H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 35
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In the absence ofbound nucleotide, a myosin head binds actin tightly in a “rigor”state. Step 1 : Binding of ATP opens the cleft in the myosinhead, disrupting the actin-binding site and weakening theinteraction with actin. Step 2 : Freed of actin, the myosin headhydrolyzes ATP, causing a conformational change in the head thatmoves it to a new position, closer to the () end of the actinfilament, where it rebinds to the filament.
Step 3 : As phosphate(Pi) dissociates from the ATP-binding pocket, the myosin headundergoes a second conformational change—the power stroke—which restores myosin to its rigor conformation. Because myosinis bound to actin, this conformational change exerts a force thatcauses myosin to move the actin filament.
Step 4 : Release ofADP completes the cycle. [Adapted from R. D. Vale and R. A. Milligan,2002, Science 288:88.]Head pivots andbinds a newactin subunitFocus Animation: Myosin Crossbridge CycleMEDIA CONNECTIONSADP•PiKEY CONCEPTS OF SECTION 3.4Molecular Motors and the Mechanical Work of CellsPi releaseMotor proteins are mechanochemical enzymes that convert energy released by ATP hydrolysis into either linearor rotary movement (see Figure 3-22).■3PiADPHead pivots andmoves filament(power stroke)Linear motor proteins (myosins, kinesins, and dyneins)move along cytoskeletal fibers carrying bound cargo,which includes vesicles, chromosomes, thick filaments inmuscle, and microtubules in eukaryotic flagella.■Myosin II consists of two heavy chains and several lightchains.
Each heavy chain has a head (motor) domain,which is an actin-activated ATPase; a neck domain, whichis associated with light chains; and a long rodlike tail domain that organizes the dimeric molecule and binds to thickfilaments in muscle cells (see Figure 3-24).■ADP release4ADPsmall conformational change in the head domain that isamplified into a large movement of the neck region. Thesmall conformational change in the head domain is localized to a “switch” region consisting of the nucleotide- andactin-binding sites. A “converter” region at the base of thehead acts like a fulcrum that causes the leverlike neck tobend and rotate.Homologous switch, converter, and lever arm structuresin kinesin are responsible for the movement of kinesin motorproteins along microtubules.
The structural basis for dyneinmovement is unknown because the three-dimensional structure of dynein has not been determined.Movement of myosin relative to an actin filament resultsfrom the attachment of the myosin head to an actin filament, rotation of the neck region, and detachment in acyclical ATP-dependent process (see Figure 3-25). The samegeneral mechanism is thought to account for all myosinand kinesin-mediated movement.■3.5 Common Mechanismsfor Regulating Protein FunctionMost processes in cells do not take place independently ofone another or at a constant rate. Instead, the catalytic activity of enzymes or the assembly of a macromolecular complex is so regulated that the amount of reaction product orthe appearance of the complex is just sufficient to meet theneeds of the cell.
As a result, the steady-state concentrations3.5 • Common Mechanisms for Regulating Protein Functionof substrates and products will vary, depending on cellularconditions. The flow of material in an enzymatic pathway iscontrolled by several mechanisms, some of which also regulate the functions of nonenzymatic proteins.One of the most important mechanisms for regulatingprotein function entails allostery. Broadly speaking, allosteryrefers to any change in a protein’s tertiary or quaternarystructure or both induced by the binding of a ligand, whichmay be an activator, inhibitor, substrate, or all three.
Allosteric regulation is particularly prevalent in multimeric enzymes and other proteins. We first explore several ways inwhich allostery influences protein function and then considerother mechanisms for regulating proteins.Cooperative Binding Increases a Protein’sResponse to Small Changes in LigandConcentrationIn many cases, especially when a protein binds several molecules of one ligand, the binding is graded; that is, the binding of one ligand molecule affects the binding of subsequentligand molecules.
This type of allostery, often called cooper-% Saturation10050P50 = 260204060p O2 (torr)p O2 in capillariesof active muscles80100p O2 in alveoliof lungs▲ EXPERIMENTAL FIGURE 3-26 Sequential binding ofoxygen to hemoglobin exhibits positive cooperativity. Eachhemoglobin molecule has four oxygen-binding sites; at saturationall the sites are loaded with oxygen. The oxygen concentration iscommonly measured as the partial pressure (pO2). P50 is the pO2at which half the oxygen-binding sites at a given hemoglobinconcentration are occupied; it is equivalent to the Km for anenzymatic reaction.
The large change in the amount of oxygenbound over a small range of pO2 values permits efficientunloading of oxygen in peripheral tissues such as muscle. Thesigmoidal shape of a plot of percent saturation versus ligandconcentration is indicative of cooperative binding. In the absenceof cooperative binding, a binding curve is a hyperbola, similar tothe simple kinetic curves in Figure 3-19. [Adapted from L. Stryer,Biochemistry, 4th ed., 1995, W.
H. Freeman and Company.]83ativity, permits many multisubunit proteins to respond moreefficiently to small changes in ligand concentration thanwould otherwise be possible. In positive cooperativity, sequential binding is enhanced; in negative cooperativity,sequential binding is inhibited.Hemoglobin presents a classic example of positive cooperative binding. Each of the four subunits in hemoglobincontains one heme molecule, which consists of an iron atomheld within a porphyrin ring (see Figure 8-16a). The hemegroups are the oxygen-binding components of hemoglobin(see Figure 3-10). The binding of oxygen to the heme molecule in one of the four hemoglobin subunits induces a localconformational change whose effect spreads to the othersubunits, lowering the Km for the binding of additional oxygen molecules and yielding a sigmoidal oxygen-binding curve(Figure 3-26).
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).
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