Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 34
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The aminoacids composing the active site are not necessarily adjacentin the amino acid sequence but are brought into proximity in the native conformation.■From plots of reaction rate versus substrate concentration, two characteristic parameters of an enzyme canbe determined: the Michaelis constant Km, a measure ofthe enzyme’s affinity for substrate, and the maximal velocity Vmax, a measure of its catalytic power (see Figure3-19).■KEY CONCEPTS OF SECTION 3.3Enzymes and the Chemical Work of CellsThe function of nearly all proteins depends on their ability to bind other molecules (ligands). Ligand-binding sites■3.4 • Molecular Motors and the Mechanical Work of CellsEnzymes in a common pathway are located within specific cell compartments and may be further associated asdomains of a monomeric protein, subunits of a multimericprotein, or components of a protein complex assembled ona common scaffold (see Figure 3-20).■3.4 Molecular Motors andthe Mechanical Work of CellsA common property of all cells is motility, the ability to movein a specified direction.
Many cell processes exhibit some type ofmovement at either the molecular or the cellular level; all movements result from the application of a force. In Brownian motion, for instance, thermal energy constantly buffets moleculesand organelles in random directions and for very short distances. On the other hand, materials within a cell are transported in specific directions and for longer distances. This typeof movement results from the mechanical work carried out byproteins that function as motors.
We first briefly describe thetypes and general properties of molecular motors and then lookat how one type of motor protein generates force for movement.Molecular Motors Convert Energy into MotionAt the nanoscale of cells and molecules, movement is effectedby much different forces from those in the macroscopic world.For example, the high protein concentration (200–300 mg/ml)of the cytoplasm prevents organelles and vesicles from diffusing faster than 100 m/3 hours.
Even a micrometer-sized bacterium experiences a drag force from water that stops itsforward movement within a fraction of a nanometer when itstops actively swimming. To generate the forces necessary formany cellular movements, cells depend on specialized enzymescommonly called motor proteins. These mechanochemical enzymes convert energy released by the hydrolysis of ATP orfrom ion gradients into a mechanical force.Motor proteins generate either linear or rotary motion(Table 3-2).
Some motor proteins are components of macro(a)molecular assemblies, but those that move along cytoskeletalfibers are not. This latter group comprises the myosins, kinesins, and dyneins—linear motor proteins that carry attached “cargo” with them as they proceed along eithermicrofilaments or microtubules (Figure 3-22a). DNA andRNA polymerases also are linear motor proteins becausethey translocate along DNA during replication and transcription. In contrast, rotary motors revolve to cause the beatof bacterial flagella, to pack DNA into the capsid of a virus,and to synthesize ATP. The propulsive force for bacterialswimming, for instance, is generated by a rotary motor protein complex in the bacterial membrane. Ions flow down anelectrochemical gradient through an immobile ring of proteins, the stator, which is located in the membrane.
Torquegenerated by the stator rotates an inner ring of proteins andthe attached flagellum (Figure 3-22b). Similarly, in the mitochondrial ATP synthase, or F0F1 complex, a flux of ionsacross the inner mitochondrial membrane is transduced bythe F0 part into rotation of the subunit, which projects intoa surrounding ring of and subunits in the F1 part. Interactions between the subunit and the subunits directs thesynthesis of ATP (Chapter 8).From the observed activities of motor proteins, we caninfer three general properties that they possess:The ability to transduce a source of energy, either ATPor an ion gradient, into linear or rotary movement■The ability to bind and translocate along a cytoskeletalfilament, nucleic acid strand, or protein complex■■Net movement in a given directionThe motor proteins that attach to cytoskeletal fibers alsobind to and carry along cargo as they translocate. The cargoin muscle cells and eukaryotic flagella consists of thick filaments and B tubules, respectively (see Figure 3-22a).
Thesemotor proteins can also transport cargo chromosomes andmembrane-limited vesicles as they move along microtubulesor microfilaments (Figure 3-23).(b)FlagellumADPMyosinordyneinIonsActin filament or A tubuleRotor▲ FIGURE 3-22 Comparison of linear and rotary molecularmotors. (a) In muscle and eukaryotic flagella, the head domainsof motor proteins (blue) bind to an actin thin filament (muscle) orthe A tubule of a doublet microtubule (flagella). ATP hydrolysis inthe head causes linear movement of the cytoskeletal fiber(orange) relative to the attached thick filament or B tubule of anadjacent doublet microtubule. (b) In the rotary motor inthe bacterial membrane, the stator (blue) is immobile inthe membrane.
Ion flow through the stator generates atorque that powers rotation of the rotor (orange) and theflagellum attached to it.MEDIA CONNECTIONSStatorVideo: Rotary Motor Action:FlagellumThick filament or B tubuleATP7980CHAPTER 3 • Protein Structure and FunctionTABLE 3-2Selected Molecular MotorsMotor*EnergySourceStructure/ComponentsCellular LocationMovement GeneratedLINEAR MOTORSDNA polymerase (4)ATPMultisubunit polymerase within replisomeNucleusTranslocation along DNAduring replicationRNA polymerase (4)ATPMultisubunit polymerasewithin transcriptionelongation complexNucleusTranslocation along DNAduring transcriptionRibosome (4)GTPElongation factor 2 (EF2)bound to ribosomeCytoplasm/ERmembraneTranslocation along mRNAduring translationMyosins (3, 19)ATPHeavy and light chains;head domains with ATPaseactivity and microfilamentbinding siteCytoplasmTransport of cargovesicles; contractionKinesins (20)ATPHeavy and light chains; headdomains with ATPase activityand microtubule-binding siteCytoplasmTransport of cargovesicles and chromosomesduring mitosisDyneins (20)ATPMultiple heavy, intermediate,and light chains; head domainswith ATPase activity andmicrotubule-binding siteCytoplasmTransport of cargovesicles; beating of ciliaand eukaryotic flagellaBacterial flagellarmotorH/NagradientStator and rotor proteins,flagellumPlasma membraneRotation of flagellumattached to rotorATP synthase,F0F1(8)HgradientMultiple subunits formingF0 and F1 particlesInner mitochondrialmembrane, thylakoidmembrane, bacterialplasma membraneRotation of subunitleading to ATP synthesisViral capsid motorATPConnector, proheadRNA, ATPaseCapsidRotation of connectorleading to DNA packagingROTARY MOTORS*Numbers in parentheses indicate chapters in which various motors are discussed.CargoCargo binding FIGURE 3-23 Motor protein-dependent movement ofcargo.
The head domains of myosin, dynein, and kinesin motorproteins bind to a cytoskeletal fiber (microfilaments ormicrotubules), and the tail domain attaches to one of varioustypes of cargo—in this case, a membrane-limited vesicle.Hydrolysis of ATP in the head domain causes the head domain to“walk” along the track in one direction by a repeating cycle ofconformational changes.TailNeckMotorproteinATP hydrolysisFiber bindingHeadCytoskeletal fiber3.4 • Molecular Motors and the Mechanical Work of Cells81(b) Head domain(a) Myosin IITailHead NeckNucleotidebinding siteRegulatorylight chainEssentiallight chainHeavy chainsRegulatorylight chainActinbindingsiteEssentiallight chainHeavy chain▲ FIGURE 3-24 Structure of myosin II.
(a) Myosin II is adimeric protein composed of two identical heavy chains (white)and four light chains (blue and green). Each of the head domainstransduces the energy from ATP hydrolysis into movement. Twolight chains are associated with the neck domain of each heavychain. The coiled-coil sequence of the tail domain organizesmyosin II into a dimer. (b) Three-dimensional model of a singlehead domain shows that it has a curved, elongated shape and isbisected by a large cleft. The nucleotide-binding pocket lies onone side of this cleft, and the actin-binding site lies on the otherside near the tip of the head. Wrapped around the shaft of the helical neck are the two light chains. These chains stiffen theneck so that it can act as a lever arm for the head. Shown hereis the ADP-bound conformation.All Myosins Have Head, Neck, and Tail Domainswith Distinct Functionshead, wrapped around the neck like C-clamps.
In this position, the light chains stiffen the neck region and are thereforeable to regulate the activity of the head domain.To further illustrate the properties of motor proteins, we consider myosin II, which moves along actin filaments in musclecells during contraction. Other types of myosin can transportvesicles along actin filaments in the cytoskeleton. Myosin IIand other members of the myosin superfamily are composedof one or two heavy chains and several light chains.
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