13.2 Phosphoryl Group Transfers and ATP 501 triggered by hormones or by other extracellular factors direct donation of a phosphoryl group from PEP to ADP is thermodynamically feasible The phosphate compounds found in living organisms △G°(kJ/mol can be divided somewhat arbitrarily into two groups based on their standard free energies of hydrolysis (1) PEP H20- pyruvate + Pi 61.9 (Fg.13-9). High-energy" compounds have a△°of ADP+P→ATP+H20 +30.5 hydrolysis more negative than-25 kJ/mol: "low-energy Sum: PEP ADP- pyruvate+ At compounds have a less negative AG. Based on this cri- Notice that while the overall reaction above is repre- terion, ATP, with a AG of hydrolysis of-30.5 kJ/mol sented as the algebraic sum of the first two reactions, (7.3 kcal mol), is a high-energy compound; gh the overall reaction is actually a third, distinct reaction 6-phosphate, with a AG of hydrolysis of -13.8 k that does not involve Pi: pep donates a phosphoryl (-3.3 kcal/mol), is a low-energy compound group directlyto ADP. We can describe phosphorylated The term" high-energy phosphate bond, "long used compounds as having a high or low phosphoryl group biochemists to describe the P-0 bond broken in hy- transfer potential, on the basis of their standard free en drolysis reactions, is incorrect and misleading as it ergies of hydrolysis(as listed in Table 13-6). The phos wrongly suggests that the bond itself contains the en- phoryl group transfer potential of phosphoenolpyruvate ergy. In fact, the breaking of all chemical bonds requires is very high, that of ATP is high, and that of glucose 6 an input of energy. The free energy released by hy- ph nate is low(Fig. 13-9) drolysis of phosphate compounds does not come from Much of catabolism is directed toward the synthesis the specific bond that is broken; it results from the prod- of high-energy phosphate compounds, but their forma- ucts of the reaction having a lower free-energy content tion is not an end in itself, they are the means of acti- than the reactants. For simplicity, we will sometimes use vating a very wide variety of compounds for further the term " high-energy phosphate compound"when re- chemical transformation. The transfer of a phosphoryl ferring to ATP or other phosphate compounds with a group to a compound effectively puts free energy into large, negative, standard free energy of hydrolysis that compound, so that it has more free energy to give As is evident from the additivity of free-energy up during subsequent metabolic transformations We de changes of sequential reactions, any phosphorylated scribed above how the synthesis of glucose 6-phosphate compound can be synthesized by coupling the synthe- is accomplished by phosphoryl group transfer from AtP. sis to the breakdown of another phosphorylated com- In the next chapter we see how this phosphorylation of pound with a more negative free energy of hydrolysis. glucose activates, or"primes, the glucose for catabolic ple, because cleavage of Pi from phospho- reactions that occur in nearly every living cell. Because ate(PEp)releases more energy than is of its intermediate position on the scale of group trans- needed to drive the condensation of Pi with ADP, the fer potential, ATP can carry energy from high-energy CH CHOH CReatine 3-Bisphosphoglycerate FIGURE 13-9 Ranking of biological phosphate Adenine [R@ compounds by standard free energies of hydrol- ATP Low-e donors via ATP to acceptor molecules(such as glucose and glycerol) to form their low-energy phosphate derivatives. This flow of phosphoryl Glucose 6- groups, catalyzed by enzymes called kinases. proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of low energy phosphate compounds releases Pi, which has an even lower phosphoryl group transfer
triggered by hormones or by other extracellular factors (Chapter 12). The phosphate compounds found in living organisms can be divided somewhat arbitrarily into two groups, based on their standard free energies of hydrolysis (Fig. 13–9). “High-energy” compounds have a G of hydrolysis more negative than 25 kJ/mol; “low-energy” compounds have a less negative G . Based on this criterion, ATP, with a G of hydrolysis of 30.5 kJ/mol (7.3 kcal/mol), is a high-energy compound; glucose 6-phosphate, with a G of hydrolysis of 13.8 kJ/mol (3.3 kcal/mol), is a low-energy compound. The term “high-energy phosphate bond,” long used by biochemists to describe the POO bond broken in hydrolysis reactions, is incorrect and misleading as it wrongly suggests that the bond itself contains the energy. In fact, the breaking of all chemical bonds requires an input of energy. The free energy released by hydrolysis of phosphate compounds does not come from the specific bond that is broken; it results from the products of the reaction having a lower free-energy content than the reactants. For simplicity, we will sometimes use the term “high-energy phosphate compound” when referring to ATP or other phosphate compounds with a large, negative, standard free energy of hydrolysis. As is evident from the additivity of free-energy changes of sequential reactions, any phosphorylated compound can be synthesized by coupling the synthesis to the breakdown of another phosphorylated compound with a more negative free energy of hydrolysis. For example, because cleavage of Pi from phosphoenolpyruvate (PEP) releases more energy than is needed to drive the condensation of Pi with ADP, the direct donation of a phosphoryl group from PEP to ADP is thermodynamically feasible: G (kJ/mol) (1) PEP H2O 8n pyruvate Pi 61.9 (2) ADP Pi 8n ATP H2O 30.5 Sum: PEP ADP 8n pyruvate ATP 31.4 Notice that while the overall reaction above is represented as the algebraic sum of the first two reactions, the overall reaction is actually a third, distinct reaction that does not involve Pi ; PEP donates a phosphoryl group directly to ADP. We can describe phosphorylated compounds as having a high or low phosphoryl group transfer potential, on the basis of their standard free energies of hydrolysis (as listed in Table 13–6). The phosphoryl group transfer potential of phosphoenolpyruvate is very high, that of ATP is high, and that of glucose 6- phosphate is low (Fig. 13–9). Much of catabolism is directed toward the synthesis of high-energy phosphate compounds, but their formation is not an end in itself; they are the means of activating a very wide variety of compounds for further chemical transformation. The transfer of a phosphoryl group to a compound effectively puts free energy into that compound, so that it has more free energy to give up during subsequent metabolic transformations. We described above how the synthesis of glucose 6-phosphate is accomplished by phosphoryl group transfer from ATP. In the next chapter we see how this phosphorylation of glucose activates, or “primes,” the glucose for catabolic reactions that occur in nearly every living cell. Because of its intermediate position on the scale of group transfer potential, ATP can carry energy from high-energy 13.2 Phosphoryl Group Transfers and ATP 501 G of hydrolysis (kJ/mol) Pi P O O A CHOH D 10 C M CH2 A O Creatine Phosphoenolpyruvate 70 P O 1,3-Bisphosphoglycerate Rib O P GlycerolO O P P 60 30 50 40 20 P ATP Low-energy compounds Glucose 6- P High-energy compounds Adenine COO B C A CH2 O P P Phosphocreatine O O OO OO 0 FIGURE 13–9 Ranking of biological phosphate compounds by standard free energies of hydrolysis. This shows the flow of phosphoryl groups, represented by P , from high-energy phosphoryl donors via ATP to acceptor molecules (such as glucose and glycerol) to form their low-energy phosphate derivatives. This flow of phosphoryl groups, catalyzed by enzymes called kinases, proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of lowenergy phosphate compounds releases Pi, which has an even lower phosphoryl group transfer potential (as defined in the text).
502 Chapter 13 Principles of Bioenergetics phosphate compounds produced by catabolism to com- (p. 218)involves attack at the y position of the ATP pounds such as glucose, converting them into more re- molecule. active species. atP thus serves as the universal energy Attack at the B phosphate of ATP displaces AMP and currency in all living cells transfers a pyrophosphoryl (not pyrophosphate) group One more chemical feature of ATP is crucial to its to the attacking nucleophile(Fig. 13-10b). For exam- ole in metabolism: although in aqueous solution ATP is ple, the formation of 5-phosphoribosyl-1-pyrophosphate thermodynamically unstable and is therefore a good (p. XXX), a key intermediate in nucleotide synthesis phosphoryl group donor, it is kineticallystable Because results from attack of an -OH of the ribose on the B of the huge activation energies(200 to 400 kJ/mol)re- phosphate quired for uncatalyzed cleavage of its phosphoanhydride Nucleophilic attack at the a position of ATP displaces bonds, aTP does not spontaneously donate phosphoryl PP, and transfers adenylate(5-AMP)as an adenylyl groups to water or to the hundreds of other potential group( Fig. 13-10c); the reaction is an adenylylation acceptors in the cell. Only when specific enzymes are(a-den'-i-li-la'-shun, probably the most ungainly word present to lower the energy of activation does phos- in the biochemical language). Notice that hydrolysis of phoryl group transfer from ATP proceed. The cell is the a-B phosphoanhydride bond releases considerably therefore able to regulate the disposition of the energy more energy (-46 kI/mol) than hydrolysis of the B-y carried by atP by regulating the various enzymes that bond(31 kJ/mol)(Table 13-6). Furthermore, the PP act on it formed as a byproduct of the adenylylation is hydrolyzed to two Pi by the ubiquitous enzyme inorganic pyro ATP Donates Phosphoryl, Pyrophosphory! phosphatase, releasing 19 k/mol and thereby provid and Adenylyl Groups ing a further energy"push"for the adenylylation reac- tion In effect, both phosphoanhydride bonds of ATP are The reactions of ATP are generally sn2 nucleophilic dis- split in the overall reaction Adenylylation reactions are placements(p 11.8), in which the nucleophile may be, therefore thermodynamically very favorable. When the for example, the oxygen of an alcohol or carboxylate, or energy of ATP is used to drive a particularly unfavor a nitrogen of creatine or of the side chain of arginine or able metabolic reaction, adenylylation is often the mech- histidine. Each of the three phosphates of ATP is sus- anism of energy coupling. Fatty acid activation is a good ceptible to nucleophilic attack(Fig. 13-10), and each example of this energy-coupling strategy position of attack yields a different type of product. The first step in the activation of a fatty acid- Nucleophilic attack by an alcohol on the y phos- either for energy-yielding oxidation or for use in the syn- phate(Fig. 13-10a)displaces ADP and produces a new thesis of more complex lipids-is the formation of its phosphate ester. Studies with 0-labeled reactants thiol ester(see Fig. 17-5). The direct condensation of have shown that the bridge oxygen in the new Cor a fatty acid with coenzyme a is endergonic, but the for pound is derived from the alcohol, not from ATP: the mation of fatty acyl-CoA is made exergonic by stepwise group transferred from ATP is a phosphoryl(P03 ), removal of two phosphoryl groups from ATP. First, not a phosphate(-0P0S ) Phosphoryl group transfer adenylate(AMP)is transferred from ATP to the car- from ATP to glutamate(Fig. 13-8) or to glucose boxyl group of the fatty acid, forming a mixed anhydride Three positions on ATP for attack by the nucleophile rO FIGURE 13-10 Nucleophilic displacement reac tions of ATP. Any of the three P atoms (a, B, or y) P-0-P-0- RibH Adenine may serve as the electrophilic target for nucleophilic attack-in this case, by the labeled R180R180 nucleophile R-1BO: The nucleophile may be an alcohol(ROH), a carboxyl group(RCoo), or a phosphoanhydride(a nucleoside mono-or diphosphate, for example). (a)When the oxygen R0=一0R0-0-—0-R0=0 RibHAdenine] the group transferred from atP is a phosphory (P03-), not a phosphate(--OPO3-)(b)Attack on the B position displaces AMP and leads to the transfer of a pyrophosphoryl (not pyrophosphate Phosphoryl Adenylyl group to the nucleophile. (c)Attack on the a position displaces PP and transfers the adenylyl group to the nucleophile
phosphate compounds produced by catabolism to compounds such as glucose, converting them into more reactive species. ATP thus serves as the universal energy currency in all living cells. One more chemical feature of ATP is crucial to its role in metabolism: although in aqueous solution ATP is thermodynamically unstable and is therefore a good phosphoryl group donor, it is kinetically stable. Because of the huge activation energies (200 to 400 kJ/mol) required for uncatalyzed cleavage of its phosphoanhydride bonds, ATP does not spontaneously donate phosphoryl groups to water or to the hundreds of other potential acceptors in the cell. Only when specific enzymes are present to lower the energy of activation does phosphoryl group transfer from ATP proceed. The cell is therefore able to regulate the disposition of the energy carried by ATP by regulating the various enzymes that act on it. ATP Donates Phosphoryl, Pyrophosphoryl, and Adenylyl Groups The reactions of ATP are generally SN2 nucleophilic displacements (p. II.8), in which the nucleophile may be, for example, the oxygen of an alcohol or carboxylate, or a nitrogen of creatine or of the side chain of arginine or histidine. Each of the three phosphates of ATP is susceptible to nucleophilic attack (Fig. 13–10), and each position of attack yields a different type of product. Nucleophilic attack by an alcohol on the phosphate (Fig. 13–10a) displaces ADP and produces a new phosphate ester. Studies with 18O-labeled reactants have shown that the bridge oxygen in the new compound is derived from the alcohol, not from ATP; the group transferred from ATP is a phosphoryl (OPO3 2), not a phosphate (OOPO3 2). Phosphoryl group transfer from ATP to glutamate (Fig. 13–8) or to glucose (p. 218) involves attack at the position of the ATP molecule. Attack at the phosphate of ATP displaces AMP and transfers a pyrophosphoryl (not pyrophosphate) group to the attacking nucleophile (Fig. 13–10b). For example, the formation of 5 -phosphoribosyl-1-pyrophosphate (p. XXX), a key intermediate in nucleotide synthesis, results from attack of an OOH of the ribose on the phosphate. Nucleophilic attack at the position of ATP displaces PPi and transfers adenylate (5 -AMP) as an adenylyl group (Fig. 13–10c); the reaction is an adenylylation (a-den -i-li-la- -shun, probably the most ungainly word in the biochemical language). Notice that hydrolysis of the – phosphoanhydride bond releases considerably more energy (~46 kJ/mol) than hydrolysis of the – bond (~31 kJ/mol) (Table 13–6). Furthermore, the PPi formed as a byproduct of the adenylylation is hydrolyzed to two Pi by the ubiquitous enzyme inorganic pyrophosphatase, releasing 19 kJ/mol and thereby providing a further energy “push” for the adenylylation reaction. In effect, both phosphoanhydride bonds of ATP are split in the overall reaction. Adenylylation reactions are therefore thermodynamically very favorable. When the energy of ATP is used to drive a particularly unfavorable metabolic reaction, adenylylation is often the mechanism of energy coupling. Fatty acid activation is a good example of this energy-coupling strategy. The first step in the activation of a fatty acid— either for energy-yielding oxidation or for use in the synthesis of more complex lipids—is the formation of its thiol ester (see Fig. 17–5). The direct condensation of a fatty acid with coenzyme A is endergonic, but the formation of fatty acyl–CoA is made exergonic by stepwise removal of two phosphoryl groups from ATP. First, adenylate (AMP) is transferred from ATP to the carboxyl group of the fatty acid, forming a mixed anhydride 502 Chapter 13 Principles of Bioenergetics O P O O Rib Adenine O Pyrophosphoryl transfer (b) Phosphoryl transfer (a) Adenylyl transfer (c) Rib Adenine R18O R18O R18O R18O R18O R18O R18O ADP AMP PPi O P O O O P O O O P O O O P O O O P O O O P O O Three positions on ATP for attack by the nucleophile FIGURE 13–10 Nucleophilic displacement reactions of ATP. Any of the three P atoms (, , or ) may serve as the electrophilic target for nucleophilic attack—in this case, by the labeled nucleophile RO18O:. The nucleophile may be an alcohol (ROH), a carboxyl group (RCOO), or a phosphoanhydride (a nucleoside mono- or diphosphate, for example). (a) When the oxygen of the nucleophile attacks the position, the bridge oxygen of the product is labeled, indicating that the group transferred from ATP is a phosphoryl (OPO3 2), not a phosphate (OOPO3 2). (b) Attack on the position displaces AMP and leads to the transfer of a pyrophosphoryl (not pyrophosphate) group to the nucleophile. (c) Attack on the position displaces PPi and transfers the adenylyl group to the nucleophile.
13.2 Phosphoryl Group Transfers and ATP (fatty acyl adenylate)and liberating PP. The thiol group for the breakage of these bonds, or-456 kJ/mol of coenzyme a then displaces the adenylate group and(19.2)kJ/mol forms a thioester with the fatty acid. The sum of these two reactions is energetically equivalent to the exer- ATP+2H20→→AMP+2P△G°=-648 k/mol gonic hydrolysis of ATP to AMP and PPi(AG=-456 The activation of amino acids before their kJ/mol) and the endergonic formation of fatty acyl-Coa ization into proteins(see Fig. 27-14)is (AG=31.4 k/mol). The formation of fatty acyl-Coa by an analogous set of reactions in which a transfer rna is made energetically favorable by hydrolysis of the PPi molecule takes the place of coenzyme A. an interesting by inorganic pyrophosphatase. Thus, in the activation use of the cleavage of ATP to AMP and PP, occurs in of a fatty acid, both phosphoanhydride bonds of ATP are the firefly, which uses ATP as an energy source to pro- roken. The resulting AG is the sum of the AG values duce light flashes(Box 13-2) BOX 13-2 THE WORLD OF BIOCHEMISTRY Firefly Flashes: Glowing Reports of ATP pyrophosphate cleavage of ATP to form luciferyl Bioluminescence requires considerable amounts of adenylate. In the presence of molecular oxygen and energy. In the firefly, ATP is used in a set of reactions luciferase, the luciferin undergoes a multistep oxida- that converts chemical energy into light energy. In the tive decarboxylation to oxyluciferin. This process is 1950s, from many thousands of fireflies collected by accompanied by emission of light. The color of the children in and around Baltimore, William McElroy ght flash differs with the firefly species and appears and his colleagues at The Johns Hopkins University to be determined by differences in the structure of the isolated the principal biochemical components: lu- luciferase. Luciferin is regenerated from oxyluciferin iferin, a complex carboxylic acid, and luciferase, an in a subsequent series of reactions In the laboratory, pure firefly luciferin and lu vation of luciferin by an enzymatic reaction involving ciferase are used to measure minute quantities of ATP by the intensity of the light flash produced. As little as a few picomoles(10mol)of ATP can be meas- ured in this way. An enlightening extension of the studies in luciferase was the cloning of the luciferase gene into tobacco plants. When watered with a solu tion containing luciferin, the plants glowed in the dark AMP Luciferyl adenylate The firefly, a beetle of the Lampyridae famil H regenerating firefly bioluminescence cycle Oxyluciferin reactions
(fatty acyl adenylate) and liberating PPi . The thiol group of coenzyme A then displaces the adenylate group and forms a thioester with the fatty acid. The sum of these two reactions is energetically equivalent to the exergonic hydrolysis of ATP to AMP and PPi (G 45.6 kJ/mol) and the endergonic formation of fatty acyl–CoA (G 31.4 kJ/mol). The formation of fatty acyl–CoA is made energetically favorable by hydrolysis of the PPi by inorganic pyrophosphatase. Thus, in the activation of a fatty acid, both phosphoanhydride bonds of ATP are broken. The resulting G is the sum of the G values for the breakage of these bonds, or 45.6 kJ/mol (19.2) kJ/mol: ATP 2H2O 88n AMP 2Pi G 64.8 kJ/mol The activation of amino acids before their polymerization into proteins (see Fig. 27–14) is accomplished by an analogous set of reactions in which a transfer RNA molecule takes the place of coenzyme A. An interesting use of the cleavage of ATP to AMP and PPi occurs in the firefly, which uses ATP as an energy source to produce light flashes (Box 13–2). 13.2 Phosphoryl Group Transfers and ATP 503 BOX 13–2 THE WORLD OF BIOCHEMISTRY Firefly Flashes: Glowing Reports of ATP Bioluminescence requires considerable amounts of energy. In the firefly, ATP is used in a set of reactions that converts chemical energy into light energy. In the 1950s, from many thousands of fireflies collected by children in and around Baltimore, William McElroy and his colleagues at The Johns Hopkins University isolated the principal biochemical components: luciferin, a complex carboxylic acid, and luciferase, an enzyme. The generation of a light flash requires activation of luciferin by an enzymatic reaction involving pyrophosphate cleavage of ATP to form luciferyl adenylate. In the presence of molecular oxygen and luciferase, the luciferin undergoes a multistep oxidative decarboxylation to oxyluciferin. This process is accompanied by emission of light. The color of the light flash differs with the firefly species and appears to be determined by differences in the structure of the luciferase. Luciferin is regenerated from oxyluciferin in a subsequent series of reactions. In the laboratory, pure firefly luciferin and luciferase are used to measure minute quantities of ATP by the intensity of the light flash produced. As little as a few picomoles (1012 mol) of ATP can be measured in this way. An enlightening extension of the studies in luciferase was the cloning of the luciferase gene into tobacco plants. When watered with a solution containing luciferin, the plants glowed in the dark (see Fig. 9–29). A S O P O O N HO S C Oxyluciferin regenerating reactions CO2 AMP luciferase light O2 AMP N H H ATP S N HO S COO N Adenine O Rib PPi Luciferin Luciferyl adenylate O H H H H S N HO S N O The firefly, a beetle of the Lampyridae family. Important components in the firefly bioluminescence cycle.
504 Chapter 13 Principles of Bioenergetics Assembly of Informational Macromolecules relax into a second conformation until another molecule Requires Energy of ATP binds. The binding and subsequent hydrolysis of ATP(by myosin ATPase) provide the energy that forces When simple precursors are assembled into high mo- cyclic changes in the conformation of the myosin head lecular weight polymers with defined sequences (DNA, The change in conformation of many individual myosin RNA, proteins), as described in detail in Part Il, energy is required both for the condensation of monomeric units and for the creation of ordered sequences. The precursors for DNA and RNa synthesis are nucleoside triphosphates, and polymerization is accompanied by RNA cleavage of the phosphoanhydride linkage between the a and B phosphates, with the release of PPi(Fig. 13-11) The moieties transferred to the growing polymer in Base these reactions are adenylate(AMP), guanylate(GMP cytidylate(CMP), or uridylate(UMP) for RNa synthe sis, and their deoxy analogs(with TMP in place of UMP) for DNa synthesis. As noted above, the activation of OHOH amino acids for protein synthesis involves the donation of adenylate groups from ATP, and we shall see in Chap- ter 27 that several steps of protein synthesis on the ri- bosome are also accompanied by gtP hydrolysis. In all these cases, the exergonic breakdown of a nucleoside triphosphate is coupled to the endergonic process of first anhydride synthesizing a polymer of a specific sequence AlP Energizes Active Transport and muscle contraction ATP can supply the energy for transporting an ion or a molecule across a membrane into another aqueous com- partment where its concentration is higher(see Fig 11-36). Transport processes are major consumers of en- ergy, in human kidney and brain, for example, as much as two-thirds of the energy consumed at rest is used to pump Na and K across plasma membranes via the NaK ATPase. The transport of Na and K is driven by cyclic phosphorylation and dephosphorylation of the ansporter protein, with ATP as the phosphor donor(see Fig. 11-37). Nat-dependent phosphorylation conformation, and K-dependent dephosphorylation ors return to the original conformation. Each cycle in the transport process results in the conversion of ATP to ADP and Pi, and it is the free-energy change of ATP formation that result in the electrogenic pumping of Na and Kt note that in this case atp interacts covalent by phosphoryl group transfer to the enzyme, not the ubstrate In the contractile system of skeletal muscle cells, OHOH myosin and actin are specialized to transduce the chem- FIGURE 13-11 Nucleoside triphosphates in RNA synthesis. With cal energy of ATP into motion(see Fig, 5-33). ATP each nucleoside monophosphate added to the growing chain, one Pp. binds tightly but noncovalently to one conformation of is released and hydrolyzed to two Ph The hydrolysis of two phosph myosin, holding the protein in that conformation. When anhydride bonds for each nucleotide added provides the energy for myosin catalyzes the hydrolysis of its bound ATP, the forming the bonds in the RNA polymer and for assembling a specific ADP and Pi dissociate from the protein, allowing it to sequence of nucleotides
Assembly of Informational Macromolecules Requires Energy When simple precursors are assembled into high molecular weight polymers with defined sequences (DNA, RNA, proteins), as described in detail in Part III, energy is required both for the condensation of monomeric units and for the creation of ordered sequences. The precursors for DNA and RNA synthesis are nucleoside triphosphates, and polymerization is accompanied by cleavage of the phosphoanhydride linkage between the and phosphates, with the release of PPi (Fig. 13–11). The moieties transferred to the growing polymer in these reactions are adenylate (AMP), guanylate (GMP), cytidylate (CMP), or uridylate (UMP) for RNA synthesis, and their deoxy analogs (with TMP in place of UMP) for DNA synthesis. As noted above, the activation of amino acids for protein synthesis involves the donation of adenylate groups from ATP, and we shall see in Chapter 27 that several steps of protein synthesis on the ribosome are also accompanied by GTP hydrolysis. In all these cases, the exergonic breakdown of a nucleoside triphosphate is coupled to the endergonic process of synthesizing a polymer of a specific sequence. ATP Energizes Active Transport and Muscle Contraction ATP can supply the energy for transporting an ion or a molecule across a membrane into another aqueous compartment where its concentration is higher (see Fig. 11–36). Transport processes are major consumers of energy; in human kidney and brain, for example, as much as two-thirds of the energy consumed at rest is used to pump Na and K across plasma membranes via the NaK ATPase. The transport of Na and K is driven by cyclic phosphorylation and dephosphorylation of the transporter protein, with ATP as the phosphoryl group donor (see Fig. 11–37). Na-dependent phosphorylation of the NaK ATPase forces a change in the protein’s conformation, and K-dependent dephosphorylation favors return to the original conformation. Each cycle in the transport process results in the conversion of ATP to ADP and Pi , and it is the free-energy change of ATP hydrolysis that drives the cyclic changes in protein conformation that result in the electrogenic pumping of Na and K. Note that in this case ATP interacts covalently by phosphoryl group transfer to the enzyme, not the substrate. In the contractile system of skeletal muscle cells, myosin and actin are specialized to transduce the chemical energy of ATP into motion (see Fig. 5–33). ATP binds tightly but noncovalently to one conformation of myosin, holding the protein in that conformation. When myosin catalyzes the hydrolysis of its bound ATP, the ADP and Pi dissociate from the protein, allowing it to relax into a second conformation until another molecule of ATP binds. The binding and subsequent hydrolysis of ATP (by myosin ATPase) provide the energy that forces cyclic changes in the conformation of the myosin head. The change in conformation of many individual myosin 504 Chapter 13 Principles of Bioenergetics GTP CH2 P A A O OH H O H H H OH O Guanine O P A O A O O A O O O P P O P O O A OH H H H H CH2 O Base RNA chain lengthened by one nucleotide P A O A O O A O O OH H H H H OH CH2 O Base O P A O O O O O O O B B A O P P P O 2Pi A OH H O H H H OH O Guanine A RNA chain A O O P O P A O O O O O O B B A O O O CH2 O PPi first anhydride bond broken second anhydride bond broken S FIGURE 13–11 Nucleoside triphosphates in RNA synthesis. With each nucleoside monophosphate added to the growing chain, one PPi is released and hydrolyzed to two Pi. The hydrolysis of two phosphoanhydride bonds for each nucleotide added provides the energy for forming the bonds in the RNA polymer and for assembling a specific sequence of nucleotides.
13.2 Phosphoryl Group Transfers and ATP molecules results in the sliding of myosin fibrils along intermediate; then the phosphoryl group is transferred actin filaments(see Fig. 5-32), which translates into from the(-His residue to an NDP acceptor. Because macroscopic contraction of the muscle fiber the enzyme is nonspecific for the base in the NDP and As we noted earlier, this production of mechanical works equally well on dNDPs and NDPs, it can synthe- motion at the expense of ATP is one of the few cases in size all NTPs and dNTPs, given the corresponding NDPs which ATP hydrolysis per se, rather than group trans- and a supply of ATP fer from ATP, is the source of the chemical energy in a Phosphoryl group transfers from ATP result in an coupled process accumulation of ADP; for example, when muscle is con- tracting vigorously, ADP accumulates and interferes Transphosphorylations between Nucleotides with ATP-dependent contraction. During periods of in- Occur in All Cell Types tense demand for atp the cell lowers the adp con- centration, and at the same time acquires ATP, by the Although we have focused on ATP as the cells energy action of adenylate kinase currency and donor of phosphoryl groups, all other nu- cleoside triphosphates(GTP, UTP, and CTP)and all the deoxynucleoside triphosphates(dATP, dGTP, dTTP, and 2 ADP F ATP+ AMP AG°=0 dCTP)are energetically equivalent to ATP. The free. This reaction is fully reversible, so after the intense de energy changes associated with hydrolysis of their mand for ATP ends, the enzyme can recycle AMP by phosphoanhydride linkages are very nearly identical converting it to ADP, which can then be phosphorylated with those shown in Table 13-6 for ATP. In preparation to ATP in mitochondria. A similar enzyme, b of ATP. By for their various biological roles, these other nucleotides nase, converts GMP to GDP at the expense are generated and maintained as the nucleoside triphos. pathways such as these, energy conserved in the cata hate(NTP) forms by phosphoryl group transfer to the bolic production of ATP is used to supply the cell with corresponding nucleoside diphosphates(NDPs) and all required NTPs and dNTPs monophosphates(NI Phosphocreatine(Fig. 13-5), also called creatine ATP is the primary high-energy phosphate com- phosphate, serves as a ready source of phosphoryl pound produced by catabolism, in the processes of gly- groups for the quick synthesis of ATP from ADP. The colysis, oxidative phosphorylation, and, in photosyn. phosphocreatine(PCr) concentration in skeletal mus- thetic cells, photophosphorylation. Several enzymes cle is approximately 30 mM, nearly ten times the con- hen carry phosphoryl groups from atP to the other nu- centration of ATP, and in other tissues such as smooth cleotides. Nucleoside diphosphate kinase, found in muscle, brain, and kidney[PCr] is 5 to 10 mM. The en all cells, catalyzes the reaction zyme creatine kinase catalyzes the reversible reaction ATP+ NDP (or dNDP ADP+ NTP (or dNTP ADP PCr ATP+Cr△G°=-12.5 k/mol When a sudden demand for energy depletes ATP, the Although this reaction is fully reversible, the relatively PCr reservoir is used to replenish ATP at a rate consid- high (ATPM(] ratio in cells normally drives the re- erably faster than ATP can be synthesized by catabolic action to the right, with the net formation of NTPs and pathways. When the demand for energy slackens, ATP dNTPs. The enzyme actually catalyzes a two-step phos- produced by catabolism is used to replenish the PCr phoryl transfer, which is a classic case of a double-dis reservoir by reversal of the creatine kinase reaction Or placement(Ping-Pong) mechanism(Fig. 13-12: see also ganisms in the lower phyla employ other PCr-like mole ig. 6-13b). First, phosphoryl group transfer from ATP cules(collectively called phosphagens) as phosphoryl to an active-site His residue produces a phosphoenzyme Adenosine①、cn (ATP TP or dNTP Ping P Enzhi Nucleoside① FIGURE 13-12 Ping-Pong mechanism of nucleoside diphosphate phate replaces it, and this is converted to the corresponding triphos. kinase. The enzyme binds its first substrate (ATP in our example), and phate by transfer of the phosphoryl group from the phosphohistidine a phosphoryl group is transferred to the side chain of a His residue. residue. ADP departs, and another nucleoside (or deoxynucleoside) diphos-
molecules results in the sliding of myosin fibrils along actin filaments (see Fig. 5–32), which translates into macroscopic contraction of the muscle fiber. As we noted earlier, this production of mechanical motion at the expense of ATP is one of the few cases in which ATP hydrolysis per se, rather than group transfer from ATP, is the source of the chemical energy in a coupled process. Transphosphorylations between Nucleotides Occur in All Cell Types Although we have focused on ATP as the cell’s energy currency and donor of phosphoryl groups, all other nucleoside triphosphates (GTP, UTP, and CTP) and all the deoxynucleoside triphosphates (dATP, dGTP, dTTP, and dCTP) are energetically equivalent to ATP. The freeenergy changes associated with hydrolysis of their phosphoanhydride linkages are very nearly identical with those shown in Table 13–6 for ATP. In preparation for their various biological roles, these other nucleotides are generated and maintained as the nucleoside triphosphate (NTP) forms by phosphoryl group transfer to the corresponding nucleoside diphosphates (NDPs) and monophosphates (NMPs). ATP is the primary high-energy phosphate compound produced by catabolism, in the processes of glycolysis, oxidative phosphorylation, and, in photosynthetic cells, photophosphorylation. Several enzymes then carry phosphoryl groups from ATP to the other nucleotides. Nucleoside diphosphate kinase, found in all cells, catalyzes the reaction ATP NDP (or dNDP) ADP NTP (or dNTP) DG 0 Although this reaction is fully reversible, the relatively high [ATP]/[ADP] ratio in cells normally drives the reaction to the right, with the net formation of NTPs and dNTPs. The enzyme actually catalyzes a two-step phosphoryl transfer, which is a classic case of a double-displacement (Ping-Pong) mechanism (Fig. 13–12; see also Fig. 6–13b). First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme Mg2 3:::4 intermediate; then the phosphoryl group is transferred from the P –His residue to an NDP acceptor. Because the enzyme is nonspecific for the base in the NDP and works equally well on dNDPs and NDPs, it can synthesize all NTPs and dNTPs, given the corresponding NDPs and a supply of ATP. Phosphoryl group transfers from ATP result in an accumulation of ADP; for example, when muscle is contracting vigorously, ADP accumulates and interferes with ATP-dependent contraction. During periods of intense demand for ATP, the cell lowers the ADP concentration, and at the same time acquires ATP, by the action of adenylate kinase: 2ADP ATP AMP DG 0 This reaction is fully reversible, so after the intense demand for ATP ends, the enzyme can recycle AMP by converting it to ADP, which can then be phosphorylated to ATP in mitochondria. A similar enzyme, guanylate kinase, converts GMP to GDP at the expense of ATP. By pathways such as these, energy conserved in the catabolic production of ATP is used to supply the cell with all required NTPs and dNTPs. Phosphocreatine (Fig. 13–5), also called creatine phosphate, serves as a ready source of phosphoryl groups for the quick synthesis of ATP from ADP. The phosphocreatine (PCr) concentration in skeletal muscle is approximately 30 mM, nearly ten times the concentration of ATP, and in other tissues such as smooth muscle, brain, and kidney [PCr] is 5 to 10 mM. The enzyme creatine kinase catalyzes the reversible reaction ADP PCr ATP Cr DG 12.5 kJ/mol When a sudden demand for energy depletes ATP, the PCr reservoir is used to replenish ATP at a rate considerably faster than ATP can be synthesized by catabolic pathways. When the demand for energy slackens, ATP produced by catabolism is used to replenish the PCr reservoir by reversal of the creatine kinase reaction. Organisms in the lower phyla employ other PCr-like molecules (collectively called phosphagens) as phosphoryl reservoirs. Mg2 3:::4 Mg2 3:::4 13.2 Phosphoryl Group Transfers and ATP 505 Adenosine P P P (ATP) Adenosine P P (ADP) P P P P Enz His Enz His Ping Pong Nucleoside (any NTP or dNTP) Nucleoside P P (any NDP or dNDP) FIGURE 13–12 Ping-Pong mechanism of nucleoside diphosphate kinase. The enzyme binds its first substrate (ATP in our example), and a phosphoryl group is transferred to the side chain of a His residue. ADP departs, and another nucleoside (or deoxynucleoside) diphosphate replaces it, and this is converted to the corresponding triphosphate by transfer of the phosphoryl group from the phosphohistidine residue.