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If, for example, a compartment occupies a total of 10% of the volume of the cell, the concentration of reactants in that compartment may beincreased by 10 times compared with a cell with the same number of enzymesand substrate molecules, but no compartmentalization. Reactions limited bythe speed of diffusion can thereby be speeded up by a factor of 10.the CatalyticActivitiesof its EnzymesTheCellRegulatesmany of which operate at the sameA living cell contains thousands of enz).rynes,time and in the same small volume of the c1'tosol.By their catalytic action, theseenzymes generate a complex web of metabolic pathways, each composed ofchains of chemical reactions in which the product of one enzyme becomes thesubstrate of the next.
In this maze of pathways, there are many branch points(nodes) where different enzymes compete for the same substrate.The system isso complex (see Figure 2-88) that elaborate controls are required to regulatewhen and how rapidly each reaction occurs.8 t r i m e r so fl i p o a m i d er e d u c t a s e transacetylase+ 1 2 m o l e c u l e so fdihydrolipoyldehydrogenase+24 moleculeosfpyruvatedecarboxylaseFigure3-55 The structure of pyruvateThisenzymecomplexdehydrogenase.catalyzesthe conversionof pyruvatetoacetylCoA,as part of the pathwaythatoxidizessugarsto COzand HzO(seeFigure2-79).lt is an exampleof a largemultienzymecomplexin which reactionintermediatesare passeddirectlyfrom oneenzymeto another.17OChapter3: ProteinsRegulation occurs at many levels.At one level, the cell controls how manymolecules of each enzyme it makes by regulating the expressionof the gene thatencodes that enzyme (discussedin chapter 7).
The cell also controls enzymaticactivities by confining sets of enzymes to particular subcellular compartments,enclosed by distinct membranes (discussedin chapters 12 and 14). As will bediscussed later in this chapter, enzymes are frequently covalently modified tocontrol their activity.
The rate ofprotein destruction by targeted proteolysis represents yet another important regulatory mechanism (seep. 395). But the mostgeneral process that adjusts reaction rates operates through a direct, reversiblechange in the activity of an enzyme in response to the specific small moleculesthat it encounters.The most common type of control occurs when a molecule other than oneof the substrates binds to an enzyme at a special regulatory site outside theactive site, thereby altering the rate at which the enzyme converts its substratesto products. For example, in feedback inhibition a product produced late in areaction pathway inhibits an enzyme that acts earlier in the pathway.
Thus,whenever large quantities of the final product begin to accumulate, this productbinds to the enzyme and slows down its catalytic action, thereby limiting the further entry of substrates into that reaction pathway (Figure g-s6). \Mhere pathways branch or intersect, there are usually multiple points of control by different final products, each of which works to regulate its own synthesis (Figure3-57).
Feedback inhibition can work almost instantaneously, and it is rapidlvreversedwhen the level of the product falls.Figure3-56 Feedbackinhibitionof asinglebiosyntheticpathway.TheendproductZ inhibitsthe firstenzymethat isuniqueto its synthesisand therebycontrolsits own levelin the cell.Thisis anexampleof negativeregulation.aspartateIIIImethionineFigure3-57 Multiplefeedbackinhibition.In this example,which showsthe biosyntheticpathwaysfor fourdifferentaminoacidsin bacteria,the redarrowsindicatepositionsat whichproductsfeed backto inhibitenzymes.Eachaminoacidcontrolsthe firstenzymespecificto its own synthesis,therebycontrollingits own levelsand avoidingawasteful,or evendangerous,buildupofintermediates.The productscanalsoseparatelyinhibitthe initialset ofreactionscommonto all the syntheses;inthis case,three differentenzymescatalyzethe initialreaction,eachinhibitedbv a differentoroduct.PROTEINFUNCTIONFeedback inhibition is negatiueregulation: it prevents an enzyme from acting.
Enzymes can also be subject to positiue regulation, in which a regulatorymolecule stimulates the enzyme's activity rather than shutting the enzymedown. Positive regulation occurs when a product in one branch of the metabolicnetwork stimulates the activity of an enzyme in another pathway. As one example, the accumulation of ADP activates several enzymes involved in the oxidation of sugar molecules, thereby stimulating the cell to convert more ADP to AIPAllostericEnzymesHaveTwoor MoreBindingSitesThatInteractA striking feature of both positive and negative feedback regulation is that the regulatory molecule often has a shape totally different from the shape of the substrateof the enz].ryne.This is why the effect on a protein is termed allostery (from theGreekwords allos,meaning"other," andstereos,meaning"solid" or"three-dimensional").
As biologists learned more about feedback regulation, they recognizedthat the enzyrnes involved must have at least two different binding sites on theirsurface-an active site that recognizes the substrates, and a regulatory site thatrecognizes a regulatory molecule. These two sites must somehow communicate sothat the catalytic events at the active site can be influenced by the binding of theregulatory molecule at its separatesite on the protein's surface.The interaction between separated sites on a protein molecule is nowknor,tmto depend on a conformational changein the protein: binding at one ofthe sites causesa shift from one folded shape to a slightly different folded shape.During feedback inhibition, for example, the binding of an inhibitor at one siteon the protein causesthe protein to shift to a conformation that incapacitates itsactive site, located elsewherein the protein.It is thought that most protein molecules are allosteric.
They can adopt twoor more slightly different conformations, and a shift from one to another causedby the binding of a ligand can alter their activity. This is true not only forenzymes but also for many other proteins, including receptors, structural proteins, and motor proteins. In all instances of allosteric regulation, each conformation of the protein has somewhat different surface contours, and the protein'sbinding sites for ligands are altered when the protein changes shape. Moreoveras we discuss next, each ligand will stabilize the conformation that it binds tomost strongly, and thus-at high enough concentrations-will tend to "switch'the protein toward the conformation that the ligand prefers.TwoLigandsWhoseBindingSitesAreCoupledMustReciprocallyAffectEachOther'sBindingThe effects of ligand binding on a protein follow from a fundamental chemicalprinciple knor.vnas linkage.
Suppose,for example, that a protein that binds glucose also binds another molecule, X, at a distant site on the protein's surface. Ifthe binding site for X changes shape as part of the conformational changeinduced by glucosebinding, the binding sites for X and for glucose are said to becoupled. Vy'henevertwo ligands prefer to bind to the same conformation of anallosteric protein, it follows from basic thermodynamic principles that each ligand must increasethe affinity of the protein for the other.
Thus, if the shift of theprotein in Figure 3-58 to the closed conformation that binds glucose best alsocauses the binding site for X to fit X better, then the protein will bind glucosemore tightly when X is present than when X is absent.Conversely,linkage operates in a negative way if two ligands prefer to bindto dffirent conformations of the same protein.
In this case,the binding of thefirst ligand discouragesthe binding of the second ligand. Thus, if a shape changecaused by glucose binding decreasesthe affinity of a protein for molecule X, thebinding of X must also decreasethe protein's affinity for glucose (Figure 3-59).The linkage relationship is quantitatively reciprocal, so that, for example, if glucose has a very large effect on the binding of X, X has a very large effect on thebinding of glucose.171172Chapter3: ProteinsINACTIVEFigure3-58 Positiveregulation causedby conformationalcoupling betweentwo distantbinding sites.In thisexample,both glucoseand moleculeXbind bestto the c/osedconformationof aproteinwith two domains.Becausebothglucoseand moleculeX drivethe proteintoward its closedconformation,eachligandhelpsthe otherto bind.Glucoseand moleculeX arethereforesaidto bindcooperativelyto the protein.moleculeX?Ipositiver e gu l a t i o nACTIVE10% active100% activeThe relationships sho'o.rnin Figures 3-58 and 3-59 apply to all proteins, andthey underlie all of cell biology.
They seem so obvious in retrospect that we nowtake it for granted. But the discovery of linkage in studies of a few enzymes in the1950s,followed by an extensive analysis of allosteric mechanisms in proteins inthe early 1960s, had a revolutionary effect on our understanding of biology.Since molecule X in these examples binds at a site on the enzyme that is distinctfrom the site where catalysis occurs, it need have no chemical relationship toglucose or to any other ligand that binds at the active site. Moreover, as we havejust seen, for enzymes that are regulated in this way, molecule X can either turnthe enzyme on (positive regulation) or turn it off (negative regulation). By sucha mechanism, allosteric proteins serve as general switches that, in principle,allow one molecule in a cell to affect the fate of anv other.SymmetricProteinAssembliesProduceCooperativeAllostericTransitionsA single-subunit enzyme that is regulated by negative feedback can at mostdecreasefrom 90% to about l0% activity in responseto a 1O0-foldincreasein theconcentration of an inhibitory ligand that it binds (Figure 3-60, red line).Responsesof this type are apparently not sharp enough for optimal cell regulation, and most enzymes that are turned on or off by ligand binding consist ofs).rynmetricassemblies of identical subunits.
with this arrangement, the bindingof a molecule of ligand to a single site on one subunit can promote an allosteriichange in the entire assembly that helps the neighboring subunits bind thesame ligand. As a result, a cooperatiue allosteric transition occurs (Figure 3-60,blue line), allowing a relatively small change in ligand concentration in the cellto switch the whole assembly from an almost fully active to an almost fully inactive conformation (or vice versa).CmoleculeXI{negativeregu lation100%active1 0 %a c t i v eFigure3-59 Negativeregulation causedby conformationalcoupling betweentwo distant binding sites.The schemehereresemblesthat in the oreviousfigure,but here moleculeX preferstheopen conformation,while glucoseprefersthe c/osedconformation.Becauseglucoseand moleculeX drivethe proteintowardoppositeconformations(closedandopen, respectively),the presenceofeitherligandinterfereswith the bindingof the other.173PROTEINFUNCTIONIIoa ^_EU)Ncoooo5i n h i b i t o rc o n c e n t r a t i o n-The principles involved in a cooperative "all-or-none" transition are thesame for all proteins, whether or not they are enzymes.But they are perhaps easiest to visualize for an enzyme that forms a s).rynmetricdimer.
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