Molecular Biochemistry I

Biochemical Energetics

Contents of this page:
Free energy changes & equilibrium constants
Energy coupling
"High energy" bonds
Thermodynamics vs kinetics
Introduction to oxidation/reduction

The free energy change (DG) of a reaction determines its spontaneity. The free energy change (DG), and its relation to equilibrium constant, are discussed on p. 57-59 of Biochemistry 3rd Edition by Voet & Voet. A reaction is spontaneous if DG is negative (if the free energy of the products is less than the free energy of the reactants).

DG = change in free energy,
DGo' = standard free energy change (with 1 M reactants and products, at pH 7),
R = gas constant, T = absolute temperature.

Note that the standard free energy change (DGo') of a reaction may be positive, for example, and the actual free energy change (DG) negative, depending on cellular concentrations of reactants and products. Many reactions for which DGo' is positive are spontaneous because other reactions cause depletion of products or maintenance of high substrate concentrations.

At equilibrium, DG equals zero. Solving for DGo' yields the relationship at left.

K'eq, the ratio [C][D]/[A][B] at equilibrium, is called the equilibrium constant.

An equilibrium constant greater than one (more products than reactants at equilibrium) indicates a spontaneous reaction (negative DG').

The variation of equilibrium constant with DGo' is shown in the table below.

Keq DGo' (kJ/mol)

Starting with 1 M reactants and products, the reaction:

104 – 23 proceeds forward (spontaneous)
102 – 11 proceeds forward (spontaneous)
100 = 1 0 is at equilibrium
10–2 + 11 proceeds in reverse
10–4 + 23 proceeds in reverse

Energy coupling is discussed on p. 59-60 & 566-567.

Examples of different types of coupling:

A. Some enzyme-catalyzed reactions are interpretable as two coupled half-reactions, one spontaneous and the other non-spontaneous. At the enzyme active site, the coupled reaction is kinetically facilitated, while the individual half-reactions are prevented. The free energy changes of the half-reactions may be summed, to yield the free energy of the coupled reaction.

For example, in the reaction catalyzed by the Glycolysis enzyme Hexokinase, the two half-reactions are:

Coupled reaction: ATP + glucose ADP + glucose-6-P .. DGo' = -17 kJoules/mol
The structure of the enzyme active site, from which water is excluded, prevents the individual hydrolytic reactions, while favoring the coupled reaction. 

B. Two separate enzyme-catalyzed reactions occurring in the same cellular compartment, one spontaneous and the other non-spontaneous, may be coupled by a common intermediate (reactant or product).

A hypothetical, but typical, example involving pyrophosphate:

Overall: A + ATP + H2O B + AMP  +  2Pi ... DGo' = –18 kJ/mol

Pyrophosphate (PPi) is often the product of a reaction that needs a driving force. Its spontaneous hydrolysis, catalyzed by Pyrophosphatase enzyme, drives the reaction for which PPi is a product. For an example of such a reaction, see the discussion of cAMP formation below.

C. Ion transport may be coupled to a chemical reaction, e.g., hydrolysis or synthesis of ATP.

In the diagram at right and below, water is not shown. It should be recalled that the ATP hydrolysis/synthesis reaction is ATP + H2O ADP + Pi.

Equivalent to equation 20-3 on p. 727, the free energy change (electrochemical potential difference) associated with transport of an ion S across a membrane from side 1 to side 2 is represented below.

R = gas constant, T = temperature, Z = charge on the ion, F = Faraday constant, and DY = voltage across the membrane.

Since free energy changes are additive, the spontaneous direction for the coupled reaction will depend on the relative magnitudes of:

Two examples of such coupling are:

1. Active transport. Spontaneous ATP hydrolysis (negative DG) is coupled to (drives) ion flux against a gradient (positive DG). For an example, see the discussion of SERCA.

2. ATP synthesis in mitochondria. Spontaneous H+ flux across a membrane (negative DG) is coupled to (drives) ATP synthesis (positive DG). See the discussion of the ATP Synthase.

"High Energy" Bonds

The structure of ATP is shown below at right (see also p. 566). Anhydride bonds (in red) link the terminal phosphates.

Phosphoanhydride bonds (formed by splitting out water between two phosphoric acids or between a carboxylic acid and a phosphoric acid) tend to have a large negative DG of hydrolysis, and are thus said to be "high energy" bonds. It is important to realize that the bond energy is not necessarily high, just the free energy of hydrolysis.

"High energy" bonds are often represented by the "~" symbol (squiggle), with ~P representing a phosphate group with a high free energy of hydrolysis.

Compounds with "high energy" bonds are said to have high group transfer potential. For example, Pi may be spontaneously removed from ATP for transfer to another compound (e.g., to a hydroxyl group on glucose).

Potentially two "high energy" bonds can be cleaved, as two phosphates are released by hydrolysis from ATP (adenosine triphosphate), yielding ADP (adenosine diphosphate), and ultimately AMP (adenosine monophosphate). This may be represented as follows (omitting waters of hydrolysis):

Alternatively, as discussed above:

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 (non-spontaneous) reaction, as in the examples presented above.

AMP functions as an energy sensor and regulator of metabolism. When ATP production does not keep up with needs, a higher portion of a cell's adenine nucleotide pool is in the form of AMP. AMP then stimulates metabolic pathways that produce ATP.

Artificial ATP analogs have been designed that are resistant to cleavage of the terminal phosphate by hydrolysis, e.g., AMPPNP, depicted at right.

Such analogs have been used to study the dependence of coupled reactions on ATP hydrolysis. In addition, they have made it possible to crystallize an enzyme that catalyzes ATP hydrolysis with an ATP analog at the active site.

A reaction that is 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 the mitochondrial ATP Synthase, is ADP, while some cellular reactions dephosphorylate ATP all the way to AMP.

The enzyme Nucleoside Diphosphate Kinase (NuDiKi) equilibrates ~P among the various nucleotides that are needed, e.g., for synthesis of DNA and RNA. NuDiKi catalyzes reversible reactions such as:

ATP + GDP ADP + GTP  ,   ATP + UDP ADP + UTP  , etc.

Many organisms store energy as inorganic polyphosphate, a chain of many phosphate residues linked by phosphoanhydride bonds. It may be represented as: 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. For example, 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 free energy of hydrolysis? Contributing factors for ATP and PPi are thought to include:

Phosphocreatine (also called creatine phosphate), another compound with a "high energy" phosphate linkage, is used in nerve and muscle cells for storage of ~P bonds.

Creatine Kinase catalyzes:  

phosphocreatine + ADP ATP + creatine

This is a reversible reaction, though the equilibrium constant slightly favors phosphocreatine formation. Phosphocreatine is produced when ATP levels are high. When ATP is depleted during exercise in muscle, phosphate is transferred from phosphocreatine to ADP, to replenish ATP.

Phosphoenolpyruvate (PEP), involved in production of ATP in Glycolysis, has a larger negative DG of phosphate hydrolysis than ATP.

Removal of phosphate from the ester linkage in PEP is spontaneous because the enol product spontaneously converts to a ketone.

The ester linkage in PEP is an exception. Generally phosphate esters, formed by splitting out water between a phosphoric acid and a hydroxyl group, have a low but negative DG of hydrolysis. Examples, shown below, include:

  • the linkage between the first phosphate and the ribose hydroxyl of ATP.
  • the linkage between phosphate and a hydroxyl group in glucose-6-phosphate or glycerol-3-phosphate.
  • the linkage between phosphate and the hydroxyl group of an amino acid residue in a protein (serine, threonine or tyrosine). Regulation of proteins by phosphorylation and dephosphorylation will be discussed later. An example mentioned above is AMP-Activated Protein Kinase.

ATP has special roles in energy coupling and phosphate transfer. The free energy of hydrolysis of phosphate from ATP is intermediate among the examples listed in the table below (more complete table p. 566). ATP can thus act as a phosphate donor, and ATP can be synthesized by transfer of phosphate from other compounds, such as phosphoenolpyruvate (PEP).

Compound

DGo' 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

Some other "high energy" bonds:

A thioester forms between a carboxylic acid and a thiol (SH) group, e.g., the thiol of coenzyme A (abbreviated CoA-SH). 

Thioesters are "high energy" linkages. In contrast to phosphate esters, thioesters have a large negative DG of hydrolysis.

The thiol of coenzyme A can react with a carboxyl group of acetic acid (yielding acetyl-CoA) or a fatty acid (yielding fatty acyl-CoA). 

The spontaneity of thioester cleavage is essential to the role of coenzyme A as an acyl group carrier. Like ATP, acyl-coenzyme A has a high group transfer potential.

Coenzyme A includes b-mercaptoethylamine, in amide linkage to the carboxyl group of the B vitamin pantothenate

The hydroxyl of pantothenate is in ester linkage to a phosphate of ADP-3'-phosphate

The functional group is the thiol (SH) of b-mercaptoethylamine.

3',5'-Cyclic AMP (abbreviated cAMP), shown at right and below, is used by cells as a transient signal.

Adenylate Cyclase (Adenylyl Cyclase) catalyzes cAMP synthesis:
ATP
cAMP + PPi.
The reaction is highly spontaneous due to the production of PPi, which spontaneously hydrolyzes.

Phosphodiesterase catalyzes catalyzes hydrolytic cleavage of one of the phosphate ester linkages (in red), converting cAMP 5'-AMP. This is a highly spontaneous reaction, because cAMP is sterically constrained by having a phosphate with ester linkages to two hydroxyls of the same ribose. The lability of cAMP to hydrolysis makes it an excellent transient signalSignal roles of cAMP will be discussed separately.

Explore at right the structure of cAMP. The coordinate file for this display was obtained using CHEM 3D with MM2 energy minimization.

Right click to change the display to sticks or ball & stick.
Drag the image to view the molecular conformation, and compare to the structure diagram above. 

Identify the bond that is cleaved when cAMP is hydrolyzed to AMP.
Why is the cleavage of this bond by hydrolysis spontaneous?


   C   O   N   P   H   

Distinction between thermodynamics and kinetics: A high activation energy barrier usually causes  hydrolysis of a "high energy bond" to be very slow in the absence of an enzyme catalyst. This "kinetic stability" is essential to the role of ATP and other compounds with ~ bonds. If ATP would rapidly hydrolyze in the absence of a catalyst, it could not serve its important roles in energy metabolism and phosphate transfer. Phosphate is removed from ATP only when the reaction is coupled via enzyme catalysis to some other reaction useful to the cell, such as transport of an ion, phosphorylation of glucose, or regulation of an enzyme by phosphorylation of a serine residue.

Problems relating to bioenergetics are on the page with potential test questions.

Oxidation & reduction will be covered in more detail later. A brief introduction to selected topics will be presented here. The evolution of photosynthesis, and the generation of the oxygen that is now plentiful in our environment, allowed development of metabolic pathways that derive energy from transfer of electrons from various reductants ultimately to molecular oxygen.

Oxidation of an iron atom involves loss of an electron (to some acceptor atom):

Fe++ (reduced) Fe+++ (oxidized) + e-

For a carbon compound, increased oxidation means increased number of C-O bonds. Since electrons in a C-O bond are associated more with the oxygen, the C becomes relatively electron deficient as you go from hydrocarbon to CO2.

Oxidation of carbon is spontaneous (energy yielding).

Two important electron carriers in metabolism are NAD+ and FAD.

NAD+ (Nicotinamide Adenine Dinucleotide) functions as an electron acceptor in catabolic pathways. 

The nicotinamide ring  of NAD+, which is derived from the vitamin niacin, accepts 2 e- and one H+ (a hydride) in going to the reduced state, as NAD+ becomes NADH. See also p. 461 & 555.

NADP+/NADPH is similar, except for an additional phosphate esterified to a hydroxyl group on the adenosine ribose. NADPH functions as an electron donor in synthetic pathways.

The electron transfer reaction may be summarized as:

NAD+ + 2 e- + H+  NADH

It may also be written as:

NAD+ + 2 e- + 2H+  NADH + H+

FAD (Flavin Adenine Dinucleotide) also functions as an electron acceptor. The portion of FAD that undergoes reduction/oxidation is the dimethylisoalloxazine ring, derived from the vitamin riboflavin. See p. 556.

FAD normally accepts 2 e- and 2 H+ in going to its reduced state:
       FAD + 2 e- + 2 H+ 
  FADH2

Copyright 1998-2007 by Joyce J. Diwan. All rights reserved.

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