Adenosine triphosphate (ATP) 1
High energy bonds ATP adenosine triphosphate N NH 2 N -O O P O O P O- O- O O P O- O CH 2 H O H N N adenine phosphoanhydride bonds (~) H OH ribose H OH Phosphoanhydride bonds (formed by splitting out H 2 O between 2 phosphoric acids or between carboxylic & phosphoric acids) have a large negative ΔG of hydrolysis 2
Phosphoanhydride linkages are said to be "high energy" bonds. Bond energy is not high, just ΔG of hydrolysis. "High energy" bonds are represented by the "~" symbol. ~P represents a phosphate group with a large negative ΔG of hydrolysis. Compounds with high energy bonds are said to have high group transfer potential. For example, P i may be spontaneously cleaved from ATP for transfer to another compound (e.g., to a hydroxyl group on glucose). 3
Potentially, 2 ~P bonds can be cleaved, as 2 phosphates are released by hydrolysis from ATP. AMP~P~P AMP~P + P i (ATP ADP + P i ) AMP~P AMP + P i (ADP AMP + P i ) Alternatively: AMP~P~P AMP + P~P (ATP AMP + PP i ) P~P 2 P i (PP i 2P i )
ATP often serves as an energy source. Hydrolytic cleavage of one or both of the "high energy" bonds of ATP is coupled to an energy-requiring reaction. AMP functions as an energy sensor & regulator of metabolism. When ATP production does not keep up with needs, a higher portion of a cell's adenine nucleotide pool is AMP. AMP stimulates metabolic pathways that produce ATP. Some examples of this role involve direct allosteric activation of pathway enzymes by AMP. Some regulatory effects of AMP are mediated by the enzyme AMP-Activated Protein Kinase.
A reaction important for equilibrating ~P among within a cell is that catalyzed by Adenylate Kinase: adenine nucleotides ATP + AMP 2 ADP The Adenylate Kinase reaction is also important because the substrate for ATP synthesis, e.g., by mitochondrial ATP Synthase, is ADP, while some cellular reactions dephosphorylate ATP all the way to AMP. The enzyme Nucleoside Diphosphate Kinase equilibrates ~P among the various nucleotides that are needed, e.g., for synthesis of DNA & RNA. NuDiKi catalyzes reversible reactions such as: ATP + GDP ADP + GTP, ATP + UDP ADP + UTP, etc.
Inorganic polyphosphate Many organisms store energy as inorganic polyphosphate, a chain of many phosphate residues linked by phosphoanhydride bonds: P~P~P~P~P... Hydrolysis of P i residues from polyphosphate may be coupled to energydependent reactions. Depending on the organism or cell type, inorganic polyphosphate may have additional functions. E.g., it may serve as a reservoir for P i, a chelator of metal ions, a buffer, or a regulator.
Why do phosphoanhydride linkages have a high ΔG of hydrolysis? Contributing factors for ATP & PP i include: Resonance stabilization of products of hydrolysis exceeds resonance stabilization of the compound itself. Electrostatic repulsion between negatively charged phosphate oxygen atoms favors phosphates. separation of the
Phosphoryl group transfer and ATP Living cells obtain free energy in a chemical form by the catabolism of nutrient molecules They use that energy to make ATP from ADP and Pi. ATP donates some of its chemical energy to 1. Endergonic processes such as the synthesis of metabolic intermediates and macromolecules from smaller precursors 9
2. The transport of substances across membranes against concentration gradients 3. Mechanical motion (muscle contraction) This donation of energy from ATP can occur in the two form A) ATP ADP+ Pi or B) ATP AMP+ 2 Pi 10
ATP is frequently the donor of the phosphate in the biosynthesis of phosphate esters. 11
The free energy change for ATP hydrolysis is large and negative 12
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 (P i ) 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 oxygen's. 13
(Some degree of resonance stabilization also occurs in phosphates involved in ester or anhydride linkages, but fewer resonance forms are possible than for P i ) 3. A third factor 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. 14
Although the hydrolysis of ATP is highly exergonic (ΔG = -30,5 kj/mol), the ATP is stable at ph 7, because the activation energy for ATP hydrolysis is relatively high. Rapid hydrolysis of ATP 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 change (ΔG) of ATP hydrolysis in living cells is very different. 15
The cellular concentrations of ATP, ADP and Pi are not same and are much lower than the 1 M standard conditions. In addition, Mg 2+ in the cytosol binds to ATP and ADP and for most enzymatic reactions that involve ATP as phosphoryl group donor, the true substrate is MgATP -2. The relevant ΔG is therefore that for MgATP -2 hydrolysis. 16
ΔG of ATP Hydrolysis Is Mg ++ Dependent Mg 2+ and ATP. Formation of Mg 2+ complexes partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP. 17
Phosphorylated compounds Phosphoenolpyruvate 1,3-bisphosphoglycerate Phosphocreatine Thioesters ATP AMP PP i Glucose 1-phosphate Fructose 6-phosphate Glucose 6-phosphate 18
Phosphoenolpyruvate (PEP) Involved in ATP synthesis in Glycolysis, has a very high ΔG = -61,9 kj/mol of P i hydrolysis. Phosphoenolpyruvate contains a phosphate ester bond. Removal of P i from ester linkage in PEP is spontaneous because the enol spontaneously converts to a ketone (tautomerization). 19
20
1,3-bisphosphoglycerate 1,3-bisphosphoglycerate contains an anhydride bond between the carboxyl group at C-1 and phosphoric acid. The direct product of hydrolysis 1,3-bisphosphoglycerate 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 P i contributes to the negative free energy change. further 21
Hydrolysis of acyl phosphate (1,3-bisphosphoglycerate) is accompanied by a large, negative, standard free energy change (ΔG' = -49,3 kj/mol) 22
Phosphocreatine The P-N bond can be hydrolyzed to generate free creatine and Pi. The release of P i and the resonance stabilization of creatine favor the forward reaction. The standard free energy change of phosphocreatine is large and negative (ΔG' = -43.0 kj/mol). 23
Thioesters (Acetyl COA) In thioesters a sulfur atom is replaced the usual oxygen in the ester bond. Thioesters have large, negative standard free energy change of hydrolysis. Acetyl coenzyme A is the one of important thioesters in metabolism. The acyl group in these compounds is activated for trans-acylation, condensation or oxidation-reduction reactions. 24
Hydrolysis of the ester bond generates a carboxylic acid which can ionize and assume several resonance forms. ΔG' = -31,4 kj/mol for acetyl-coa hydrolysis 25
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. 26
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 ionization, as for ATP, acyl phosphates, thioesters. 3. The products are stabilized by isomerization (tautomerization) as for phosphoenolpyruvate 4. The products are stabilized by resonance as for creatine released from phosphocreatine, carboxylate ion released from acyl phosphates and thioesters and phosphate released from anhydride or ester linkages. 27
The phosphate compounds found in living organisms divided into two groups based on their standard free energy changes of hydrolysis. High-energy compounds have a ΔG' of hydrolysis more negative than -25 kj/mol (ATP, with a ΔG' of hydrolysis of -30 kj/mol). Low-energy compounds have a less negative ΔG' (glucose 6-phosphate with a (ΔG' of hydrolysis of -13,8 kj/mol). 28
The flow of phosphoryl groups, represented by,, 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 low energy phosphate compounds releases Pi, which has an even lower phosphoryl group transfer potential 29
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. 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 standard free energy change of hydrolysis. 30
PEP + H2O Pyruvate + P i -61,9 ADP+ P i ATP+ H2O +30,5 PEP + ADP Pyruvate + ATP -31,4 Cleavage of P i from PEP releases more energy than is needed to drive to condensation of P i with ADP, the direct donation of a phosphoryl group from PEP to ADP is thermodynamically feasible. 31
The overall reaction above is represented as the algebraic sum of first two reactions, the overall reaction (third) does not involve P i ; 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 energy changes of hydrolysis 32
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 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. 33
Because of its intermediate position on the scale of group transfer potential, ATP can carry energy from high-energy phosphate compounds produced by catabolism to compounds such as glucose, converting them into more reactive species. ATP serves as the universal energy currency in all living cells 34
One more chemical feature of ATP is crucial to its role in metabolism: although in aqueous solution ATP is thermo-dynamically unstable and is therefore a good phosphoryl group donor, it is kinetically stable. Because of high activation energies required for uncatalyzed reaction ATP does not spontaneously donate phosphoryl groups to water or to the other potential acceptors in the cell. 35
ATP hydrolysis occurs only when specific enzymes which lower the energy of activation are present The cell is therefore able to regulate the disposition of the energy carried by ATP by regulating the various enzymes that act on ATP Each of the three phosphates of ATP is susceptible to nucleophilic attack and each position of attack yields a different type of product 36
Any of the three P atoms (α, β, or γ) may serve as the electrophilic target for nucleophilic attack in this case, by the labeled nucleophile R- 18 O:. The nucleophile may be an alcohol (ROH), a carboxyl group (RCOO - ), or a phosphoanhydride (a nucleoside mono- or diphosphate. 37
For example (a) When the oxygen of the nucleophile attacks the γ position, the bridge oxygen of the product is labeled, indicating that the group transferred from ATP is a phosphoryl (-PO 2-3 ), not a phosphate (-OPO 2-3 ). (b) Attack on the β position displaces AMP and leads to the transfer of a pyrophosphoryl (not pyrophosphate) group to the nucleophile. (c) Attack on the α position displaces PP i and transfers the adenylyl group to the nucleophile. 38