Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 33
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Subsequent to phosphoryltransfer from the bound ATP to the bound peptide sequence,the glycine lid must rotate back to the open position beforeADP can be released. Kinetic measurements show that the rateof ADP release is 20-fold slower than that of phosphoryl transfer, indicating the influence of the glycine lid on the rate of kinase reactions. Mutations in the glycine lid that inhibit itsflexibility slow catalysis by protein kinase A even further.Phosphoryl Transfer by Protein Kinases After substrates havebound and the catalytic core of protein kinase A has assumedthe closed conformation, the phosphorylation of a serine orthreonine residue on the target peptide can take place (Figure3-18). As with all chemical reactions, phosphoryl transfer catalyzed by protein kinase A proceeds through a transition statein which the phosphate group to be transferred and the acceptor hydroxyl group are brought into close proximity. Binding and stabilization of the intermediates by protein kinase Areduce the activation energy of the phosphoryl transfer reaction, permitting it to take place at measurable rates under themild conditions present within cells (see Figure 3-16).
Formation of the products induces the enzyme to revert to its openconformational state, allowing ADP and the phosphorylatedtarget peptide to diffuse from the active site.Vmax and Km Characterize an Enzymatic ReactionOpenClosed▲ FIGURE 3-17 Protein kinase A and conformationalchange induced by substrate binding. (a) Model of thecatalytic subunit of protein kinase A with bound substrates; theconserved kinase core is indicated as a molecular surface. Anoverhanging glycine-rich sequence (blue) traps ATP (green) in adeep cleft between the large and small domains of the core.Residues in the large domain bind the target peptide (red).
Thestructure of the kinase core is largely conserved in othereukaryotic protein kinases. (b) Schematic diagrams of open andclosed conformations of the kinase core. In the absence ofsubstrate, the kinase core is in the open conformation. Substratebinding causes a rotation of the large and small domains thatbrings the ATP- and peptide-binding sites closer together andcauses the glycine lid to move over the adenine residue of ATP,thereby trapping the nucleotide in the binding cleft.
The model inpart (a) is in the closed conformation.group from the bound ATP. After the phosphorylation reaction has been completed, the presence of the products causesthe domains to rotate to the open position, from which theproducts are released.The rotation from the open to the closed position alsocauses movement of the glycine lid over the ATP-binding cleft.The glycine lid controls the entry of ATP and release of ADP atThe catalytic action of an enzyme on a given substrate can bedescribed by two parameters: Vmax, the maximal velocity ofthe reaction at saturating substrate concentrations, and Km(the Michaelis constant), a measure of the affinity of an enzyme for its substrate (Figure 3-19). The Km is defined as thesubstrate concentration that yields a half-maximal reaction1rate (i.e., 2 Vmax).
The smaller the value of Km, the moreavidly an enzyme can bind substrate from a dilute solutionand the smaller the substrate concentration needed to reachhalf-maximal velocity.The concentrations of the various small molecules in acell vary widely, as do the Km values for the different enzymes that act on them. Generally, the intracellular concentration of a substrate is approximately the same as or greaterthan the Km value of the enzyme to which it binds.Enzymes in a Common Pathway Are OftenPhysically Associated with One AnotherEnzymes taking part in a common metabolic process (e.g.,the degradation of glucose to pyruvate) are generally locatedin the same cellular compartment (e.g., in the cytosol, at amembrane, within a particular organelle).
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.
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