Adenosine triphosphate (ATP)

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“High energy” bonds

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N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPOP-O

O

O- O-

O O

O-

adenine

ribose

ATP

adenosine triphosphate

phosphoanhydride

bonds (~)

Phosphoanhydride bonds (formed by splitting out H2O between 2phosphoric acids or between carboxylic & phosphoric acids) have alarge negative ΔG of hydrolysis

• 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, Pi may be

spontaneously cleaved from ATP for transfer to another

compound (e.g., to a hydroxyl group on glucose).

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Potentially, 2 ~P bonds can be cleaved, as 2 phosphates are

released by hydrolysis from ATP.

AMP~P~P AMP~P + Pi (ATP ADP + Pi)

AMP~P AMP + Pi (ADP AMP + Pi)

Alternatively:

AMP~P~P AMP + P~P (ATP AMP + PPi)

P~P 2 Pi (PPi 2Pi)

• 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 ofmetabolism.

When ATP production does not keep up with needs, ahigher portion of a cell's adenine nucleotide pool is AMP.

AMP stimulates metabolic pathways that produce ATP.

• Some examples of this role involve direct allostericactivation of pathway enzymes by AMP.

• Some regulatory effects of AMP are mediated by theenzyme AMP-Activated Protein Kinase.

A reaction important for equilibrating ~P among adenine nucleotides

within a cell is that catalyzed by Adenylate Kinase:

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 Pi residues from polyphosphate may be coupled to energy-

dependent reactions.

Depending on the organism or cell type, inorganic polyphosphate may

have additional functions.

E.g., it may serve as a reservoir for Pi, a chelator of metal ions, a buffer,

or a regulator.

Why do phosphoanhydride linkages have a high ΔG of

hydrolysis? Contributing factors for ATP & PPi include:

• Resonance stabilization of products of hydrolysis exceeds

resonance stabilization of the compound itself.

• Electrostatic repulsion between negatively charged

phosphate oxygen atoms favors separation of the

phosphates.

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

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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

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ATP is frequently the donor of the phosphate in the

biosynthesis of phosphate esters.

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The free energy change for ATP hydrolysis is large and

negative

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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 oxygen's.

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(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. 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.

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• 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.

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• The cellular concentrations of ATP, ADP and Pi are

not same and are much lower than the 1 M standard

conditions.

• In addition, Mg2+ 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.

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ΔG°´ of ATP Hydrolysis Is Mg++ Dependent

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Mg2+ and ATP. Formation of Mg2+ complexespartially shields the negative charges andinfluences the conformation of the phosphategroups in nucleotides such as ATP and ADP.

Phosphorylated compounds

• Phosphoenolpyruvate

• 1,3-bisphosphoglycerate

• Phosphocreatine

• Thioesters

• ATP

• AMP

• PPi

• Glucose 1-phosphate

• Fructose 6-phosphate

• Glucose 6-phosphate

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Phosphoenolpyruvate (PEP)

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• Involved in ATP synthesis in Glycolysis, has a very high ΔG´°= -61,9

kJ/mol of Pi hydrolysis.

• Phosphoenolpyruvate contains a phosphate ester bond.

• Removal of Pi from ester linkage in PEP is spontaneous because

the enol spontaneously converts to a ketone (tautomerization).

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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 Pi further

contributes to the negative free energy change.

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Hydrolysis of acyl phosphate (1,3-bisphosphoglycerate) isaccompanied by a large, negative, standard free energychange (ΔG'° = -49,3 kJ/mol)

Phosphocreatine

The P-N 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 is large and negative (ΔG'° = -43.0 kJ/mol).

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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.

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Hydrolysis of the ester bond generates a carboxylic acid whichcan ionize and assume several resonance forms.ΔG'° = -31,4 kJ/mol for acetyl-CoA hydrolysis

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• Free energy of hydrolysis for thioesters and oxygen esters. Theproducts of both types of hydrolysis reaction have about the samefree-energy content (G), but the thioester has a higher free-energycontent than the oxygen ester.

• Orbital overlap between the O and C atoms allows resonancestabilization in oxygen esters; orbital overlap between S and C atoms ispoorer and provides little resonance stabilization.

• 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.

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• 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).

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The flow of phosphoryl groups, represented by ,, from high-energyphosphoryl donors via ATP to acceptor molecules (such as glucose andglycerol) to form their low-energy phosphate derivatives. This flow ofphosphoryl groups, catalyzed by enzymes called kinases, proceeds with anoverall loss of free energy under intracellular conditions. Hydrolysis of lowenergy phosphate compounds releases Pi, which has an even lowerphosphoryl group transfer potential

• 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.

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PEP + H2O Pyruvate + Pi -61,9

ADP+ Pi ATP+ H2O +30,5

PEP + ADP Pyruvate + ATP -31,4

Cleavage of Pi from PEP releases more energy than is

needed to drive to condensation of Pi with ADP, the direct

donation of a phosphoryl group from PEP to ADP is

thermodynamically feasible.

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• The overall reaction above is represented as the algebraic

sum of first two reactions, the overall reaction (third) 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 energy changes of hydrolysis

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• 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.

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• 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

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• 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.

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• 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

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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-18O:. 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 γ position, the bridge oxygen of the product is labeled,

indicating that the group transferred from ATP is a

phosphoryl (-PO32-), not a phosphate (-OPO3

2-).

(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 PPi and transfers the

adenylyl group to the nucleophile.

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