Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 36
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Consequently, the sequential binding of oxygen is facilitated, permitting hemoglobin to load more oxygen in peripheral tissues than it otherwise could at normaloxygen concentrations.Ligand Binding Can Induce Allosteric Releaseof Catalytic Subunits or Transition to a Statewith Different ActivityPreviously, we looked at protein kinase A to illustrate binding and catalysis by the active site of an enzyme.
This enzymecan exist as an inactive tetrameric protein composed of twocatalytic subunits and two regulatory subunits. Each regulatory subunit contains a pseudosubstrate sequence that bindsto the active site in a catalytic subunit. By blocking substratebinding, the regulatory subunit inhibits the activity of thecatalytic subunit.Inactive protein kinase A is turned on by cyclic AMP(cAMP), a small second-messenger molecule. The binding ofcAMP to the regulatory subunits induces a conformationalchange in the pseudosubstrate sequence so that it can nolonger bind the catalytic subunit.
Thus, in the presence ofcAMP, the inactive tetramer dissociates into two monomericactive catalytic subunits and a dimeric regulatory subunit(Figure 3-27). As discussed in Chapter 13, the binding of various hormones to cell-surface receptors induces a rise in theintracellular concentration of cAMP, leading to the activation of protein kinase A.
When the signaling ceases and thecAMP level decreases, the activity of protein kinase A isturned off by reassembly of the inactive tetramer. The binding of cAMP to the regulatory subunits exhibits positive cooperativity; thus small changes in the concentration of thisallosteric molecule produce a large change in the activity ofprotein kinase A.Many multimeric enzymes undergo allosteric transitionsthat alter the relation of the subunits to one another but donot cause dissociation as in protein kinase A.
In this typeof allostery, the activity of a protein in the ligand-boundstate differs from that in the unbound state. An example isthe GroEL chaperonin discussed earlier. This barrel-shaped84CHAPTER 3 • Protein Structure and Function(a)Catalytic siteNucleotidebinding sitePseudosubstrateCC+CRRC+Inactive PKARRActive PKAcAMPNH2(b)CNCHCCNCHNOCH2HOHHOPONHOOHcyclic AMP(cAMP)▲ FIGURE 3-27 Ligand-induced activation of protein kinaseA (PKA). At low concentrations of cyclic AMP (cAMP), the PKAis an inactive tetramer.
Binding of cAMP to the regulatory (R)subunits causes a conformational change in these subunits thatpermits release of the active, monomeric catalytic (C) subunits.(b) Cyclic AMP is a derivative of adenosine monophosphate. Thisintracellular signaling molecule, whose concentration rises inresponse to various extracellular signals, can modulate theactivity of many proteins.100-fold by the release of Ca2 from ER stores or by its import from the extracellular environment.
This rise in cytosolic Ca2 is sensed by Ca2-binding proteins, particularlythose of the EF hand family, all of which contain the helixloop-helix motif discussed earlier (see Figure 3-6a).The prototype EF hand protein, calmodulin, is found inall eukaryotic cells and may exist as an individualmonomeric protein or as a subunit of a multimeric protein. Adumbbell-shaped molecule, calmodulin contains four Ca2binding sites with a KD of ≈106 M. The binding of Ca2 tocalmodulin causes a conformational change that permitsCa2/calmodulin to bind various target proteins, therebyswitching their activity on or off (Figure 3-28).
Calmodulinand similar EF hand proteins thus function as switch proteins, acting in concert with Ca2 to modulate the activityof other proteins.Switching Mediated by Guanine Nucleotide–BindingProteins Another group of intracellular switch proteins constitutes the GTPase superfamily. These proteins includemonomeric Ras protein (see Figure 3-5) and the G subunit ofthe trimeric G proteins. Both Ras and G are bound to theplasma membrane, function in cell signaling, and play a keyrole in cell proliferation and differentiation. Other membersEF1EF3EF2protein-folding machine comprises two back-to-back multisubunit rings, which can exist in a “tight” peptide-bindingstate and a “relaxed” peptide-releasing state (see Figure3-11). The binding of ATP and the co-chaperonin GroES toone of the rings in the tight state causes a twofold expansionof the GroEL cavity, shifting the equilibrium toward the relaxed peptide-folding state.EF4TargetpeptideCa2+Calcium and GTP Are Widely Used to ModulateProtein ActivityIn the preceding examples, oxygen, cAMP, and ATP cause allosteric changes in the activity of their target proteins (hemoglobin, protein kinase A, and GroEL, respectively).
Twoadditional allosteric ligands, Ca2 and GTP, act through twotypes of ubiquitous proteins to regulate many cellularprocesses.Calmodulin-Mediated Switching The concentration ofCa2 free in the cytosol is kept very low (≈107 M) by membrane transport proteins that continually pump Ca2 out ofthe cell or into the endoplasmic reticulum. As we learn inChapter 7, the cytosolic Ca2 level can increase from 10- to▲ FIGURE 3-28 Switching mediated by Ca2/calmodulin.Calmodulin is a widely distributed cytosolic protein that containsfour Ca2-binding sites, one in each of its EF hands.
Each EFhand has a helix-loop-helix motif. At cytosolic Ca2+ concentrationsabove about 5 107 M, binding of Ca2 to calmodulin changesthe protein’s conformation. The resulting Ca2/calmodulin wrapsaround exposed helices of various target proteins, therebyaltering their activity.3.5 • Common Mechanisms for Regulating Protein FunctionActiveActive ("on")RGTPaseGDPGEFsGTP+++−GAPsRGSsGDIsGTPaseGDPOHPiATPProteinphosphataseProteinkinaseInactive ("off ")GTP85OH2OROPADPO−O−Inactive▲ FIGURE 3-29 Cycling of GTPase switch proteins betweenthe active and inactive forms. Conversion of the active into theinactive form by hydrolysis of the bound GTP is accelerated byGAPs (GTPase-accelerating proteins) and RGSs (regulators of Gprotein–signaling) and inhibited by GDIs (guanine nucleotidedissociation inhibitors).
Reactivation is promoted by GEFs(guanine nucleotide–exchange factors).▲ FIGURE 3-30 Regulation of protein activity bykinase/phosphatase switch. The cyclic phosphorylation anddephosphorylation of a protein is a common cellular mechanismfor regulating protein activity. In this example, the target proteinR is inactive (light orange) when phosphorylated and active (darkorange) when dephosphorylated; some proteins have theopposite pattern.of the GTPase superfamily function in protein synthesis, thetransport of proteins between the nucleus and the cytoplasm,the formation of coated vesicles and their fusion with targetmembranes, and rearrangements of the actin cytoskeleton.All the GTPase switch proteins exist in two forms (Figure3-29): (1) an active (“on”) form with bound GTP (guanosinetriphosphate) that modulates the activity of specific targetproteins and (2) an inactive (“off”) form with bound GDP(guanosine diphosphate).
The GTPase activity of theseswitch proteins hydrolyzes bound GTP to GDP slowly, yielding the inactive form. The subsequent exchange of GDP withGTP to regenerate the active form occurs even more slowly.Activation is temporary and is enhanced or depressed byother proteins acting as allosteric regulators of the switchprotein. We examine the role of various GTPase switch proteins in regulating intracellular signaling and other processesin several later chapters.Nearly 3 percent of all yeast proteins are protein kinases orphosphatases, indicating the importance of phosphorylationand dephosphorylation reactions even in simple cells. Allclasses of proteins—including structural proteins, enzymes,membrane channels, and signaling molecules—are regulatedby kinase/phosphatase switches.
Different protein kinases andphosphatases are specific for different target proteins and canthus regulate a variety of cellular pathways, as discussed inlater chapters. Some of these enzymes act on one or a few target proteins, whereas others have multiple targets. The latterare useful in integrating the activities of proteins that are coordinately controlled by a single kinase/phosphatase switch.Frequently, another kinase or phosphatase is a target, thus creating a web of interdependent controls.Cyclic Protein Phosphorylationand Dephosphorylation RegulateMany Cellular FunctionsAs noted earlier, one of the most common mechanisms forregulating protein activity is phosphorylation, the additionand removal of phosphate groups from serine, threonine, ortyrosine residues.
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