496 Chapter 13 Principles of Bioenergetics 13.2 Phosphoryl Group Transfers and AIP 0-P⊥0-P-0-P-0 Having developed some fundamental principles of en- O-RibHAdenine ergy changes in chemical systems, we can now exam H-0 ine the energy cycle in cells and the special role of ATP as the energy currency that links catabolism and an- abolism(see Fig. 1-28). Heterotrophic cells obtain free energy in a chemical form by the catabolism of nutrient molecules, and they use that energy to make ATP from of charge ADP and P: AtP then donates some of its chemical en- ergy to endergonic processes such as the synthesis of metabolic intermediates and macromolecules from smaller precursors, the transport of substances across membranes against concentration gradients, and me- chanical motion. This donation of energy from ATP gen 60-P-08H erally involves the covalent participation of ATP in the reaction that is to be driven with the eventual result that atP is converted to ADP and Pi or, in some reac- tions, to amP and 2 Pi. We discuss here the chemical HO→P-0→P 6-0-Rib ADenine] basis for the large free-energy changes that accompany hydrolysis of ATP and other high-energy phosphate ADIz compounds, and we show that most cases of energy donation by atP involve group transfer, not simple hy drolysis of ATP. To illustrate the range of energy trans- ductions in which ATP provides the energy, we consider the synthesis of information-rich macromolecules, the transport of solutes across membranes, and motion pro- duced by muscle contraction ATP-+H20- ADP3-+P2-+H* The Free-Energy Change for AlP Hydrolysis FIGURE 13-1 Chemical basis for the large free-energy change asso Figure 13-1 summarizes the chemical basis for the rel- ciated with ATP hydrolysis. O The charge separation that results from atively large, negative, standard free energy of hydrol- hydrolysis relieves electrostatic repulsion among the four negative ysis of ATP. The hydrolytic cleavage of the terminal charges on ATP.@ The product inorganic phosphate(P)is stabilized phosphoric acid anhydride(phosphoanhydride)bond in by formation of a resonance hybrid, in which each of the four phos- ATP separates one of the three negatively charged phorus-oxygen bonds has the same degree of double-bond charact phosphates and thus relieves some of the electrostatic and the hydrogen ion is not permanently associated with any one of repulsion in ATP: the Pi(HPOA)released is stabilized the oxygens. (Some degree of resonance stabilization also occurs in by the formation of several resonance forms not possi- phosphates involved in ester or anhydride linkages, but fewer reso- ble in ATP: and ADP2-, the other direct product of nance forms are possible than for P- )3 The product ADP2-imme- hydrolysis, immediately ionizes, releasing H* into a diately ionizes, releasing a proton into a medium of very low(H*I medium of very low [H*](10-7M). Because the con- (pH 7. A fourth factor (not shown)that favors ATP hydrolysis is the centrations of the direct products of ATP hydrolysis are, greater degree of solation (hydration) of the products P, and ADF Table 13-5), mass action favors the hydrolysis reaction acln ATP, which further stabilizes the products relative to the re- in the cell, far below the concentrations at equilibrium in the cell. Although the hydrolysis of ATP is highly exergonic and Pi are not identical and are much lower than the (AG=-30.5 k/mol), the molecule is kinetically sta- 1.0M of standard conditions(Table 13-5). Furthermore, ble at pH 7 because the activation energy for ATP Mg in the cytosol binds to ATP and ADP(Fig. 13-2) phoanhydride bonds occurs only when catalyzed by an phosphoryl group donor, the true substrate is MgATP R hydrolysis is relatively high Rapid cleavage of the phos- and for most enzymatic reactions that involve ATP enzyme The relevant AG is therefore that for MgATP" hy The free-energy change for ATP hydrolysis drolysis. Box 13-1 shows how AG for ATP hydrolysis in 30.5 kJ/mol under standard conditions, but the actual the intact erythrocyte can be calculated from the data free energy of hydrolysis(4G) of ATP in living cells is in Table 13-5. In intact cells, AG for ATP hydrolysis very different: the cellular concentrations of ATP, ADP, usually designated AGp is much more negative than
13.2 Phosphoryl Group Transfers and ATP Having developed some fundamental principles of energy changes in chemical systems, we can now examine the energy cycle in cells and the special role of ATP as the energy currency that links catabolism and anabolism (see Fig. 1–28). Heterotrophic cells obtain free energy in a chemical form by the catabolism of nutrient molecules, and they use that energy to make ATP from ADP and Pi . ATP then donates some of its chemical energy to endergonic processes such as the synthesis of metabolic intermediates and macromolecules from smaller precursors, the transport of substances across membranes against concentration gradients, and mechanical motion. This donation of energy from ATP generally involves the covalent participation of ATP in the reaction that is to be driven, with the eventual result that ATP is converted to ADP and Pi or, in some reactions, to AMP and 2 Pi . We discuss here the chemical basis for the large free-energy changes that accompany hydrolysis of ATP and other high-energy phosphate compounds, and we show that most cases of energy donation by ATP involve group transfer, not simple hydrolysis of ATP. To illustrate the range of energy transductions in which ATP provides the energy, we consider the synthesis of information-rich macromolecules, the transport of solutes across membranes, and motion produced by muscle contraction. The Free-Energy Change for ATP Hydrolysis Is Large and Negative Figure 13–1 summarizes the chemical basis for the relatively large, negative, standard free energy of hydrolysis of ATP. The hydrolytic cleavage of the terminal phosphoric acid anhydride (phosphoanhydride) bond in ATP separates one of the three negatively charged phosphates and thus relieves some of the electrostatic repulsion in ATP; the Pi (HPO4 2) released is stabilized by the formation of several resonance forms not possible in ATP; and ADP2, the other direct product of hydrolysis, immediately ionizes, releasing H into a medium of very low [H] (~107 M). Because the concentrations of the direct products of ATP hydrolysis are, in the cell, far below the concentrations at equilibrium (Table 13–5), mass action favors the hydrolysis reaction in the cell. Although the hydrolysis of ATP is highly exergonic (G 30.5 kJ/mol), the molecule is kinetically stable at pH 7 because the activation energy for ATP hydrolysis is relatively high. Rapid cleavage of the phosphoanhydride bonds occurs only when catalyzed by an enzyme. The free-energy change for ATP hydrolysis is 30.5 kJ/mol under standard conditions, but the actual free energy of hydrolysis (G) of ATP in living cells is very different: the cellular concentrations of ATP, ADP, and Pi are not identical and are much lower than the 1.0 M of standard conditions (Table 13–5). Furthermore, Mg2 in the cytosol binds to ATP and ADP (Fig. 13–2), and for most enzymatic reactions that involve ATP as phosphoryl group donor, the true substrate is MgATP2. The relevant G is therefore that for MgATP2 hydrolysis. Box 13–1 shows how G for ATP hydrolysis in the intact erythrocyte can be calculated from the data in Table 13–5. In intact cells, G for ATP hydrolysis, usually designated Gp, is much more negative than 496 Chapter 13 Principles of Bioenergetics ADP3 Pi 2 H G 30.5 kJ/mol ATP4 H2O A B OP P O O B A O O O O O OO Rib Adenine O HO O ADP2 A B O O O OO Rib O Adenine ADP3 OP OP O O B A O O O H OP B A O O O OP O O B A O O O A O B P O O O O OO Rib O Adenine ATP4 H H O Pi OOPO A O O O OOPO B A O O 3 OH resonance stabilization A H 2 3 ionization hydrolysis, with relief of charge repulsion 1 FIGURE 13–1 Chemical basis for the large free-energy change associated with ATP hydrolysis. 1 The charge separation that results from hydrolysis relieves electrostatic repulsion among the four negative charges on ATP. 2 The product inorganic phosphate (Pi) is stabilized by formation of a resonance hybrid, in which each of the four phosphorus–oxygen bonds has the same degree of double-bond character and the hydrogen ion is not permanently associated with any one of the oxygens. (Some degree of resonance stabilization also occurs in phosphates involved in ester or anhydride linkages, but fewer resonance forms are possible than for Pi.) 3 The product ADP2 immediately ionizes, releasing a proton into a medium of very low [H] (pH 7). A fourth factor (not shown) that favors ATP hydrolysis is the greater degree of solvation (hydration) of the products Pi and ADP relative to ATP, which further stabilizes the products relative to the reactants.
13.2 Phosphoryl Group Transfers and ATP 497 TABLE 13-5 Adenine Nucleotide, Inorganic Phosphate, and Phosphocreatine Concentrations in Some Cells Concentration(m AMP P Rat hepatocyte 3.38 0 Rat myocyte 8050.930.0480528 Rat neuron 590.73 Human erythrocyte 25 0.25002 E coli cell 1.04 For erythrocytes the concentrations are those of the cytosol (human erythrocytes lack a nucleus and mitochondria). In the her types of cels the data are far the entire cell contents, although the cy osdl and the mitochondria have very differe concentrations of ADP PCr s phosphocreatine, discussed on p. 489 'This vale reflects total concentration the true value for free ADP may be much lower(see Box 13-1) △G°, ranging from-50to-65kJ/ moL AGp is often called the phosphorylation potential. In the follow ing discussions we use the standard free-energy change for ATP hydrolysis, because this allows comparison, on 0.0 the same basis, with the energetics of other cellular MgATP eactions. Remember, however, that in living cells AG is the relevant quantity-for ATP hydrolysis and all other reactions-and may be quite different from AG O→P-0-P Other Phosphorylated Compounds and Thioesters MeAD Also Have Large Free Energies of Hydrolysis Phosphoenolpyruvate(Fig. 13-3)contains a phosphate FIGURE 13-2 Mg*and AT. Formation of Mg*complexes partially ester bond that undergoes hydrolysis to yield the enol Shields the negative Charges and intluences the conformation of the form of pyruvate, and this direct product can immedi- phosphate groups in nucleotides such as AlP and ADP ately tautomerize to the more stable keto form of pyru- vate. Because the reactant(phosphoenolpyruvate)has only one form(enol)and the product(pyruvate) has two possible forms, the product is stabilized relative to the -49.3 kJ/mol), which can, again, be explained in terms eactant. This is the greatest contributing factor to of the structure of reactant and products. When H20 is the high standard free energy of hydrolysis of phospho- added across the anhydride bond of 1, 3-bisphospho enolpyruvate:△G°=-61.9k/ glycerate, one of the direct products, 3-phosphoglyceric Another three-carbon compound, 1, 3-bisphospho- acid, can immediately lose a proton to give the car glycerate(Fig. 13-4), contains an anhydride bond be- boxylate ion, 3-phosphoglycerate, which has two equally een the carboxyl group at C-1 and phosphoric acid. probable resonance forms(Fig. 13-4). Removal of the Hydrolysis of this acyl phosphate is accompanied by a direct product (3-phosphoglyceric acid) and formation of large, negative, standard free-energy change (AG the resonance- stabilized ion favor the forward reaction URE 13-3 Hydrolysis of phosphoenol pyruvate( PEP). Catalyzed by pyruvate kinase, this reaction is followed by spontaneous CHs tautomerization of the product, pyruvate nol form) m) thus the products of hydrolysis are stabilized the reactants. Resonance PEP+ H20 stabilization of Pi also occurs, as shown AG°=-619kJ/mol
G , ranging from 50 to 65 kJ/mol. Gp is often called the phosphorylation potential. In the following discussions we use the standard free-energy change for ATP hydrolysis, because this allows comparison, on the same basis, with the energetics of other cellular reactions. Remember, however, that in living cells G is the relevant quantity—for ATP hydrolysis and all other reactions—and may be quite different from G . Other Phosphorylated Compounds and Thioesters Also Have Large Free Energies of Hydrolysis Phosphoenolpyruvate (Fig. 13–3) contains a phosphate ester bond that undergoes hydrolysis to yield the enol form of pyruvate, and this direct product can immediately tautomerize to the more stable keto form of pyruvate. Because the reactant (phosphoenolpyruvate) has only one form (enol) and the product (pyruvate) has two possible forms, the product is stabilized relative to the reactant. This is the greatest contributing factor to the high standard free energy of hydrolysis of phosphoenolpyruvate: G 61.9 kJ/mol. Another three-carbon compound, 1,3-bisphosphoglycerate (Fig. 13–4), contains an anhydride bond between the carboxyl group at C-1 and phosphoric acid. Hydrolysis of this acyl phosphate is accompanied by a large, negative, standard free-energy change (G 13.2 Phosphoryl Group Transfers and ATP 497 Concentration (mM)* ATP ADP† AMP Pi PCr Rat hepatocyte 3.38 1.32 0.29 4.8 0 Rat myocyte 8.05 0.93 0.04 8.05 28 Rat neuron 2.59 0.73 0.06 2.72 4.7 Human erythrocyte 2.25 0.25 0.02 1.65 0 E. coli cell 7.90 1.04 0.82 7.9 0 Adenine Nucleotide, Inorganic Phosphate, and Phosphocreatine Concentrations in Some Cells TABLE 13–5 *For erythrocytes the concentrations are those of the cytosol (human erythrocytes lack a nucleus and mitochondria). In the other types of cells the data are for the entire cell contents, although the cytosol and the mitochondria have very different concentrations of ADP. PCr is phosphocreatine, discussed on p. 489. † This value reflects total concentration; the true value for free ADP may be much lower (see Box 13–1). OP Mg2 A O PO O B A O O OO Rib O Adenine MgADP OP B Mg2 A O O O OP O O B A O O O O B P O O O O OO Rib O Adenine MgATP2 ø ø ø ø O O O O B A FIGURE 13–2 Mg2 and ATP. Formation of Mg2 complexes partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP. O A tautomerization C J O G Pyruvate (keto form) O C CH3 H2O Pi B O P O G J D C G 61.9 kJ/mol D PEP3 H2O J O G PEP G O pyruvate Pi 2 CH2 O B OC D OH J O G Pyruvate (enol form) O C CH2 O J hydrolysis O C O FIGURE 13–3 Hydrolysis of phosphoenolpyruvate (PEP). Catalyzed by pyruvate kinase, this reaction is followed by spontaneous tautomerization of the product, pyruvate. Tautomerization is not possible in PEP, and thus the products of hydrolysis are stabilized relative to the reactants. Resonance stabilization of Pi also occurs, as shown in Figure 13–1. 49.3 kJ/mol), which can, again, be explained in terms of the structure of reactant and products. When H2O is added across the anhydride bond of 1,3-bisphosphoglycerate, one of the direct products, 3-phosphoglyceric acid, can immediately lose a proton to give the carboxylate ion, 3-phosphoglycerate, which has two equally probable resonance forms (Fig. 13–4). Removal of the direct product (3-phosphoglyceric acid) and formation of the resonance-stabilized ion favor the forward reaction.
FIGURE 13-4 Hydrolysis of 1,3- phosphoglycerate. The 00 0 OH product of hydrolysis is 3-phospho- glyceric acid, with an undissociated carboxylic acid group, but CHOH CHOH dissociation occurs immediately. CH This ionization and the resonance structures it makes possible stabilize hydrolysis the product relative to the reactants O-P=0 O-P=0 Resonance stabilization of Pi further contributes to the negative free. 1, 3-Bisphosphoglycerate 3-Phosphoglycerate 1.3-Bisphosphoglycerate-+H20-3-phosphoglycerate-+P?-+H+ △G°=-49.3kJ/mol BOX 13-1 WORKING IN BIOCHEMISTRY The Free Energy of Hydrolysis of ATP within Cells: much larger than the standard free-energy change The Real Cost of Doing Metabolic Business (-30.5 kJ/mol). By the same token, the free energy The standard free energy of hydrolysis of ATP is equired to synthesize ATP from ADP and Pi under of ATP, ADP, and P; are not only unequal but much 52 kJ/mol prevailing in the erythrocyte would be 30.5 kJ/mol. In the cell. however, the concentrations lower than the standard 1 m concentrations(see Table Because the concentrations of ATP, ADP, and Pi 13-5). Moreover, the cellular pH may differ somewhat differ from one cell type to another(see Table 13-5) from the standard pH of 7.0. Thus the actual free AGp for ATP hydrolysis likewise differs among cells energy of hydrolysis of ATP under intracellular con- Moreover, in any given cell, AGp can vary from time ditions(AGp) differs from the standard free-energy to time, depending on the metabolic conditions in the ange.△G. We can easily calculate△G cell and how they influence the concentrations of aTP tions of ATP, ADP, and P, are 2. 25, 0. 25, and 1.65 mM. free-energy change for any given metabolic reaction respectively. Let us assume for simplicity that the pH as it occurs in the cell, providing we know the con- is 7.0 and the temperature is 25C, the standard pH centrations of all the reactants and products of the re- and temperature. The actual free energy of hydrolysis action and know about the other factors(such as pH, temperature, and concentration of Mg ) that may af- of ATP in the erythrocyte under these conditions is fect the AG and thus the calculated free-energy given by the relationship change,△G1 Gp=AG+RTIn-oPIPI To further complicate the issue, the total concen- trations of ATP, ADP, Pi, and H may be substantially higher than the free concentrations, which are the Substituting the appropriate values we obtain thermodynamically relevant values. The difference is △Gp=-30.5 kJ/mol+ due to tight binding of ATP, ADP, and Pi to cellular proteins. For example, the concentration of fi ee ADP (8.315 J/molK)(298 s K)In (0.25x 102 ) 1.65 x 10-2 in resting muscle has been variously estimated at be- tween 1 and 37 uM. Using the value 25 uM in the cal- =-30.5 kJ/mol+(248 kJ/mol) In1.8×10-4 culation outlined above, we get a AGp of -58 k/mol. 30.5 KJ/mol 21 kj/mol Calculation of the exact value of A G, is perhaps 52 k/mol less instructive than the generalization about actual free-energy changes: in vivo, the energy Thus AGp, the actual free-energy change for ATP hy- released by atP hydrolysis is greater than the stan- drolysis in the intact erythrocyte(-52 kJ/mol), is dard free-energy change, AG
498 Chapter 13 Principles of Bioenergetics 3-Phosphoglyceric acid hydrolysis A O M CHOH CH2 D A A A A P O P O C O O OH O H H2O Pi ionization 1,3-Bisphosphoglycerate 3-Phosphoglycerate D P O G O O O A O M O CHOH CH2 D A A A A P O P O C O 3 1 2 J OG G resonance stabilization A O CHOH CH2 D A A A A P O P O C O O O O O G 49.3 kJ/mol 1,3-Bisphosphoglycerate4 H2O 3-phosphoglycerate3 P i 2 H FIGURE 13–4 Hydrolysis of 1,3- bisphosphoglycerate. The direct product of hydrolysis is 3-phosphoglyceric acid, with an undissociated carboxylic acid group, but dissociation occurs immediately. This ionization and the resonance structures it makes possible stabilize the product relative to the reactants. Resonance stabilization of Pi further contributes to the negative freeenergy change. BOX 13–1 WORKING IN BIOCHEMISTRY The Free Energy of Hydrolysis of ATP within Cells: The Real Cost of Doing Metabolic Business The standard free energy of hydrolysis of ATP is 30.5 kJ/mol. In the cell, however, the concentrations of ATP, ADP, and Pi are not only unequal but much lower than the standard 1 M concentrations (see Table 13–5). Moreover, the cellular pH may differ somewhat from the standard pH of 7.0. Thus the actual free energy of hydrolysis of ATP under intracellular conditions (Gp) differs from the standard free-energy change, G . We can easily calculate Gp. In human erythrocytes, for example, the concentrations of ATP, ADP, and Pi are 2.25, 0.25, and 1.65 mM, respectively. Let us assume for simplicity that the pH is 7.0 and the temperature is 25 C, the standard pH and temperature. The actual free energy of hydrolysis of ATP in the erythrocyte under these conditions is given by the relationship Gp G RT ln [A [ D A P T ] P [P ] i ] Substituting the appropriate values we obtain Gp 30.5 kJ/mol (8.315 J/mol K)(298 K) ln 30.5 kJ/mol (2.48 kJ/mol) ln 1.8 104 30.5 kJ/mol 21 kJ/mol 52 kJ/mol Thus Gp, the actual free-energy change for ATP hydrolysis in the intact erythrocyte (52 kJ/mol), is much larger than the standard free-energy change (30.5 kJ/mol). By the same token, the free energy required to synthesize ATP from ADP and Pi under the conditions prevailing in the erythrocyte would be 52 kJ/mol. Because the concentrations of ATP, ADP, and Pi differ from one cell type to another (see Table 13–5), Gp for ATP hydrolysis likewise differs among cells. Moreover, in any given cell, Gp can vary from time to time, depending on the metabolic conditions in the cell and how they influence the concentrations of ATP, ADP, Pi , and H (pH). We can calculate the actual free-energy change for any given metabolic reaction as it occurs in the cell, providing we know the concentrations of all the reactants and products of the reaction and know about the other factors (such as pH, temperature, and concentration of Mg2) that may affect the G and thus the calculated free-energy change, Gp. To further complicate the issue, the total concentrations of ATP, ADP, Pi , and H may be substantially higher than the free concentrations, which are the thermodynamically relevant values. The difference is due to tight binding of ATP, ADP, and Pi to cellular proteins. For example, the concentration of free ADP in resting muscle has been variously estimated at between 1 and 37 M. Using the value 25 M in the calculation outlined above, we get a Gp of 58 kJ/mol. Calculation of the exact value of Gp is perhaps less instructive than the generalization we can make about actual free-energy changes: in vivo, the energy released by ATP hydrolysis is greater than the standard free-energy change, G . (0.25 103 )(1.65 103 ) 2.25 103
13.2 Phosphoryl Group Transfers and ATP C00 FIGURE 13-5 Hydrolysis of phospho creatine Breakage of the P-N bond H,O in phosphocreatine produces creatine, 0-P-N-C-N-CH3 时yBNC--CH1←→”)=NCH2 which is stablized by formation of a NH2 resonance hybrid. The other product, Phosphocreatin Creatine Pi is also resonance stabilized sphocreatine+ H2O ine+P2 △G°=-43.0kJ/mol In phosphocreatine(Fig. 13-5), the P-N bond can these compounds is activated for transacylation,con- be hydrolyzed to generate free creatine and P:. The re- densation, or oxidation-reduction reactions. Thioesters lease of Pi and the resonance stabilization of creatine undergo much less resonance stabilization than do oxy favor the forward reaction. The standard free-energy gen esters; consequently, the difference in free energy change of phosphocreatine hydrolysis is again large, between the reactant and its hydrolysis products, which -43. 0 k/mol are resonance-stabilized, is greater for thioesters than In all these phosphate-releasing reactions, the sev- for comparable oxygen esters(Fig. 13-7). In both cases eral resonance forms available to Pi(Fig. 13-1)stabi- hydrolysis of the ester generates a carboxylic acid lize this product relative to the reactant, contributing to which can ionize and assume several resonance forms an already negative free-energy change. Table 13-6 lists Together, these factors result in the large, negative AG the standard free energies of hydrolysis for a number of (-31 kJ/mol) for acetyl-CoA hydrolysis phosphorylated compounds To summarize, for hydrolysis reactions with large, Thioesters, in which a sulfur atom replaces the negative, standard free-energy changes, the products usual oxygen in the ester bond, also have large, nega- are more stable than the reactants for one or more of tive, standard free energies of hydrolysis. Acetyl-coen- the following reasons: (1) the bond strain in reactants zyme A, or acetyl-CoA(Fig. 13-6), is one of many due to electrostatic repulsion is relieved by charge sep thioesters important in metabolism. The acyl group in aration, as for ATP; (2)the products are stabilized by TABLE 13-6 Standard Free Energies of Hydrolysis of Some Phosphorylated Compounds and Acetyl-CoA(a Thioester) Acetyl-CoA kl/mol)(kcal/mol) H20Yhydrolysis CoASH 1, 3-bisphosphoglyce Acetic acid 3-phosphoglycerate P)-493 11.8 OH 10.3 ADP(→AMP+ P 7.8 AIP(→ADP+P H AP(→AMP+Pp 109 AMP(→ adenosine+P Acetate P(→2P) Glucose 1-phosphate 209-5.0 Glucose 6-phosphate Glycerol 1-phosphate -2.2 Acetyl-CoA H20 -,acetate CoA +H Acetyl-COA 314 -7.5 FIGURE 13-6 Hydrolysis of acetyl-coenzyme A. Acetyl-CoA is a Source: Data mostly from lencks, WP(1976)in Hanaboo and Molecular thioester with a large, negative, standard free energy of hydrolysis Biolgy 3rd edn(Fasman, G.D., ed), Physical and Chemic CRC Press, Boca Raton, FL. The value for the free energy Thioesters contain a sulfur atom in the position occupied by an oxy- PA.& Arabshahi, A. (1995)Standard free-energy change for the hydrolysis of the a-B gen atom in oxygen esters. The complete structure of coenzyme A phosphoanhydride bridge in AlP. Biochemistry 34, 11, 307-11 310. (CoA, Or CoASH) is shown in Figure 8-41
In phosphocreatine (Fig. 13–5), the PON bond can be hydrolyzed to generate free creatine and Pi . The release of Pi and the resonance stabilization of creatine favor the forward reaction. The standard free-energy change of phosphocreatine hydrolysis is again large, 43.0 kJ/mol. In all these phosphate-releasing reactions, the several resonance forms available to Pi (Fig. 13–1) stabilize this product relative to the reactant, contributing to an already negative free-energy change. Table 13–6 lists the standard free energies of hydrolysis for a number of phosphorylated compounds. Thioesters, in which a sulfur atom replaces the usual oxygen in the ester bond, also have large, negative, standard free energies of hydrolysis. Acetyl-coenzyme A, or acetyl-CoA (Fig. 13–6), is one of many thioesters important in metabolism. The acyl group in these compounds is activated for transacylation, condensation, or oxidation-reduction reactions. Thioesters undergo much less resonance stabilization than do oxygen esters; consequently, the difference in free energy between the reactant and its hydrolysis products, which are resonance-stabilized, is greater for thioesters than for comparable oxygen esters (Fig. 13–7). In both cases, hydrolysis of the ester generates a carboxylic acid, which can ionize and assume several resonance forms. Together, these factors result in the large, negative G (31 kJ/mol) for acetyl-CoA hydrolysis. To summarize, for hydrolysis reactions with large, negative, standard free-energy changes, the products are more stable than the reactants for one or more of the following reasons: (1) the bond strain in reactants due to electrostatic repulsion is relieved by charge separation, as for ATP; (2) the products are stabilized by 13.2 Phosphoryl Group Transfers and ATP 499 NH2 COO resonance stabilization Pi hydrolysis A B O OOO O A CH2 N H2O O B NH2 COO O CH3 A P B OOO A A CH2 N H C Creatine H2N H2N C N COO A A CH2 CH3 C CH3 O N O G 43.0 kJ/mol creatine P i 2 Phosphocreatine2 H2O Phosphocreatine H2N FIGURE 13–5 Hydrolysis of phosphocreatine. Breakage of the PON bond in phosphocreatine produces creatine, which is stabilized by formation of a resonance hybrid. The other product, Pi, is also resonance stabilized. G (kJ/mol) (kcal/mol) Phosphoenolpyruvate 61.9 14.8 1,3-bisphosphoglycerate (n 3-phosphoglycerate Pi ) 49.3 11.8 Phosphocreatine 43.0 10.3 ADP (n AMP Pi ) 32.8 7.8 ATP (n ADP Pi ) 30.5 7.3 ATP (n AMP PPi ) 45.6 10.9 AMP (n adenosine Pi ) 14.2 3.4 PPi (n 2Pi ) 19.2 4.0 Glucose 1-phosphate 20.9 5.0 Fructose 6-phosphate 15.9 3.8 Glucose 6-phosphate 13.8 3.3 Glycerol 1-phosphate 9.2 2.2 Acetyl-CoA 31.4 7.5 Standard Free Energies of Hydrolysis of Some Phosphorylated Compounds and Acetyl-CoA (a Thioester) TABLE 13–6 Source: Data mostly from Jencks, W.P. (1976) in Handbook of Biochemistry and Molecular Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. I, pp. 296–304, CRC Press, Boca Raton, FL. The value for the free energy of hydrolysis of PPi is from Frey, P.A. & Arabshahi, A. (1995) Standard free-energy change for the hydrolysis of the –- phosphoanhydride bridge in ATP. Biochemistry 34, 11,307–11,310. CH3 G 31.4 kJ/mol acetate CoA C O OH Acetate O AcetylH2O resonance stabilization CoASH hydrolysis ionization G J S-CoA CH3 C O O G J CH3 C O O Acetyl-CoA Acetic acid D G H O CoA H2O H FIGURE 13–6 Hydrolysis of acetyl-coenzyme A. Acetyl-CoA is a thioester with a large, negative, standard free energy of hydrolysis. Thioesters contain a sulfur atom in the position occupied by an oxygen atom in oxygen esters. The complete structure of coenzyme A (CoA, or CoASH) is shown in Figure 8–41.
Extra stabilization t CH3- ester FIGURE 13-7 Free energy of hydrolysis for thioesters and oxygen esters. The products of both types of hydrolysis reaction have about the same free-energy content(G), but the thioester has a higher △ Gfor oxygen free-energy content than the oxygen ester. Orbital overlap between the O and C atoms allows resonance stabilization CH +R→SH +R→OH in oxygen esters; orbital overlap betwee S and C atoms is poorer and provides ionization, as for ATP, acyl phosphates, and thioesters: chanical motion. This occurs in muscle contraction and (3) the products are stabilized by isomerization(tau- in the movement of enzymes along DNA or of ribosomes tomerization), as for phosphoenolpyruvate: and/or(4) along messenger RNA. The energy-dependent reactions the products are stabilized by resonance, as for creatine catalyzed by helicases, Reca protein, and some topo. released from phosphocreatine, carboxylate ion re- isomerases( Chapter 25)also involve direct hydrolysis leased from acyl phosphates and thioesters, and phos- of phosphoanhydride bonds. GTP-binding proteins that phate(P) released from anhydride or ester linkages. act in signaling pathways directly hydrolyze Gtp to drive conformational changes that terminate signals ATP Provides Energy by Group Transfers Not by simple Hydrolysis (a) written as a one-step reaction Throughout this book you will encounter reactions or C00 processes for which ATP supplies energy, and the con- tribution of ATP to these reactions is commonly indi H3N-CH ATP ADP +P HN cated as in Figure 13-8a, with a single arrow showing the conversion of ATP to ADP and Pi (or, in some cases, nH of ATP to AMP and pyrophosphate, PPi). When writter this way. these reactions of ATP appear to be simple hy drolysis reactions in which water displaces Pi (or PP) and one is tempted to say that an ATP-dependent re- Glutamate Glutamine action is"driven by the hydrolysis of ATP. " This is not the case. ATP hydrolysis per se usually accomplishes ADP nothing but the liberation of heat, which cannot drive HON-CH chemical process in an isothermal system. A single re- action arrow such as that in Figure 13-8a almost in- variably represents a two-step process( Fig. 13-8b)in which part of the ATP molecule, a phosphoryl or py- rophosphoryl group or the adenylate moiety(AMP),is first transferred to a substrate molecule or to an amino 000 acid residue in an enzyme, becoming covalently at tached to the substrate or the enzyme and raising its free-energy content. Then, in a second step, the phos- glutamyl phosphate phate-containing moiety transferred in the first step is displaced, generating Pi, PPi, or AMP. Thus ATP partic (b)Actual two-step reaction ipates covalently in the enzyme-catalyzed reaction to which it contributes free energy FIGURE 13-8 ATP hydrolysis in two steps (a) The contribution of Some processes do involve direct hydrolysis of ATP ATP to a reaction is often shown as a single step, but is almost always (or GTP), however. For example, noncovalent binding two-step process. (b) Shown here is the reaction catalyzed by ATP- of ATP(or of GTP), followed by its hydrolysis to ADP dependent glutamine synthetase. A phosphoryl group is transferred (or GDP) and P;, can provide the energy to cycle some from ATP to glutamate, then @2 the phosphoryl group is displaced by proteins between two conformations, producing me- NH3 and released as Pp
ionization, as for ATP, acyl phosphates, and thioesters; (3) the products are stabilized by isomerization (tautomerization), as for phosphoenolpyruvate; and/or (4) the products are stabilized by resonance, as for creatine released from phosphocreatine, carboxylate ion released from acyl phosphates and thioesters, and phosphate (Pi ) released from anhydride or ester linkages. ATP Provides Energy by Group Transfers, Not by Simple Hydrolysis Throughout this book you will encounter reactions or processes for which ATP supplies energy, and the contribution of ATP to these reactions is commonly indicated as in Figure 13–8a, with a single arrow showing the conversion of ATP to ADP and Pi (or, in some cases, of ATP to AMP and pyrophosphate, PPi ). When written this way, these reactions of ATP appear to be simple hydrolysis reactions in which water displaces Pi (or PPi ), and one is tempted to say that an ATP-dependent reaction is “driven by the hydrolysis of ATP.” This is not the case. ATP hydrolysis per se usually accomplishes nothing but the liberation of heat, which cannot drive a chemical process in an isothermal system. A single reaction arrow such as that in Figure 13–8a almost invariably represents a two-step process (Fig. 13–8b) in which part of the ATP molecule, a phosphoryl or pyrophosphoryl group or the adenylate moiety (AMP), is first transferred to a substrate molecule or to an amino acid residue in an enzyme, becoming covalently attached to the substrate or the enzyme and raising its free-energy content. Then, in a second step, the phosphate-containing moiety transferred in the first step is displaced, generating Pi , PPi , or AMP. Thus ATP participates covalently in the enzyme-catalyzed reaction to which it contributes free energy. Some processes do involve direct hydrolysis of ATP (or GTP), however. For example, noncovalent binding of ATP (or of GTP), followed by its hydrolysis to ADP (or GDP) and Pi , can provide the energy to cycle some proteins between two conformations, producing mechanical motion. This occurs in muscle contraction and in the movement of enzymes along DNA or of ribosomes along messenger RNA. The energy-dependent reactions catalyzed by helicases, RecA protein, and some topoisomerases (Chapter 25) also involve direct hydrolysis of phosphoanhydride bonds. GTP-binding proteins that act in signaling pathways directly hydrolyze GTP to drive conformational changes that terminate signals 500 Chapter 13 Principles of Bioenergetics CH2 NH2 H3N CH ATP ADP C O A O J G A A A CH2 COO CH2 H3N O G J A A NH3 Glutamate CH2 H3N CH C O A O J G A A A CH2 COO NH3 G C O J O O G O ATP ADP Pi CH A O A CH2 COO D P glutamyl phosphate O Enzyme-bound Pi Glutamine 1 2 (a) Written as a one-step reaction (b) Actual two-step reaction FIGURE 13–8 ATP hydrolysis in two steps. (a) The contribution of ATP to a reaction is often shown as a single step, but is almost always a two-step process. (b) Shown here is the reaction catalyzed by ATPdependent glutamine synthetase. 1 A phosphoryl group is transferred from ATP to glutamate, then 2 the phosphoryl group is displaced by NH3 and released as Pi. O CH3 C CH3 O C O Thioester J O O R OH Extra stabilization of oxygen ester by resonance CH3 C G J O O O OR R OH CH3 C G J O O S OSH Free energy, G resonance stabilization Oxygen ester CH3 C G J O O OH R OR O G G for oxygen ester hydrolysis G for thioester hydrolysis FIGURE 13–7 Free energy of hydrolysis for thioesters and oxygen esters. The products of both types of hydrolysis reaction have about the same free-energy content (G), but the thioester has a higher free-energy content than the oxygen ester. Orbital overlap between the O and C atoms allows resonance stabilization in oxygen esters; orbital overlap between S and C atoms is poorer and provides little resonance stabilization.