B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 58

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In this case, the binding of thefirst ligand discourages the binding of the second ligand. Thus, if a shape changecaused by glucose binding decreases the affinity of a protein for molecule X, thebinding of X must also decrease the protein’s affinity for glucose (Figure 3–58).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.151152Chapter 3: ProteinsFigure 3–57 Positive regulation causedby conformational coupling betweentwo separate binding sites. In thisexample, both glucose and molecule Xbind best to the closed conformation of aprotein with two domains.

Because bothglucose and molecule X drive the proteintoward its closed conformation, eachligand helps the other to bind. Glucoseand molecule X are therefore said to bindcooperatively to the protein.INACTIVEmoleculeXmoleculeXpositiveregulationglucoseACTIVE10% active100% activeThe relationships shown in Figures 3–57 and 3–58 apply to all proteins, andthey underlie all of cell biology. The principle seems so obvious in retrospectthat we now take it for granted. But the discovery of linkage in studies of a fewMBoC6 m3.58/3.53enzymes in the 1950s, followedby an extensive analysis of allosteric mechanismsin proteins in the early 1960s, had a revolutionary effect on our understanding ofbiology.

Since molecule X in these examples binds at a site on the enzyme thatis distinct from the site where catalysis occurs, it need not have any chemicalrelationship to the substrate 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 such amechanism, allosteric proteins serve as general switches that, in principle, canallow one molecule in a cell to affect the fate of any other.Symmetric Protein Assemblies Produce Cooperative AllostericTransitionsA single-subunit enzyme that is regulated by negative feedback can at mostdecrease from 90% to about 10% activity in response to a 100-fold increase inthe concentration of an inhibitory ligand that it binds (Figure 3–59, red line).Responses of this type are apparently not sharp enough for optimal cell regulation,and most enzymes that are turned on or off by ligand binding consist of symmetric assemblies of identical subunits.

With this arrangement, the binding of a molecule of ligand to a single site on one subunit can promote an allosteric change inthe entire assembly that helps the neighboring subunits bind the same ligand. Asa result, a cooperative allosteric transition occurs (Figure 3–59, blue line), allowingACTIVEmoleculeXmoleculeXnegativeregulationglucoseINACTIVE100% active10% activeFigure 3–58 Negative regulation causedby conformational coupling betweentwo separate binding sites. The schemehere resembles that in the previous figure,but here molecule X prefers the openconformation, while glucose prefers theclosed conformation.

Because glucoseand molecule X drive the protein towardopposite conformations (closed and open,respectively), the presence of either ligandinterferes with the binding of the other.PROTEIN FUNCTION153percent enzyme activity1005001 subunit2 subunits4 subunits5inhibitor concentration10a relatively small change in ligand concentration in the cell to switch the wholeassembly from an almost fully active to an almost fully inactive conformation (orvice versa).The principles involved in a cooperative“all-or-none” transition are the sameMBoC6 m3.60/3.55for all proteins, whether or not they are enzymes.

Thus, for example, they are critical for the efficient uptake and release of O2 by hemoglobin in our blood. Butthey are perhaps easiest to visualize for an enzyme that forms a symmetric dimer.In the example shown in Figure 3–60, the first molecule of an inhibitory ligandbinds with great difficulty since its binding disrupts an energetically favorableinteraction between the two identical monomers in the dimer.

A second moleculeof inhibitory 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 induced fit model for a cooperative allosteric transition, we can view such a symmetric enzyme as having only two possible conformations, corresponding to the “enzyme on” and “enzyme off” structures in Figure3–60. In this view, ligand binding perturbs an all-or-none equilibrium betweenthese two states, thereby changing the proportion of active molecules. Both models represent true and useful concepts.Figure 3–59 Enzyme activity versusthe concentration of inhibitory ligandfor single-subunit and multisubunitallosteric enzymes.

For an enzyme with asingle subunit (red line), a drop from 90%enzyme activity to 10% activity (indicatedby the two dots on the curve) requires a100-fold increase in the concentration ofinhibitor. The enzyme activity is calculatedfrom the simple equilibrium relationshipK = [IP]/[I][P], where P is active protein,I is inhibitor, and IP is the inactive proteinbound to inhibitor. An identical curveapplies to any simple binding interactionbetween two molecules, A and B. Incontrast, a multisubunit allosteric enzymecan respond in a switchlike manner to achange in ligand concentration: the steepresponse is caused by a cooperativebinding of the ligand molecules, asexplained in Figure 3–60.

Here, the greenline represents the idealized result expectedfor the cooperative binding of two inhibitoryligand molecules to an allosteric enzymewith two subunits, and the blue line showsthe idealized response of an enzyme withfour subunits. As indicated by the two dotson each of these curves, the more complexenzymes drop from 90% to 10% activityover a much narrower range of inhibitorconcentration than does the enzymecomposed of a single subunit.Many Changes in Proteins Are Driven by Protein PhosphorylationProteins are regulated by more than the reversible binding of other molecules. Asecond method that eukaryotic cells use extensively to regulate a protein’s function is the covalent addition of a smaller molecule to one or more of its amino acidside chains.

The most common such regulatory modification in higher eukaryotesis the 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 threeimportant 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, attracting acluster of positively charged amino acid side chains. This can, in turn, affect thebinding of ligands elsewhere on the protein surface, dramatically changing theFigure 3–60 A cooperative allosteric transition in an enzyme composed oftwo identical subunits. This diagram illustrates how the conformation of onesubunit can influence that of its neighbor.

The binding of a single molecule ofan inhibitory ligand (yellow) to one subunit of the enzyme occurs with difficultybecause it changes the conformation of this subunit and thereby disrupts thesymmetry of the enzyme. Once this conformational change has occurred,however, the energy gained by restoring the symmetric pairing interactionbetween the two subunits makes it especially easy for the second subunitto bind the inhibitory ligand and undergo the same conformational change.Because the binding of the first molecule of ligand increases the affinity withwhich the other subunit binds the same ligand, the response of the enzyme tochanges in the concentration of the ligand is much steeper than the responseof an enzyme with only one subunit (see Figure 3–59 and Movie 3.10).ENZYME ONinhibitorDIFFICULTTRANSITIONsubstrateEASYTRANSITIONENZYME OFF154Chapter 3: Proteinsprotein’s activity.

When a second enzyme removes the phosphate group, the protein returns to its original conformation and restores its initial activity.Second, an attached phosphate group can form part of a structure that thebinding sites of other proteins recognize. As previously discussed, the SH2 domainbinds to a short peptide sequence containing a phosphorylated tyrosine sidechain (see Figure 3–40B). More than ten other common domains provide bindingsites for attaching their protein to phosphorylated peptides in other protein molecules, each recognizing a phosphorylated amino acid side chain in a differentprotein context. Third, the addition of a phosphate group can mask a binding sitethat otherwise holds two proteins together, and thereby disrupt protein–proteininteractions.

As a result, protein phosphorylation and dephosphorylation veryoften drive the regulated assembly and disassembly of protein complexes (see, forexample, Figure 15–11).Reversible protein phosphorylation controls the activity, structure, and cellular localization of both enzymes and many other types of proteins in eukaryoticcells. In fact, this regulation is so extensive that more than one-third of the 10,000or so proteins in a typical mammalian cell are thought to be phosphorylated atany given time—many with more than one phosphate. As might be expected, theaddition and removal of phosphate groups from specific proteins often occur inresponse to signals that specify some change in a cell’s state. For example, thecomplicated series of events that takes place as a eukaryotic cell divides is largelytimed in this way (discussed in Chapter 17), and many of the signals mediatingcell–cell interactions are relayed from the plasma membrane to the nucleus by acascade of protein phosphorylation events (discussed in Chapter 15)._OATPOADPOHA Eukaryotic Cell Contains a Large Collection of Protein Kinasesand Protein PhosphatasesProtein phosphorylation involves the enzyme-catalyzed transfer of the terminalphosphate group of an ATP molecule to the hydroxyl group on a serine, threonine,or tyrosine side chain of the protein (Figure 3–61).

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