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In the examplesholtryrin Figure 3-61, the first molecule of an inhibitory ligand binds with greatdifficulty since its binding disrupts an energetically favorable interactionbetween the two identical monomers in the dimer. A second molecule ofinhibitory ligand now binds more easily,however, because its binding restoresthe energetically favorable monomer-monomer contacts of a symmetric dimer(this also completely inactivates the enzyme).As an alternative to this inducedfirmodel for a cooperative allosteric transition, we can view such a symmetrical enzyme as having only two possible conformations, corresponding to the "enzyme on" and "enzyme off" structures inFigure 3-61.

In this view, ligand binding perturbs an all-or-none equilibriumbetween these two states,thereby changing the proportion of active molecules.Both models represent true and useful concepts; it is the second model that weshall describe next.Figure3-60 Enzymeactivity versustheconcentrationof inhibitoryligandforsingle-subunitand multisubunitallostericenzymes.Foran enzymewith asinglesubunit (redline),a drop from 900/oactivity(indicatedenzymeactivityto 10o/oby the two dots on the curve)requiresaofin the concentration10O-foldincreaseTheenzymeactivityisinhibitor.from the simpleequilibriumcalculatedwhereP isl( = tlPl/tlltPl,relationshipactiveprotein,I is inhibitor,and lP is theinactiveoroteinboundto inhibitor.Anidenticalcurveappliesto any simplebindinginteractionbetweentwoA and B.In contrast,amolecules,enzymecanmultisubunitallostericrespondin a switchlikemannerto athe steepchangein ligandconcentration:is causedby a cooperativeresponseasbindingof the ligandmolecules,explainedin Figure3-61.Here,the green/ine representsthe idealizedresultexpectedfor the cooperativebinding ofto antwo inhibitoryligandmoleculesenzymewith two subunits,andallostericthe blueline showsthe idealizedof an enzymewith fourresponsesubunits.As indicatedby the two dots oneachof thesecurves,the morecomplexactivityenzymesdrop from 90o/oro10o/oovera much narrowerrangeof inhibitorthan doesthe enzymeconcentrationcomposedof a singlesubunit.lsTranscarbamoylasein AspartateTheAllostericTransitionUnderstoodin AtomicDetailOne enzyme used in the early studies of allosteric regulation was aspartatetranscarbamoylase from E coli.

lt catalyzesthe important reaction that beginsthe synthesisof the pyrimidine ring of C, U, and T nucleotides: carbamoyl phosphate + aspartate -+ ly'-carbamoylaspartate.One of the final products of thispathway, cltosine triphosphate (CTP),binds to the enzyme to turn it off whenever CTP is plentiful.Aspartate transcarbamoylaseis a large complex of six regulatory and six catalyic subunits. The catalyic subunits form two trimers, each arranged in theshape of an equilateral triangle; the two trimers face each other and are heldFigure3-61 A cooperativeallosterictransitionin an enzymecomposedofhow the conformationof two identicalsubunits.ThisdiagramillustratesThe bindingof a singleone subunitcan influencethat of its neighbor.moleculeof an inhibitoryligand (yellow)to one subunitof the enzymeof this subunitoccurswith difficultybecauseit changesthe conformationand therebydisruptsthe symmetryof the enzyme.Oncethisthe energygainedbychangehasoccurred,however,conformationalrestoringthe symmetricpairinginteractionbetweenthe two subunitsmakesit especiallyeasyfor the secondsubunitto bind the inhibitorythe bindingchange.Becauseligandand undergothe sameconformationalthe affinitywith whichthe otherof the firstmoleculeof ligandincreasesof the enzymeto changesinsubunitbindsthe sameligand,the responseof anof the ligandis much steeperthan the responsethe concentrationenzymewith only one subunit(seeFigure3-60).ONENZYMEinhibitorEASYTRANSITIONtr174Chapter3: ProteinsINACTIVE ENZYME: T STATEcata lytics ub u n i t sCTPe#5nmACTIVEENZYME:R STATEtogether by three regulatory dimers that form a bridge between them.

The entiremolecule is poised to undergo a concerted, all-or-none, allosteric transitionbetween two conformations, designated as T (tense) and R (relaxed)states (Figure 3-62).The binding of substrates (carbamoyl phosphate and aspartate) to the catalytic trimers drives aspartate transcarbamoylase into its catalytically active Rstate, from which the regulatory crP molecules dissociate.

By contrast, thebinding of crP to the regulatory dimers converts the enzyme to the inactive Tstate, from which the substrates dissociate. This tug-of-war between crp andsubstratesis identical in principle to that described previously in Figure 3-59 fora simpler allosteric protein. But because the tug-of-war occurs in a symmetricmolecule with multiple binding sites, the enzyme undergoes a cooperativeallosteric transition that will turn it on suddenly as substratesaccumulate (forming the R state) or shut it off rapidly when crp accumulates (forming the T state).A combination of biochemistry and x-ray crystallography has revealedmanyfascinating details of this allosteric transition. Each regulatory subunit has twodomains, and the binding of crP causes the two domains to move relative toeach other, so that they function like a lever that rotates the two catalytic trimersand pulls them closer together into the T state (see Figure 3-62).

\.4rhenthisoccurs, hydrogen bonds form between opposing catal)'tic subunits. This helpswiden the cleft that forms the active site within each catalytic subunit, therebydisrupting the binding sites for rhe substrates (Figure 3-63). Adding largeamounts of substrate has the opposite effect, favoring the R state by binding inthe cleft of each catalytic subunit and opposing the above conformationalchange. conformations that are intermediate between R and T are unstable, sothat the enzyme mostly clicks back and forth between its R and T forms, producing a mixture of these two speciesin proportions that depend on the relitiveconcentrations of CTP and substrates.Figure3-62 The transition betweenR and T statesin the enzyme aspartatetranscarbamoylase.<CTAA>The enzymeconsistsof a complexof sixcatalyticsubunitsand six regulatorysubunits,andthe structuresof its inactive(T state)andactive(Rstate)forms havebeendeterminedby x-raycrystallography.Theenzymeis turned off by feedbackinhibitionwhen CTPconcentrationsrise.Eachregulatorysubunitcan bind onemoleculeof CTP,which is one of the finalproductsin the pathway.By meansof thisnegativefeedbackregulation,the pathwayis preventedfrom producingmore CTPthan the cell needs.(Basedon K.L.Krause,K.W.Volzand W.N.Lipscomb,Proc.NatlAcad.Sci.U.5.A.82:1643-1647, 1985.With permissionfrom NationalAcademyof Sciences.)175PROTEINFUNCTIONArg 167Ws164(g,rcsArg229rg 234G l u2 3 9T state (inactive)in ProteinsAre Drivenby ProteinPhosphorylationManyChangesProteins are regulated by more than the reversible binding of other molecules.Asecond method that eucaryotic cells use to regulate a protein's function is thecovalent addition of a smaller molecule to one or more of its amino acid sidechains.

The most common such regulatory modification in higher eucaryotes isthe addition of a phosphate group. We shall therefore use protein phosphorylation to illustrate some of the general principles involved in the control of proteinfunction through the modification of amino acid side chains.A phosphorylation event can affect the protein that is modified in twoimportant ways.

First, because each phosphate group carries two negativecharges,the enzyme- catalyzed addition of a phosphate group to a protein cancause a major conformational change in the protein by, for example, attractinga cluster of positively charged amino acid side chains. This can, in turn, affectthe binding of ligands elsewhere on the protein surface, dramatically changingthe protein's activity. \A/trena second enzyme removes the phosphate group, theprotein returns to its original conformation and restoresits initial activity.Second, an attached phosphate group can form part of a structure that thebinding sites of other proteins recognize.As previously discussed,certain protein domains, sometimes referred to as modules, appear very frequently as partsof larger proteins. One such module is the SH2 domain, described earliel whichbinds to a short peptide sequence containing a phosphorylated tyrosine sidechain (seeFigure 3-398).

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