermatt (elevation1660m) to the top of the Matterhorn (4478m, Figure Q2-4), or might END-OF-CHAPTER PROBLEMS 105 FigureQ2-4 The Matterhorn(Problem 2-22).(Courtesyof ZermattTourism.) IAI-^O fert a\l-CHr-CH2-CH2-CH2-CH2-CH2-CH2-C .o,,no'Jrna ll \t' -l cH2-C excreted compound tt' work (D = mass (kg)x g (m/sec2;x height gained (m) where gis accelerationdue to gravity (9.8m/sec2).Onejoule is 1 kg m2lsec2and there are 4.lB kf per kcal. \Alhatassumptionsmade here will greatlyunderestimate how much candy you need? 2-23 At first glance, fermentation of pyruvate to lactate appearsto be an optional add-on reaction to glycolysis.After all, could cells growing in the absenceof oxygen not simply discard pyruvate as a waste product? In the absenceof fermentation, which products derived from glycolysis would accumulate in cells under anaerobic conditions? Could the metabolism of glucosevia the glycoll'tic pathway continue in the absenceof oxygenin cells that cannot carry out fermentation?\.A/hyor why not? 2-24 In the absenceof oxygen,cellsconsumeglucoseat a high, steadyrate.When oxygenis added,glucoseconsumption drops precipitouslyand is then maintained at the lower rate.\.Vhyis glucoseconsumedat a high rate in the absence of oxygenand at a low rate in its presence? 2-25 The liver provides glucose to the rest of the body betweenmeals.It doesso by breakingdown glycogen,forming glucose6-phosphatein the penultimate step.Glucose6phosphateis convertedto glucoseby splitting offthe phosphate (AG' = -3.3 kcal/mole).\Mhydo you supposethe liver removesthe phosphateby hydrolysis,rather than reversing the reactionby which glucose6-phosphate(G6P)is formed from glucose (glucose + AIP -+ G6P + ADB AG' = -4.0 kcal/mole)?By reversingthis reactionthe liver could generate both glucoseand AIP //o \o_ phenylacetate o *^^ n'\-cur-cH2-cH2-cH2-cH2-cH2-c comoound ll \at you need to stop at Hdrnli Hut (3260m) to eat another one? Imagine that you and your gear have a mass of 75 kg, and that all of your work is done against gravity (that is, you are just climbing straight up). Rememberfrom your introductory physicscoursethat -| o- seven-carbonchain o excreted ri\. com -l ' o o u n dl l \4. o benzoate fattyacid to analyze labeling experiment FigureQ2-5Theoriginal (Problem of an derivatives 2-26).( ) Fedandexcreted oxidation derivatives of an fattyacidchain.(B)Fedandexcreted even-number fattyacidchain. odd-number Can you explainthe reasoningthat led him to concludethat two-carbon fragments, as opposed to any other number, were removed, and that degradation was from the carboxylic acid end, as opposedto the other end? 2-27 Pathways for synthesis of amino acids in microorganismswere worked out in part by cross-feedingexperiments among mutant organisms that were defective for individual steps in the pathway. Results of cross-feeding experiments for three mutants defective in the trlptophan TrpIl, and TrpE-are sholrryt in Figure parhway-TrpF, Q2-6. The mutants were streaked on a Petri dish and allowed to grow briefly in the presence of a very small amount of tryptophan, producing three pale streaks.As shown, heavier growth was observed at points where some streakswere close to other streaks.These spots of heavier growth indicate that one mutant can cross-feed(supply an intermediate)to the other one. From the pattern of cross-feedingshor,trrin Figure Q2-6, deducethe order ofthe stepscontrolled by the products of the TrpB, TrpD, and TrpE genes.Explain your reasoning. C R O S S - F E E D IRNEGS U L T TrpD- TrpE- 2-26 In 1904Franz Knoop performed what was probably the first successfullabeling experiment to study metabolic pathways. He fed many different fatty acids labeled with a terminal benzene ring to dogs and analyzedtheir urine for excreted benzene derivatives.\Alheneverthe fatty acid had an even number of carbon atoms, phenylacetate was excreted (Figure Q2-5A). lVhenever the fatty acid had an odd number of carbon atoms, benzoatewas excreted(Figure Q2-58). From theseexperimentsKnoop deducedthat oxidation of fatty acidsto CO2and H2Oinvolved the removalof two-carbon fragments from the carboxylic acid end of the chain. o- eight-carbonchain TrDB' using FigureQ2-6 Definingthe pathwayfor tryptophansynthesis (Problem2-27).Results of a crossexperiments cross-feeding feedingexperimentamongmutantsdefectivefor stepsin the tryptophan biosyntheticpathway.Darkoreason the Petridish show regionsof cellgrowth. CARBONSKELETONS Carbonhasa unique role in the cell becauseof its abilityto form strongcovalentbondswith other carbonatoms.Thuscarbonatomscan ioin to form chains. \/ ,/ \/ CCCC " " \ \ _ r,, LLLL /\ \/ \/ /\ ^,.. "\ /\ \,, \/ -t\ - -\ ^,r- or branchedtrees ^,,. /\ also written as C-C- -c/ ,21 alsowritten as ./c- \a /\ X written arso r, al-) COVALENTBONDS HYDROCARBONS A covalent bond forms when two atoms come very close together and shareone or more of their electrons.In a single bond one electronfrom eachof the two atoms is shared;in a double bond a total of four electronsare shared. Eachatom forms a fixed number of covalentbonds in a definedspatialarrangement.For example,carbonforms four singlebondsarrangedtetrahedrally,whereasnitrogenforms three singlebondsand oxygenforms two singlebondsarranged as shown below. Carbonand hydrogencombine together to make stable compounds(or chemicalgroups) calledhydrocarbons. Theseare nonpolar,do not form hydrogenbonds,and are generallyinsolublein water. Atomsjoined by two or more covalentbonds cannot rotate freely around the bond axis. This restriction is a major influenceon the three-dimensional shaoe of many macromolecules. HrC. CH, ALTERNATING DOUBLEBONDS The carbonchain can includedouble bonds.lf theseare on alternatecarbon atoms,the bonding electronsmove within the molecule,stabilizingthe structureby a phenomenoncalled reS0nance. HrC. Alternatingdouble bonds in a ring can generate a very stable structure. CHt HrC. CH, HrC. CH, H,C CH, Hzc. the truth is sbmewhereberween these two structures 9H, H:C oftenwritten., O part of the hydrocarbon"tail" of a fatty acid molecule C_OCHEMICAL GROUPS GROUPS C-N CHEMICAL Many biologicalcompoundscontaina carbon bondedto an oxygen.For example, Aminesand amidesare two important examplesof compoundscontaininga carbonlinkedto a nitrogen. Aminesin water combinewith an H+ ion to become positivelycharged. lrHt,H _ ct_/ l(/ +u* * _q_n_H* r\nr\H Amidesare formed by combiningan acidand an amine.Unlikeamines,amidesare unchargedin water. An exampleisthe peptide bond that joins amino acids in a orotein. o -c/ The -COOHis calleda carboxylgroup. In water this losesan H+ion to become-COO-. esters Estersare formed by a condensationreaction betweenacid and an alcohol. '.Or,O _[_r/+Ho_l___Lr/ I' acid \ | oH alcohol | \ l+H20 o-c- ester I acid \o" + H"N-J- I amtne Nitrogenalsooccursin severalring compounds,including important constituentsof nucleicacids:purinesand pyrimidines. )n' ^4rtn I o/"C (a pyrimidine) ll cytosine tN-C-H H group the sulfhydryl group.In the aminoacidcysteine iscalleda sulfhydryl form,-t-Sn mayexistin the reduced SULFHYDRYL GROUP lrl form, -C-S-S-Cor more rarelyin an oxidized,cross-bridging PHOSPHATES Inorganicphosphateis a stableion formed from phosphoricacid,H3PO4. lt is often written as Pi. Phosphateesterscan form between a phosphateand a free hydroxyl group. Phosphategroups are often attached to proteins in this way. carboxylgroup, or two or more phosphategroups,givesan acid anhydride. Hzo l* T Hzo also written as high-energyacyl phosphate bond (carboxylic-phosphoric acidanhydride)found in some metabolites -t_ o ^, o-P Hzo L T Hzo phosphoanhydride-ahighenergybond found in moleculessuchas ATP -o alsowritten as WATER WATERSTRUCTURE Two atoms, connected by a covalent bond, may exert different attractionsfor the electronsof the bond. In suchcasesthe bond is polar,with one end slightlynegativelycharged(6-) and the other slightlypositivelycharged(6+). Moleculesof water join together transiently in a hydrogen-bondedlattice.Evenat 37oC, 15o/o oI the water moleculesare joined to four othersin a short-livedassemblvknown as a "flickeringcluster." .:tt:'i l,::,: electronegative regron Although a water moleculehasan overallneutralcharge(havingthe same numberof electronsand protons),the electronsare asymmetrically distributeo, which makesthe moleculepolar.The oxygennucleusdrawselectronsaway from the hydrogennuclei,leavingthesenucleiwith a smallnet positivecharge. The excessof electron density on the oxygen atom createsweakly negative regionsat the other two cornersof an imaginarytetrahedron. H Y D R O G EB NO N D S Because they are polarized,two adjacentH2Omoleculescan form a linkageknown as a hydrogen bond. Hydrogenbondshave only about 1/20the strength of a covalentbond. Hydrogenbondsare strongestwhen the three atoms lie in a straightline. 6- H ""!,,, l,u H The cohesivenature of water is responsible for many of its unusual properties,suchas high surfacetension, specificheat, and heat of vaporization. bond lengths hydrogenbond 0 . 2 7n m Q rrrrrrrrrrrrrrrrrrr H -Oo-.tor'tt hydrogen bond covalentbond H Y D R O P H I LM I CO L E C U L E S H Y D R O P H O BM I CO L E C U L E S Substances that dissolvereadilyin water are termed hydrophilic.Theyare composedof ionsor polar moleculesthat attractwater moleculesthrough electricalchargeeffects.Water moleculessurroundeachion or polar molecule on the surfaceof a solidsubstanceand carrvit into solution. Moleculesthat containa preponderance of nonpolarbondsare usuallyinsolublein water and are termed hydrophobic.Thisis true, especially, of hydrocarbons, which containmany C-H bonds.Water molecules are not attractedto suchmoleculesand so have little tendencyto surroundthem and carrythem into solution. H HH \/ C .\ \H -C lonicsubstances suchas sodiumchloride dissolvebecausewater moleculesare attracted to the positive(Na+)or negative (Cl) chargeof eachion. H / Polarsubstances suchas urea dissolvebecausetheir molecules form hydrogenbondswith the surroundingwater molecules. H IHH o\ . H C ,/\ WATERAS A SOLVENT Many substances, suchas householdsugar,dissolvein water. That is,their moleculesseparatefrom eachother, eachbecomingsurroundedby water molecules. in a When a substancedissolves liquid,the mixture is termed a solution. The dissolvedsubstance(in this case sugar)isthe solute,and the liquid that doesthe dissolving(in this casewater) is the solvent.Water is an excellent solvent for many substancesbecause of its polar bonds. sugarcrystal s u g a rm o l e c u l e ACIDS ION EXCHANGE HYDROGEN Substances that releasehydrogenionsinto solution a r e c a l l e da c i d s . Positivelychargedhydrogenions(H+)can spontaneously move from one water moleculeto another,therebycreating two ionic species. HH HCI-+Cf hydrochloric acid (strongacid) hydrogen ion ion chloride oilililrH-o Many of the acidsimportant in the cellare only partially dissociated, and they are thereforeweak acids-for example, the carboxylgroup (-COOH),which dissociates to give a hydrogenion in solution ,/ n..P, +oo/ - H hydroxyl ion hydronium ion (water acting as (water acting as a weak acid) a weak base) oftenwrittenas: Hro i H* + -c/ \ H* + oHhydroxyl nrl:Xn"n Note that this is a reversible reaction. hydrogenionsare israpidlyreversible, Since the process Purewater shuttlingbetweenwatermolecules. continually of hydrogenionsand concentration a steady-state contains hydroxylions(both10-'M). pH BASES ( w e a ka c i d ) pH The acidityof a solutionis defined by the concentration of H+ ions it possesses. Forconveniencewe usethe pH scale,where 1 02 1 03 pH = log,o[H+] 101 1 2 NH:+H*-NHo* ion ammonia hydrogen ion ammonium 5 1o-4 A 1o-s 5 10-6 6 1o-7 1 08 1 0e 7 8 For pure water 1 o1 0 10 [H+] = 10-7moles/liter 1o-11 1 01 2 11 1013 1014 that reducethe number of hydrogenions in Substances solutionare calledbases.Somebases,suchas ammonia, combinedirectlywith hydrogenions. 9 12 II 14 Other bases,suchas sodiumhydroxide,reducethe number of H* ionsindirectly,by making OH- ionsthat then combine directlywith H' ionsto make H2O. NaOH s o d i u mh y d r o x i d e (strong base) Nasodium ion Many basesfound in cellsare partiallydissociatedand are termed weak bases.Thisis true of compoundsthat containan amino group (-NH2),which has a weak tendency to reversiblyaccept an H' ion from water, increasingthe quantity of free OH- ions. -NHz+H*--NH:* WEAKCHEMICAL BONDS VAN DERWAALSATTRACTIONS Organicmoleculescan interactwith other moleculesthrough three types of short-rangeattractive forces known as noncovalentbonds: van der Waals attractions,electrostaticattractions,and hydrogen bonds.The repulsionof hydrophobicgroupsfrom water is also importantfor ordering biologicalmacromolecules. lf two atoms are too closetogether they repel each other very strongly. For this reason,an atom can often be treated as a soherewith a fixed radius.The characteristic "size" lor eachatom is specifiedby a uniquevan der Waals radius.The contact distancebetween any two noncovalentlybondedatoms isthe sum of their van der Waalsradii. & 0 . 1 5n m radius Weak chemicalbondshave lessthan 1/20the strengthof a strong covalentbond. Theyare strong enough to providetight binding only when many of them are formed simultaneously. H Y D R O G EB NO N D S As already describedfor water (seePanel2-2), hydrogenbondsform when a hydrogenatom is "sandwiched " betweentwo electron-attracting atoms (usuallyoxygenor nitrogen). Hydrogenbondsare strongestwhen the three atomsare i n a s t r a i g h tl i n e : to-" nnnro\ N-H I I lt R-C-H R-C-H I c : o l l l l l l l lH l t- N lll i l i l i l i l i lH - N FFC-R I I Two bases,G and C, hydrogen-bondedin DNA or RNA. Fig H. N-Hililill \' C-C \\\ .H-. L. : /// .NilililililtH\/ N-C. ,/ \\'bllllllltn-N' ,,/ tH tl 0 . 1 5n m single-bonded caroonS HYDROGEN BONDSIN WATER Amino acidsin polypeptidechainshydrogen-bonded together. c:o At very short distancesany two atoms show a weak bonding interactiondue to their fluctuatingelectrical charges.The two atomswill be attractedto eachother in this way until the distancebetweentheir nucleiis approximatelyequalto the sum of their van der Waals radii.Although they are individuallyvery weak, van der Waalsattractionscan becomeimoortant when two macromolecularsurfacesfit very closetogether, becausemany atomsare involved. Note that when two atomsform a covalentbond, the centersof the two atoms (the two atomic nuclei)are much closertogether than the sum of the two van der W a a l sr a d i i .T h u s . |||||/ Examplesin macromolecules: I 0 . 1 4n m radius Any moleculesthat can form hydrogenbondsto eachother can alternativelyform hydrogenbondsto water molecules. Becauseof this competitionwith water molecules, the hydrogenbondsformed betweentwo moleculesdissolved in water are relativelyweak. HYDROPHOBIC FORCES Water forces hydrophobic groups together, becausedoing so minimizestheir disruptive effects on the hydrogen-bondedwater network. Hydrophobicgroupsheld together in this way are sometimessaid to be held together by "hydrophobic bonds,"eventhough the apparentattraction is actuallycausedby a repulsionfrom the water. IN ATTRACTIONS ELECTROSTATIC A Q U E O UsSO L U T I O N S Chargedgroupsare shieldedby their interactionswith water molecules. Electrostaticattractions are therefore quite weak in water. Attractive forces occur both between fully charged groups(ionicbond) and betweenthe partiallycharged groupson polar molecules. Similarly,ions in solutioncan clusteraround chargedgroupsand further weaken these attractions. CI o o ,.O -C // Na Na+ 1mm I rr.t ,rNa @ cl- H H -.-N - \], CI\ [/ In the absenceof water, electrostaticforces are very strong. They are responsible for the strengthof suchmineralsas marbleand agate,and for crystalformation in common table salt.NaCl. a crystalof salt,NaCl Cl, ^Na \ The force of attraction between the two charges,6+ and 5-, falls off rapidlyasthe distancebetweenthe chargesincreases. .H H. N 6+ '.,o. H -S ELECTROSTATIC ATTRACTIONS I H CI al\ Despitebeing weakenedby water and salt, electrostaticattractions are very important in biologicalsystems.For example,an enzymethat bindsa positivelychargedsubstratewill often have a negativelychargedamino acidsidechain at the appropriateplace. MONOSACCHARIDES Monosaccharides usuallyhavethe generalformula (CH2O)',where n can be 3, 4, 5, 6,7, or 8, and havetwo or more hydroxylgroups. Theyeither containan aldehydegroup ( -c(l ) and are calledaldosesor a ketone group ( ).:o ) and are calledketoses. 3-carbon(TRIOSES) S-carbon(PENTOSES) 6-carbon(HEXOSES) H,O \^// L H,O ta/ I I-OH I H-C -OH I H-C -OH I H-C -OH I g l y c er a1d e h y d e nbose H-C U o /ro "t H-C c' I-OH I-OH H-C H H-C {-OH I HO-C-H I H-C -OH H-C H-C H I-OH I-OH I H g Iu c o s e H H I I I H-C -OH I H-C -OH I H-C -OH I I I c:o H-C -OH H-C -OH U u Y H I H-C -OH I c*-o I-OH H-C I H H ribulose d i hydroxyacetone I HO-C-H I H-C -OH I H-C -OH H-C I -OH I H fructose RINGFORMATION ISOMERS In aqueoussolution,the aldehydeor ketone group of a sugar moleculetendsto reactwith a hydroxylgroup of the same molecule,therebyclosingthe moleculeinto a ring. Many monosaccharides differ only in the spatialarrangement of atoms-that is,they are isomers.For example,glucose, galactose,and mannosehavethe sameformula (C6H,'2Ojbut differ in the arrangementof groupsaround one or two carbon atoms. H'. zro rc -OH 2C HO;C -H H- C - O H al H -sC-OH H -CH?OH I grucose cH20H OH H'. zo ,f H -C -OH mannoSe -l H-C -OH HIC -OH -CH,OH OH OH Note that eachcarbonatom hasa number. Thesesmalldifferencesmake only minor changesin the chemicalpropertiesof the sugars.But they are recognizedby enzymesand other proteinsand thereforecan have important biologicaleffects. 0 A N DB L T N K S SUGARDERIVATIVES The hydroxylgroup on the carbonthat carriesthe aldehydeor ketone can rapidlychangefrom one positionto the other. Thesetwo positionsare calleda and B. The hydroxylgroupsof a simple can be replaced monosaccharide by other groups.Forexample, glucosamine cr hydroxyl B hydroxyl As soon as one sugaris linkedto another,the o or B form is frozen. qH2oH DISACCHARIDES The carbonthat carriesthe aldehyde or the ketone can reactwith any hydroxylgroup on a secondsugar m o l e c u l et o f o r m a d i s a c c h a r i d e . The linkageis calleda glycosidic bond. Threecommon disaccharides are maltose(glucose+ glucose) lactose(galactose+ glucose) sucrose(glucose+ fructose) The reactionforming sucroseis shown here. OLIGOSACCHARIDES AND POLYSACCHARIDES Largelinearand branchedmoleculescan be made from simplerepeatingsugar s u b u n i t sS. h o r tc h a i n sa r e c a l l e do l i g o s a c c h a r i d ewsh,i l e l o n g c h a i n sa r e c a l l e d polysaccharides. Glycogen,for example,is a polysaccharide made entirelyof glucoseunitsjoined together. glycogen COMPLEXOLIGOSACCHARI DE5 In many casesa sugarsequence is nonrepetitive.Many different moleculesare possible.Such complexoligosaccharides are usuallylinkedto proteinsor to lipids, as is this oligosaccharide, which is molecule oart of a cell-surface that definesa particularblood group. COMMON FATTY ACIDS Fatty acidsare stored as an energy reserve(fats and oils)through an esterlinkageto glycerolto form triacylglycerols, alsoknown astriglycerides. Theseare carboxylicacids with long hydrocarbontails. COOH I COOH I I CH, f", CHz CH, CH, CH, CH, CH, CH, f", CH, f*' CH CH, I I I I I I CH, I CH" I CHr I CH, I CHt I CHt I CH, I CH, I 9H, I cH. t" CH: CH, I lI I CHr I o il ),,c Hundredsof different kindsof fatty acidsexist.Somehaveone or more double bonds in their hydrocarbontail and are saidto be unsaturated.Fattyacidswith no double bondsare saturated. -oo \/ I fn, fn, fn' CHt CH CH: o tl I 9I H , CH, i", f*, ,r'C o'C CHt I CHt tI CHt fn, I ll ( COOH L- I I T h i sd o u b l eb o n d is rigid and creates . za kinkin the chain. ' ll rh" restof the chain is free to rotate about the other C-C bonds. CHt I 9H, I CHt tI CH. I CH, I CH, I CH. 3i,'J'''' t' (Crs) stear|c a c i d( C r e ) CH: oleic acid (Cre) space-fillingmodel carbonskeleton UNSATURATED CARBOXYL GROUP SATURATED P H O S P H O L I P I D S P h o s p h o l i pai dr set h e m a j o rc o n s t i t u e n t s of cellmembranes. lf free, the carboxylgroup of a fatty acidwill be ionized. &c.. ,a/ B u t m o r e u s u a l l yi t i s l i n k e dt o other groupsto form either esters WC or amides o ,1/ C \ s p a c e - f i l l i nm g o d e lo f the phospholipid p h o s p h a t i d y l c hionle In phospholipids two of the -OH groupsin glycerolare linkedto fatty acids,while the third -OH group is linked g e n e r a ls t r u c t u r e to phosphoricacid.The phosphateis further linkedto o f a p h o s p h o l i p i d one of a varietyof smallpolar groups(alcohols). LIPIDAGGREGATES POLYISOPRENOIDS long-chainpolymersof isoprene ln water they can form a surfacefilm o r f o r m s m a l lm i c e l l e s . Their derivativescan form largeraggregatesheld together by hydrophobicforces: Triglycerides can form largespherical fat dropletsin the cell cytoplasm. O T H E RL I P I D S ipid P h o s p h o l i p i dasn d g l y c o l i p i dfso r m s e l f - s e a l i nl g bilayersthat are the basisfor all cell membranes. Lioidsare defined asthe water-insoluble m o l e c u l e isn c e l l st h a t a r e s o l u b l ei n o r g a n i c solvents.Two other commontypesof lipids are steroidsand polyisoprenoids. Both are made from isopreneunits. CH. \ -CH:CHr .C ,// Lr 12 isoprene Steroidshavea common multiple-ringstructure. cholesterol-found in manv membranes GLYCOLIPIDS Likephospholipids, thesecompoundsare composedof a hydrophobic - C - NI H dolicholphosphate-used to carryactivatedsugars in the membrane-associated synthesisof glycoproteins and somepolysaccharides NUCLEOTIDES PHOSPHATES A nucleotideconsistsof a nitrogen-containing base,a five-carbonsugar,and one or more phosphategroups. The phosphatesare normallyjoined to the C5 hydroxylof the riboseor deoxyribosesugar(designated5'). Mono-, di-, and triphosphatesare common. BASICSUGAR LINKAGE BASE -o- - ililtl P- o - I " o-o-o - P- o - I P- . ) - c H ^ l-" a sI n l"'ott The phosphatemakesa nucleotide negativelycharged. The baseis linkedto the samecarbon(C1) usedin sugar-sugar bonds. NH l2 I i HclC\NH \Hu I ll U HCli\x "t-'.,-t\ llC lcvtosine nt-r-t\ o luracil The basesare nitrogen-containingring c o m p o u n d se, i t h e rp y r i m i d i n eosr p u r i n e s . H o l1 " n,.I ' \./-\** 3 ll r I thy lrl\ - t \ PENTOSE a five-carbonsugar Eachnumberedcarbonon the sugarof a nucleotideis followed by a prime mark;therefore,one speaksof the " 5 - p r i m ec a r b o n , "e t c . two kindsare used bose B-o-2-deoxyri usedin deoxvribonucleic acid NOMENCLATURE A nucleosideor nucleotideis namedaccordingto its nitrogenousbase. Singleletter abbreviationsare usedvariouslyas shorthandfor (1) the basealone,(2) the nucleoside, or (3) the whole nucleotidethe contextwill usuallymake clearwhich of the three entitiesis meant.When the context is not sufficient,we will add the terms "base", B A S E+ S U G A R= N U C L E O S I D E "nucleoside","nucleotide",or-as in the examolesbelow-use the full 3-letternucleotide cooe. AMP dAMP UDP ATP N U C L E IACC I D S = adenosinemonophosphate = deoxyadenosine monophosphate = u r i d i n ed i p h o s p h a t e = adenosinetriphosphate = NUCLEOTIDE BASE+ SUGAR+ PHOSPHATE NUCLEOTIDES HAVEMANY OTHERFUNCTIONS Nucleotidesare joined together by a phosphodiester linkagebetween 5' and 3' carbonatomsto form nucleicacids. The linearsequenceof nucleotidesin a nucleicacid chain is commonly abbreviated by a one-letter code, A-G-C-T-T-A-C-A, with the 5' end of the chain at the left. bonds. Theycarrychemicalenergy in their easilyhydrolyzedphosphoanhydride p h o s p h o a n h y d r i dbeo n d s -l ooo -o- ililtl -l - f H, l- o - P- o - P - o o- o- o- example:ATP(or OH ) OH Theycombinewith other groupsto form coenzymes. H O H H O H HS ttttlllllll H t H H H H H O H CH.H ll l l'l il r r t t _C _C _N _C _C -C _N_C _C _C -C ll -O- HO CH3H O ll P_O-P-O O- - CH 2 O- example:coenzymeA (CoA) ooH 5' endof chain I O : P- O - 5', oTheyare usedas specificsignalingmoleculesin the cell' l)Hz example:cyclicAMP (cAMP) o o II I P_ I o- ooH T H EI M P O R T A N COEF F R E E E N E R GF YO RC E L L S Life is possiblebecauseof the complexnetwork of interacting chemicalreactionsoccurringin everycell.In viewingthe metabolicpathwaysthat comprisethis network, one might suspectthat the cell has had the abilityto evolvean enzymeto carryout any reactionthat it needs.But this is not so.Although enzymesare powerful catalysts, they can speedup only those reactionsthat are thermodynamically possible;other reactions proceedin cellsonly becausethey are coupledto very favorable reactionsthat drive them. The ouestionof whether a reaction can occurspontaneously, or insteadneedsto be coupledto another reaction,is centralto cell biology. The answeris obtained by referenceto a quantity calledthe free energy: the total changein free energyduring a set of reactionsdetermines whether or not the entire reactionseouencecan occur.ln this panel we shallexplainsomeof the fundamentalideas-derived from a specialbranchof chemistryand physicscalledthermodynamics-that are requiredfor understandingwhat free energy is and why it is so important to cells. E N E R GR Y E L E A S EBDY C H A N G EISN C H E M I C ABLO N D I N G I SC O N V E R T EI N D T OH E A T of molecularcollisionsthat heat uo first the walls of the box and then the outsideworld (representedby the sea in our example).In the end, the systemreturnsto its initial temperature,by which time all the chemicalbond energy releasedin the box has been convertedinto heat energyand transferredout of the box to the surroundings. Accordingto the first law, the changein the energyin the box (AE6o", which 5EA we shalldenote as AE) must be equal and oppositeto the amount of heat energytransferred,which we shalldesignate as h: that is,AE = -h. Thus,the energy in the box (E) decreases when heat leavesthe system. Ealso can changeduring a reactionas a resultof work being done on the outsideworld. For example,supposethat there is UNIVERSE a smallincreasein the volume (Ay) of the box during a reaction. Sincethe walls of the box must pushagainstthe constant pressure(P) in the surroundingsin order to expand,this does An enclosedsystemis defined as a collectionof moleculesthat work on the outsideworld and requiresenergy.The energy doesnot exchangematter with the restof the universe(for usedis P(AV),which accordingto the first law must decrease example,the "cell in a box" shown above).Any suchsystemwill the energy in the box (E) by the sameamount. ln most reactions containmoleculeswith a total energyE. Thisenergywill be chemicalbond energy is convertedinto both work and heat. distributedin a varietyof ways:someasthe translationalenergy Enthalpy (H) is a compositefunction that includesboth of these of the molecules,someas their vibrationaland rotationalenergies, (H = E + PV).To be rigorous,it is the changein enthalpy but most asthe bonding energiesbetweenthe individualatoms (AH) in an enclosedsystem,and not the changein energy,that that make up the molecules.Supposethat a reactionoccursin is equal to the heat transferredto the outsideworld during a the system.The f irst law of thermodynamicsplacesa constraint reaction.Reactionsin which H decreases releaseheat to the on what typesof reactionsare possible:it statesthat "in any surroundingsand are saidto be "exothermic,"while reactions process, the total energyof the universeremainsconstant." in which H increases absorbheat from the surroundingsand Forexample,supposethat reactionA - B occurssomewherein are saidto be "endothermic."Thus,-h = AH. However,the the box and releases a great deal of chemicalbond energy.This volume changeis negligiblein most biologicalreactions,so to energywill initiallyincreasethe intensityof molecularmotions a good approximation (translational, vibrational,and rotational)in the system,which is equivalentto raisingits temperature.However,these increased motionswill soon be transferredout of the svstembv a series THESECOND LAW OFTHERMODYNAMICS Considera containerin which 1000coinsare all lying headsup. lf the containeris shakenvigorously,subjectingthe coinsto the typesof random motionsthat all moleculesexperiencedue to their frequent collisionswith other molecules,one will end up with about half the coinsoriented headsdown. The reasonfor this reorientationis that there is only a singleway in which the originalorderlystateof the coinscan be reinstated (everycoin mu{lie headsup), whereasthere are many different ways (about 102s8) to achievea disorderlystate in which there is an equal mixtureof headsand tails;in fact, there are more wavs to achievea 50-50statethan to achieveany other state.Each state has a probabilityof occurrencethat is proportionalto the number of ways it can be realized.The secondlaw of thermodynamicsstatesthat "systemswill changespontaneously from statesof lower probabilityto statesof higher probability." Sincestatesof lower probabilityare more "ordered" than statesof high probability,the secondlaw can be restated: "the universeconstantlychangesso as to becomemore disordered." THEENTROPY, 5 The secondlaw (but not the first law) allowsone to predictthe directionof a particularreaction.But to make it usefulfor this purpose,one needsa convenientmeasureof the probabilityor, equivalently,the degreeof disorderof a state.The entropy (5) i s s u c ha m e a s u r el.t i s a l o g a r i t h m i fcu n c t i o no f t h e p r o b a b i l i t y suchthat the changein entropy (45) that occurswhen the reactionA - B convertsone mole of A into one mole of B is A5=RInpB/pA where p4 and p, are the probabilitiesof the two statesA and B, R is the gas constant(2 cal deg 1 mole 1),and A5 is measured i n e n t r o p yu n i t s( e u ) .I n o u r i n i t i a le x a m p l eo f 1 0 0 0c o i n s t, h e relativeprobabilityof all heads(stateA) versushalf headsand half tails (stateB) is equal to the ratio of the number of different waysthat the two resultscan be obtained.One can calculate t h a t p A = 1 a n d p , = 1 0 0 0 1 ( 5 0 x0 15 0 0 1-) 1 0 2 s eT.h e r e f o r e , the entropy changefor the reorientationof the coinswhen their containeris vigorouslyshakenand an equal mixtureof heads or about 1370eu per mole of and tails is obtained is R In (102e8), We seethat, becauseA5 suchcontainers(6 x 1023containers). defined above is positivefor the transitionfrom stateA to state B (ps/p4 > 1), reactionswith a large increasein 5 (that is, for which A5 > 0) are favoredand will occurspontaneously. in Chapter2, heat energycausesthe random As discussed the transferof heat from an commotionof molecules.Because the numberof enclosedsystemto its surroundingsincreases different arrangementsthat the moleculesin the outsideworld their entropy.lt can be shown that the can have,it increases releaseof a fixed quantity of heat energyhasa greaterdisordering effect at low temperaturethan at high temperature,and that the value of A5 for the surroundings.as defined above (ASr"u), is preciselyequalto h, the amount of heattransferredto the surroundingsfrom the system,dividedby the absolute temperature(f ): T H EG I B B S FREE E N E R G YG, When dealingwith an enclosedbiologicalsystem,one would like to have a simpleway of predictingwhether a given reaction will or will not occurspontaneously in the system.We have seenthat the crucialquestionis whether the entropy changefor the universeis positiveor negativewhen that reactionoccurs. In our idealizedsystem,the cell in a box, there are two separate componentsto the entropy changeof the universe-the entropy changefor the systemenclosedin the box and the entropy changefor the surrounding"sea"-and both must be added together before any predictioncan be made.For example,it is possiblefor a reactionto absorbheat and therebydecreasethe entropy of the sea (A5r""< 0) and at the sametime to cause sucha large degreeof disorderinginsidethe box (A56o* > 0) = A5r"" + A56o,is greater than 0. In this that the total A5rn;u"rr" casethe reactionwill occurspontaneously, eventhough the seagivesup heat to the box during the reaction.An exampleof sucha reactionisthe dissolvingof sodiumchloridein a beaker containingwater (the "box"), which is a spontaneousprocess eventhough the temperatureof the water dropsasthe salt goesinto solution. Chemistshavefound it usefulto define a number of new "compositefunctions"that describecombinationsof physical propertiesof a system.The propertiesthat can be combined includethe temperature(f), pressure(P), volume (V), energy (E), and entropy (5). The enthalpy(H) is one suchcomposite function.But by far the most usefulcompositefunction for biologistsis the Gibbs free energy, G. lt servesas an accounting devicethat allowsone to deducethe entropy changeof the universeresultingfrom a chemicalreactionin the box, while avoidingany separateconsiderationof the entropychangein the sea.The definition of G is G=H-TS where, for a box of volume V, H is the enthalpydescribedabove (E + PV), r is the absolutetemperature,and 5 is the entropy. Eachof thesequantitiesappliesto the insideof the box only. The changein free energyduring a reactionin the box (the G of the productsminusthe G of the startingmaterials)is denoted asAG and, as we shallnow demonstrate,it is a direct measureof the amount of disorderthat is createdin the universewhen the reaction occurs. At constanttemperature the change in free energy (AG) during a reactionequalsAH - IA5. Rememberingthat AH = -h, the heat absorbedfrom the sea,we have But h/f is equal to the entropy change of the sea (A5r""),and the A5 in the above equation is A56o^.Therefore We concludethat the free-energychangeis a direct measure of the entropy changeof the universe.A reactionwill proceed in the directionthat causesthe changein the free energy(AG) to be lessthan zero, becausein this casethere will be a positive entropy changein the universewhen the reactionoccurs. For a complexset of coupledreactionsinvolvingmany the total free-energychangecan be comdifferent molecules, puted simplyby adding up the free energiesof all the different molecularspeciesafter the reactionand comparingthis value with the sum of free energiesbefore the reaction;for common the requiredfree-energyvaluescan be found from substances publishedtables.In this way one can predictthe directionof a reactionand thereby readilycheckthe feasibilityof any proposedmechanism. Thus,for example,from the observed proton gradient valuesfor the magnitudeof the electrochemical acrossthe inner mitochondrialmembraneand the AG for ATP hydrolysisinsidethe mitochondrion,one can be certainthat ATP synthaserequiresthe passageof more than one proton for each moleculeof ATPthat it synthesizes. The value of AG for a reactionis a direct measureof how far the reactionis from equilibrium.The large negativevaluefor ATP hydrolysisin a cell merelyreflectsthe fact that cellskeep the ATP hydrolysisreactionas much as 10 ordersof magnitude away from equilibrium.lf a reactionreachesequilibrium, AG = 0, the reactionthen proceedsat preciselyequal rates in the forward and backward direction. For ATP hydrolysis, equilibriumis reachedwhen the vast majorityof the ATP has been hydrolyzed,as occursin a dead cell' F o r e a c hs t e p ,t h e p a r t o f t h e m o l e c u l et h a t u n d e r g o e sa c h a n g ei s s h a d o w e di n b l u e , a n d t h e n a m e o J t h e e n z y m et h a t c a t a l y z etsh e r e a c t i o ni s i n a y e l l o w b o x . S T E P1 G l u c o s ei s phosphorylatedby ATPto f o r m a s u g a rp h o s p h a t e . T h e n e g a t i v ec h a r g eo f t h e phosphatepreventspassage of the sugar p h o s p h a t et h r o u g h t h e p r a s m am e m D r a n e , t r a p p i n gg l u c o s ei n s i d e the cell. o. .H \,/ 't- !r 1CH?OH I H-C.-OH l' f r o m c a r b o n1 t o H O c a r o o nz , t o r m t n g a ketosefrom an a l d o s es u g a r ( S e e Panel2-4.) *'-i? + H.)-(--H lH- OH H-C-OH 5 | -CH,OqP(openchainform) H-C-OH l5 .CH,OP ( o p e nc h a i nf o r m ) STEP3 The new hydroxyl .l g r o u p o n c a r b o n1 ' , phosphorylatedby ATP,in p r e p a r a t i o nf o r t h e f o r m a t i o n o f t w o t h r e e - c a r b o ns u g a r phosphates.The entry of sugars i n t o g l y c o l y s iiss c o n t r o l l e da t t h i s s t e p ,t h r o u g h r e g u l a t i o no f t h e enzyme p hosphof ru ctok i nase- (ringform) P O H-) |C, " - \o ( CH,O P Hr)) rLJ/l | + tt'' OH f ructose 1,6-bisphosphate CH,O P STEP 4 The s i x - c a r b o sn u g a ri s c l e a v e dt o p r o d u c e two three-carbon m o l e c u l e sO n l y t h e glyceraldehyde 3-phosphate can p r o c e e di m m e d i a t e l y through glycolysis I a-A I HO HO-C-H I +C H-C-OH I I cH2o P I H-C-OH I cH2o I ( o p e nc h a i nf o r m ) f r u c t o s e1 , 5 - b i s p h o s p h a t e CH,OH \// I H-C-OH cH2o P S T E P5 Theother product of step 4, d i hydroxyacetone p h o s p h a t ei,s isomerized to form glyceraldehyde 3-phosphate. ADP P glyceraldehyde ?-nhncnh:te C ,ro " I I H-C-OH cH2o P g l y c e r a l d e h y d3e- p h o s p h a t e t ovo + E E E E +u * f H-C-OH l cH2o P F i g u r e2 - 7 3 ) 1,3-bisphosphoglycerate g l y c e r a l d e h y d3e- p h o s p h a t e S T E P7 T h et r a n s f e r t o A D Po f t h e h i g h - e n e r g yp h o s p h a t e groupthat was generated in step 6 forms ATP. C I I + H-C-OH cH2o P 1,3-bisphosphoglycerate o. .o \// o. .o\// 'Cl STEP 8 Theremaining p h o s p h a t ee s t e rl i n k a g ei n 3-phosphoglycerate, which has a relativelylow free energy of hydrolysis,is moved from carbon 3 to carbon 2 to form 2-phosphoglycerate. C I H-C-Oi*P H-C-OH ,l 3-phosphoglycerate C C I H-C-O I P cH2oH 2 - p h o s p h olgy c e r a t e P cHz p h o s ph o e n oIp y r u v a t e C C C-O I C-O o. .o\./ o. .o\// I 2-p hosphog lycerate o. .o \./ o. .o \,/ STEP9 The removal of water from 2-phosphoglycerate c r e a t e sa h i g h - e n e r g ye n o l p h o s p h a t el i n k a g e . STEP10 The transfer to ADP of the high-energy p h o s p h a t eg r o u p t h a t w a s generated in step 9 forms A T P ,c o m p l e t i n gg l y c o l y s i s . t- cH2oH - C H r O ' .P . P cHz p h o s p h o e n oply r u v a t e N E TR E S U LO T FG L Y C O L Y S I S In addition to the pyruvate,the net productsare t w o m o l e c u l e so f A T Pa n d t w o m o l e c u l e so f N A D H I I CH: HS-CoA pyruvate The completecitricacidcycle.The two carbonsfrom acetylCoA that enter this turn of the cycle(shadowedin ) will be convertedto CO, in subsequentturns of the cycle:it is the two carbons shadowed in blue that are convertedto CO, in this cycle. (2c) acetyl CoA HS-CoA eoo *", tHO-C-COO- Step 1 (+a^) in, \*'ioo\ Po (6c) isocitrate fn, HC COO t citrate(6c) no-tH Coo- fumarate (4C) (x-ketoglutarate(5C) €oot fu 2 ' t' CH ffoo- s u c c i n a t e( 4 C ) ,rStep6 GOO- Hzo Coz CH. succinylCoA (4C) t' Ioo- I coo- 2 CH, il*p_:, t- C=O I S-CoA coz HS-CoA HS-CoA EE*t- Detailsof the eight stepsare shown below. For eachstep,the part of the moleculethat undergoesa changeis shadowedin hlue and the name of the enzymethat catalyzesthe reactionis in a yellow box. O:C -S-CoA STEP1 After the enzyme removesa proton from the CH, group on acetyl CoA, t h e n e g a t i v e l yc h a r g e d C H r - f o r m sa b o n d t o a carbonylcarbon of oxaloacetate.The s u b s e q u e nlto s sb y h y d r o l y s ios f t h e c o e n z y m e A (CoA)drivesthe reaction strongly forward. S T E P2 An isomerization reaction,in which water is f i r s t r e m o v e da n d t h e n added back, movesthe hydroxyl group from one c a r b o na t o m t o i t s n e i g h b o r I CH, cooI CHr t- HO-C-COO I 9H, l coo- t- HO-C-COO + HS-CoA + H* I CH, t- coocitrate coo- Hzo H- citrate cooI C-H I c-cootl C-H I coo- cls-aconitateintermediate H-C cooI-H I H-C -COO- I HO-C -H I coo isocitrate STEP3 ln the first of f o u r o x i d a t i o ns t e p si n t h e cycle,the carbon carrying the hydroxyl group is convertedto a carbonyl g r o u p .T h e i m m e d i a t e p r o d u c ti s u n s t a b l e l,o s i n g C O ,w h i l e s t i l l b o u n d t o the enzyme. cooI H-C -H I H-C -H I a-i I coo cooI-H H-C H_C I HO-c-H I coo- (x-ketogIutarate isocitrate STEP4 The o-ketog/utarate dehyd ro gen asecomplex closely r e s e m b l etsh e l a r g ee n z y m e complexthat convertspyruvate to acetyl co{(pyruvate dehydrogenase).lt likewise catalyzesan oxidation that producesNADH,CO2,and a h i g h - e n e r g yt h i o e s t e rb o n d t o coenzymeA (CoA). STEP5 A phosphate m o l e c u l ef r o m s o l u t i o n d i s p l a c etsh e C o A ,f o r m i n g a h i g h - e n e r g yp h o s p h a t e l i n k a g et o s u c c i n a t eT. h i s p h o s p h a t ei s t h e n p a s s e dt o G D Pt o f o r m G T P .( l n b a c t e r i a and plants,ATP is formed instead.) cooI H-C-H I-H cooI H-C -H I H-C-H I c:o I coo- H-C S succinyl-CoA (x-ketoglutarate cooH-C -H H-C I S-CoA H-C coo I-H I H-C-H s u c cni a t e cooI C-H H-C I coo f u marate S T E P8 l n t h e l a s to f J o u r o x i d a t i o ns t e p si n t h e c y c l et,h e c a r b o nc a r r y i n gt h e h y d r o x y l group is converted t o a c a r b o n y lg r o u p , regeneratingthe oxaloacetate n e e d e df o r s t e p 1 . ,, I coo- I coo S T E P7 T h e a d d i t i o no f water to fumarate placesa hydroxyl group next to a c a r b o n y lc a r b o n . coo I-H I ,, ) n-L-r I H-C-H I s u c c ln a r e succinyl-CoA S T E P6 ln the third oxidation step in the cycle,FAD removestwo hydrogen atoms from succinate. cooI HO-C -H I H-C-H I coomalate I I-CoA succinatedehydrogenase - cooI C-H /\ rl H-C I coo- f umarate cooI HO-C -H I H-C-H I coo malate cooI c:o I CH, I coo oxa loacetate + HS-CoA 124 Chapter2: CellChemistryand Biosynthesis REFERENCES General Berg,JM,Tymoczko, JL& StryerL (2006)Biochemistry, 6th ed New York:WH Freeman GarrettRH& Grisham CM (2005)Biochemistry, 3rded philadelphia: ThomsonBrooks/Cole Hortonl-1R, MoranLA,Scrimgeour et a (2005)Princip esof Bioch-.mistry 4th ed UpperSaddleRiver, NJ:prenticeHall NelsonDL& CoxMM (2004)Lehnlnger Principles of Biochemistry, 4th ed NewYork:Worth Nicholls DG& Ferguson S_l(2002)Bioenergerics,3rd ed Newyork: AcademicPress MathewsCK,van Ho de KE& AhernK G (2000)Biochemistry, 3rded 5 a ql r a r c , s c oB: e n j a rr C u m m i n g s MooreJA(1993)Sclence Asa Wayof KnowingCambridge, MA: Harvard University Press VoetD,Voet.lG& PrattCIV(2004)Fundamentals of Biochemistry, 2nd ed NewYork:Wiley The ChemicalComponentsof a Cell AbelesRH,FreyPA& Jencks WP(1992)Biochemistry Boston: Jones& Bartlett AtkinsPW('l996)Mo ecues NewYork:WH Freeman Branden C & ToozeJ (l 999) ntroduction to ProteinStructure, 2nd ed NewYork:Garland Scence Bretscher MS(,l985) Themolecules of the cel membrane5clAm 2 5 3 :01O I O 9 Burey 5K& Petsko GA(t 9BB)Weaklypolarinteractions in proteins,4dy PrateinChem39.125-1 89 De DuveC (2005)Singulanties: Landmarks on the pathways of Lif-. 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