ATP and Phosphorylation.ppt

ATP: Adenosine Triphosphate

Professor B. Z. ChowdhrySchool of ScienceRoom 202, Grenville BuildingTel: 0208-331-8208E-mail: [email protected]

ATP has the following structure:

ATP consists of: a base (adenine), a sugar (ribose) and a phosphate chain.

ATP possesses 2 phosphoanhydride bonds.

AND

ATP possesses 1 phosphoester bond

• The following statements relating to ATP are incorrect:

• a) that it can trap and store energy”• b) that it is the universal energy currency”• c) it is a high energy molecule”• d) that it can give up its stored energy in order to “drive” reactions which otherwise would be energetically favourable”• e) “that it is an unstable molecule”.

• The following statements are also untrue:

– ATP is hydrolysed to ADP and Pi

– phosphate transfer potentials account for the function of ATP

– the coupling of oxidation of substrates i.e. catabolism to anabolism is energetic not stoichiometric

What about:High energy phosphate compounds

Low energy phosphate compounds ?

Concept of high energy and low energyphosphates not useful!

ATP is claimed to have special roles in energy coupling and phosphate transfer. The free energy of hydrolysis of phosphate from ATP is intermediate among the examples listed in Table 1 (next slide). ATP thus act as a phosphate donor (true), and ATP can be synthesized by transfer of phosphate from other compounds, such as phosphoenolpyruvate (PEP).

BUT Go OF PHOSPHATE HYDROLYSIS IS IRRELEVANT

Compound Go' of phosphate hydrolysis (kJ/mol)

Phosphoenolpyruvate (PEP) 61.9

Phosphocreatine 43.1

Pyrophosphate 33.5

ATP (to ADP) 30.5

Glucose-6-phosphate 13.8

Glycerol-3-phosphate 9.2

Table 1. Standard free energies of hydrolysis

• 1) The rate of ATP turnover in cells is seconds to minutes.• 2) ATP cannot be stored.• 3) ATP is water soluble.• 4) Under physiological conditions ATP is stable.• 5) ATP in cells, is complexed to Mg2+ ions i.e.

ATP-Mg2+ complex is structurally complex (can be recognized easily and specifically without ambiguity by enzymes).

• 6) ATP is involved in reactions which are irreversible.

THE FACTS

• 8) Reactions involving ATP in vivo are never close to equilibrium.

• 9) Cellular reactions are under kinetic control

not thermodynamic control.

• 10) Free energy (Go) values apply to reactions, not reactants (or products).

• 11) ATP is a very important phosphorylating agent for both small molecular weight molecules (e.g. glucose) and proteins.

• 12) Reactions which involve ATP are usually catalysed by kinase enzymes e.g., hexokinase and phosphofructokinase reactions in glycolysis.

• 13) ATP occurs in all eukaryotic and most procaryotic cells; it is central to many cellular functions.

• ATP is produced via three main mechanisms:

• (a) substrate level phosphorylation (glycolysis and the TCA cycle)

• (b) oxidative phosphorylation ( occurs in the mitochondria of eukaryotic cells; the majority of ATP is produced via this mechanism)

• (c) the adenylate kinase reaction (mainly in muscle cells as an “emergency” supply of ATP).

• ATP can be used in a number of synthetic reactions which can be generalised as follows:

XOH + YOH + nATP + (n-1)H2O

XOY + nADP + H3PO4 (1)

• If n = 1 then equation (1) can be split into sets of hypothetical reactions:

• XOH + YOH XOY + H2O (2)• ATP + H2O ADP + H3PO4 (3) OR XOH + ATP XOP + ADP (4)• XOP + YOH XOY + H3PO4 (5)

• Which of these sets of reactions describes the in vivo synthesis of XOY?

• (i) the amount of XOY formed in the presence of enzymes catalysing (2) and (3) depends only on the equilibrium constant for reaction (2) and is independent of the presence of ATP.

• (ii) reaction (3) does not achieve anything and is very unlikely to occur in vivo because ATP is hardly ever hydrolysed!

• Reactions (4) and (5) are plausible because the amount of XOY which is formed will depend on the concentrations of XOH, YOH and ATP.

• Reactions (4) and (5) are chemically i.e. stoichiometrically linked.

• Values of free energies (Go) for the above reactions are irrelevant (cellular systems are under kinetic not thermodynamic control).

ATP is being continuously synthesized and used.

Cannot be stored

Metabolic half –life of ATP seconds to minutes

Brain cells only a few seconds supply of ATP

Living systems are not at equilibrium

Non-equilibrium thermodynamics

Oscillating thermodynamics (Complicated ??)

The real role of ATP has nothing to do with energetics and

is a gross misuse as well as misunderstanding of thermodynamic rules

……………………………………………………….. This does not detract from the fact that in cells

ATP fulfils vital functions which need to be understood if progress is going to be made in

understanding cellular function

The primary causes of the present situation in which the energetics of biological processes are discussed in misleading terms lies in two major areas:

(i)free energy (Go) values apply to reactions, not reactants (or products).

(ii)the inability to distinguish between kinetic and thermodynamic control.

All reactions in vivo are enzyme catalysed and biological systems operate by complicated sequences of integrated reactions in which very few, if any, of the individual steps are at equilibrium.

Living systems are under kinetic not thermodynamic control.

Furthermore it is likely that reactions involving ATP in vivo are never close to equilibrium.

The intermediate has to have the following properties:

(i) be stable under physiological conditions and water soluble.

(ii) have some degree of structural complexity:

Recognition Specificity

ATP should be involved in reactions which are irreversible Large fluctuations in the levels of ATP concentration would disrupt integrated metabolic pathways The directions in which metabolic reactions proceed will be under kinetic control. This is achieved by changes in concentrations of reactant(s) or product(s) or by the effects of reactants, products and positive or negative effectors.

There are many examples of coupled reactions involving ATP in vivo, these include:

(i) the synthesis of nucleotides and polysaccharides,

(ii) the chemical activation of amino acids as esters of a specific tRNA prior to their incorporation into

proteins,

(iii) the synthesis of acetylcoenzyme A in bacterial cells.

If ATP is to function in its many roles its concentration in cells has to be maintained at adequate levels.It must not be destroyed by direct hydrolysis.It is apparent that the central role of the nucleotide in metabolism is due as much to its kinetic versatility as to its thermodynamic properties.

Reminder:It is a fundamental thermodynamic property of chemical reactions that the equilibrium constant of a reaction reflects only the equilibrium concentrations of reactants and products and is completely independent of the pathway which the reaction follows.

There are many examples of coupled reactions involving ATP in vivo, these include the:(i)synthesis of nucleotides and polysaccharides.

(ii) chemical activation of amino acids as esters of a specific tRNA prior to their incorporation into proteins.

(iii)synthesis of acetylcoenzyme A in bacterial cells. It is worth examining reaction (iii) in more detail. 

Consider the following reactions: (1) CH3CO2

- + ATP4- CH3COOPO32- + ADP3-

  (2) CH3COOPO3

2- + HSCoA CH3COSCoA + HOPO32-

 Reactions (1) and (2) sum to the following reaction:

(3) CH3CO2- + ATP4- + HSCoA CH3COSCoA + ADP3- + HPO42-

The equilibrium constants for reactions (1) and (2) are: 8 x10-3 and 74, respectively.

[Note that the forward and backward reactions would be catalysed, in vivo, by different enzymes!]

For the overall reaction (3) the equilibrium constant (Keq) is equal to: 0.59 (i.e. 8 x10-3 x 74). For direct esterification Keq = 6.6 x 10-5.

Question Why is the Keq for reaction (3) much larger than that for the direct condensation reaction? 

Answer In any set of coupled reactions the elements of water (not a molecule of water as such) are removed from the reactants of the condensation and incorporated into phosphate. If we make allowances for ionisation an OH group is removed from acetic acid (CH3CO2

-) and liberated as part of ADP whilst H+ is removed from coenzyme A and liberated as part of phosphoric acid. The formation of the condensation product is therefore facilitated because water is being removed and liberated in the form of a species which has mole fractions less than unity!

CoenzymeVitamin source

Major metabolic role Mechanistic role

Adenosine triphosphate - Transfer of phosphoryl or

nucleotidyl groups

Cosubstrate

Metabolite Coenzymes: Synthesized from common metabolites Include: e.g. ATP

Methionine + ATP adenosylmethionine + PPPi (S-AM)

S-AM is required for:the conversion of norepinephrine to epinephrine

HO

HO

CH2 CH2 NH3

+

Norepinephrine

Epinephrine

HO

HO

CH2 CH2 NH2

+CH3

• Protein phosphorylation catalyzed by protein kinases plays a critical role in many cellular processes e.g. cellular signalling

Protein phosphorylation

Topic 2

Protein phosphorylation

Protein OH Protein P

ATP ADP

Pi H2O

Ser-Thr Kinases

Non-phosphorylatedprotein

Ser-Thr Phosphatases Phosphorylated protein

During protein phosphorylation by ATP phosphate moieties are usually transferred to

the serine, threonine or tyrosine (sometimes arginine) amino acid residues of proteins from ATP molecules by protein kinases

• Reversible protein phosphorylation acts as a biological regulatory mechanism

• It is thought that perhaps 30 % of the proteins encoded by the human genome contain covalently bound phosphate, and abnormal phosphorylation is now recognized as a cause or consequence of many human diseases

• Protein + ATP Protein-P +ADP Active Inactive OR

• Protein + ATP Protein-P +ADP Inactive Active

E1

E2

Signalling networksCan be very complexE.g. protein A phosphorylates protein BProtein B phosphorylates CBUTA can also phosphorylate C directlyB can phosphorylate D, which may in turn phosphorylate A……………………………………………………………………Phosphorylation of sugars: part of their catabolismallows cells to accumulate sugars……………………………………………………………………30 % of all proteins in cells may undergo phosphorylationHuman genome : 2 % of genes may be kinase genes

Disregulated kinase activity: cause of disease e.g. cancer

Kinases regulate many aspects of cell growth, movement and death

Attempts are being made to develop drugs to inhibit specific kinases to treat several diseases e.g., the drugs: Gleevec (imatinib)Iressa (gefitinib)