H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 33
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Within a compartment, products from one reaction can move by diffusionto the next enzyme in the pathway. However, diffusion entails random movement and is a slow, inefficient process for3.3 • Enzymes and the Chemical Work of Cells(a)Asp-184−Lys-72+−OOOPαATPOPβ−+ Mg2+OOOOMg2+ +Rate of formation of reactionproduct (P) (relative units)Initial stateAsp-166−OPγO H2−OCH2+Lys-168COPPATP+ MgOOMg2+ +2+OPOOCH2COO−OPO 2−POOO[E] = 0.25 unit0KmVmax0.8High-affinitysubstrate(S)0.6Low-affinitysubstrate (S’)0.4Km for S’0.20Km for SConcentration of substrate ([S] or [S’])▲ EXPERIMENTAL FIGURE 3-19 The Km and Vmax for anPhosphate transferEnd stateVmax0.51.0OO1.0Concentration of substrate [S]Rate of reactionOO[E] = 1.0 unit1.5(b)Intermediate stateOVmax2.0Ser or Thr oftarget peptideFormation oftransition stateADP772−OOPOOCH2CPhosphorylatedpeptide▲ FIGURE 3-18 Mechanism of phosphorylation by proteinkinase A.
(Top) Initially, ATP and the target peptide bind to theactive site (see Figure 3-17a). Electrons of the phosphate groupare delocalized by interactions with lysine side chains and Mg2.Colored circles represent the residues in the kinase core criticalto substrate binding and phosphoryl transfer.
Note that theseresidues are not adjacent to one another in the amino acidsequence. (Middle) A new bond then forms between the serineor threonine side-chain oxygen atom and phosphate, yielding apentavalent intermediate. (Bottom) The phosphoester bondbetween the and phosphates is broken, yielding the productsADP and a peptide with a phosphorylated serine or threonineside chain.
The catalytic mechanism of other protein kinases issimilar.enzyme-catalyzed reaction are determined from plots of theinitial velocity versus substrate concentration. The shape ofthese hypothetical kinetic curves is characteristic of a simpleenzyme-catalyzed reaction in which one substrate (S) isconverted into product (P).
The initial velocity is measuredimmediately after addition of enzyme to substrate before thesubstrate concentration changes appreciably. (a) Plots of theinitial velocity at two different concentrations of enzyme [E] as afunction of substrate concentration [S]. The [S] that yields a halfmaximal reaction rate is the Michaelis constant Km, a measure ofthe affinity of E for S. Doubling the enzyme concentration causesa proportional increase in the reaction rate, and so the maximalvelocity Vmax is doubled; the Km, however, is unaltered. (b) Plotsof the initial velocity versus substrate concentration with asubstrate S for which the enzyme has a high affinity and with asubstrate S for which the enzyme has a low affinity.
Note thatthe Vmax is the same with both substrates but that Km is higherfor S, the low-affinity substrate.moving molecules between widely dispersed enzymes (Figure3-20a). To overcome this impediment, cells have evolvedmechanisms for bringing enzymes in a common pathwayinto close proximity.In the simplest such mechanism, polypeptides with different catalytic activities cluster closely together as subunits ofa multimeric enzyme or assemble on a common “scaffold”(Figure 3-20b).
This arrangement allows the products of onereaction to be channeled directly to the next enzyme in thepathway. The first approach is illustrated by pyruvate78CHAPTER 3 • Protein Structure and Function(a)(a)ReactantsE1E2ProductsAE3CB(b)(b)ProductsReactantsAReactantsBPyruvateORCOABHSCoACH3CCOO−E1OCO2ScaffoldProductsE2CCH3CSCoAE3Acetyl CoA(c)ReactantsNAD+ProductsABNet reaction:Pyruvate + NAD+ + CoACNADH +H+CO2 + NADH + acetyl CoA▲ FIGURE 3-20 Evolution of multifunctional enzyme.In the hypothetical reaction pathways illustrated here the initialreactants are converted into final products by the sequentialaction of three enzymes: A, B, and C.
(a) When the enzymes arefree in solution or even constrained within the same cellularcompartment, the intermediates in the reaction sequence mustdiffuse from one enzyme to the next, an inherently slow process.(b) Diffusion is greatly reduced or eliminated when the enzymesassociate into multisubunit complexes. (c) The closest integrationof different catalytic activities occurs when the enzymes arefused at the genetic level, becoming domains in a single protein.▲ FIGURE 3-21 Structure and function of pyruvatedehydrogenase, a large multimeric enzyme complex thatconverts pyruvate into acetyl CoA.
(a) The complex consists of24 copies of pyruvate decarboxylase (E1), 24 copies of lipoamidetransacetylase (E2), and 12 copies of dihydrolipoyl dehydrogenase(E3). The E1 and E3 subunits are bound to the outside of the coreformed by the E2 subunits. (b) The reactions catalyzed by thecomplex include several enzyme-bound intermediates (notshown). The tight structural integration of the three enzymesincreases the rate of the overall reaction and minimizes possibleside reactions.dehydrogenase, a complex of three distinct enzymes that converts pyruvate into acetyl CoA in mitochondria (Figure 3-21).The scaffold approach is employed by MAP kinase signaltransduction pathways, discussed in Chapter 14. In yeast,three protein kinases assembled on the Ste5 scaffold proteinform a kinase cascade that transduces the signal triggered bythe binding of mating factor to the cell surface.In some cases, separate proteins have been fused togetherat the genetic level to create a single multidomain, multifunctional enzyme (Figure 3-20c).
For instance, the isomerization of citrate to isocitrate in the citric acid cycle iscatalyzed by aconitase, a single polypeptide that carries outtwo separate reactions: (1) the dehydration of citrate to formcis-aconitate and then (2) the hydration of cis-aconitate toyield isocitrate (see Figure 8-9).on proteins and the corresponding ligands are chemicallyand topologically complementary.The affinity of a protein for a particular ligand refers tothe strength of binding; its specificity refers to the preferential binding of one or a few closely related ligands.■Enzymes are catalytic proteins that accelerate the rateof cellular reactions by lowering the activation energyand stabilizing transition-state intermediates (see Figure3-16).■An enzyme active site comprises two functional parts: asubstrate-binding region and a catalytic region.
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).
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