Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.

The free energy change (
D
G ) of a reaction determines its spontaneity. A reaction is spontaneous if
D
G is negative (if the free energy of products is less than that of reactants).
For a reaction A + B  C + D
D
G =
D
G o
' + RT ln
[ C ] [ D ]
[ A ] [ B ]
D
G o ' = standard free energy change (at pH 7, 1M reactants & products); R = gas constant; T = temp.

For a reaction A + B  C + D
D
G =
D
Gº' + RT ln
[ C ] [ D ]
[ A ] [ B ]
D
G o ' of a reaction may be positive, &
D
G negative , depending on cellular concentrations of reactants and products.
Many reactions for which
D
G o ' is positive are spontaneous because other reactions cause depletion of products or maintenance of high substrate concentration.

At equilibrium
D
G = 0.
K' eq
, the ratio
[C][D] / [A][B] at equilibrium, is the equilibrium constant .
D
G =
D
Gº' + RT ln
[ C ] [ D ]
[ A ] [ B ]



=
D
Gº' + RT ln
[ C ] [ D ]
[ A ] [ B ]
D
Gº' = RTln
[ C ] [ D ]
[ A ] [ B ]
An equilibrium constant
( K' eq
) greater than one indicates a spontaneous reaction (negative
D
G

').
defining K' eq
=
D
Gº' =
[ C ] [ D ]
[ A ] [ B ]
RT ln K' eq

D
G o ' =
-
RT ln K' eq
Variation of equilibrium constant with
D
G o ‘ (25 o C)
eq
D
kJ/mol
Starting with 1 M reactants & products, the reaction:
10
4
23 proceeds forward (spontaneous)
10
2
11 proceeds forward (spontaneous)
10
0
= 1 0 is at
10
-
2 + 11 reverses to form “reactants”
10
-
4 + 23 reverses to form “reactants”


A spontaneous reaction may drive a non-spontaneous reaction.

Free energy changes of coupled reactions are additive .
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 individual half-reactions are prevented.

Free energy changes of 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 half-reactions are:
ATP + H
2
O  ADP + P i
P i
D
G o ' =
-
31 kJ/mol
+ glucose  glucose-6-P + H
2
O
D
G o ' = +14 kJ/mol
Coupled reaction:
ATP + glucose  ADP + glucose-6-P
D
G o ' =
-
17 kJ/mol
The structure of the enzyme active site, from which H
2
O is excluded, prevents the individual hydrolytic reactions, while favoring the coupled reaction.

B.
Two separate reactions, occurring in the same cellular compartment, one spontaneous and the other not, may be coupled by a common intermediate (reactant or product).
A hypothetical, but typical, example involving PP i
:
Enzyme 1:
A + ATP  B + AMP + PP i
Enzyme 2:
PP i
+ H
2
O  2 P i
Overall spontaneous reaction:
A + ATP + H
2
O  B + AMP + 2 P i
D
D
G
D
G
G o o
' = + 15 kJ/mol o ' = – 33 kJ/mol
' = – 18 kJ/mol
Pyrophosphate (PP i
) is often the product of a reaction that needs a driving force.
Its spontaneous hydrolysis, catalyzed by Pyrophosphatase enzyme, drives the reaction for which PP i is a product.

Ion Transport may be coupled to a chemical reaction, e.g., hydrolysis or synthesis of ATP.
In this diagram & below, water is not shown. It should be recalled that the ATP hydrolysis/synthesis reaction is: ATP + H
2
O

ADP + P i
.
S
1
Side 1
ADP + P i
S
2
ATP
Side 2

S
1
S
2
Side 1 Side 2
The free energy change (electrochemical potential difference) associated with transport of an ion S across a membrane from side 1 to side 2 is:

D
[S]
[S]
1
2
G = R T ln + Z F
DY
R = gas constant, T = temperature, Z = charge on the ion,
F = Faraday constant,
DY
= voltage.

Since free energy changes are additive, the spontaneous direction for the coupled reaction will depend on relative magnitudes of:
S
1
Side 1
ADP + P i
ATP
Side 2
S
2
 D
G for ion flux - varies with ion gradient & voltage.
 D
G for chemical reaction - negative
D
G o ' for ATP hydrolysis;
D
G depends also on [ATP], [ADP], [P i
].

S
1 active transport
ADP + P i
S
2
ATP
H
+
1
ATP synthesis
ADP + P i
ATP
H
+
2
Two examples:
Active Transport : Spontaneous ATP hydrolysis
(negative
D
G) is coupled to (drives) ion flux against a gradient (positive
D
G).
ATP synthesis : Spontaneous H + flux (negative
D
G) is coupled to (drives) ATP synthesis (positive
D
G).

NH
2
ATP adenosine triphosphate
N
N
N
O O O N adenine
-O P O P O P O CH
2
O
H H
OOOphosphoanhydride bonds ( ~ )
H
OH OH
H ribose
Phosphoanhydride bonds (formed by splitting out H
2
O between 2 phosphoric acids or between carboxylic & phosphoric acids) have a large negative
D
G of hydrolysis .

ATP adenosine triphosphate adenine
N
NH
2
N
-O
N
O
P
O
P
O
P O O
OOphosphoanhydride
Obonds ( ~ )
O CH
2
H
H
OH
O
H
OH
H ribose
N
Phosphoanhydride linkages are said to be "high energy" bonds .
Bond energy is not high, just
D
G of hydrolysis.
"High energy" bonds are represented by the " ~ " symbol.
~ P represents a phosphate group with a large negative
D
G 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).

Potentially, 2 ~ P bonds can be cleaved, as 2 phosphates are released by hydrolysis from ATP .
AMP ~ P ~ P

AMP ~ P + P i
AMP ~ P

AMP + P i
(ATP

ADP + 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
(non-spontaneous) reaction. (Examples presented earlier.)

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 .

NH
2
Artificial ATP analogs have been designed that are resistant to cleavage of the terminal phosphate by hydrolysis.
-O
O
P
O-
H
N
O
P
O-
O
O
P
O-
O CH
2
H
H
OH
O
H
N
N
OH
H
AMPP N P (ADP N P) ATP analog
N
Example: AMPP N P.
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.
N

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 ( NuDiKi ) 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.

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 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 P i
, a chelator of metal ions, a buffer, or a regulator.

Why do phosphoanhydride linkages have a high
D
G 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 separation of the phosphates.

Phosphocreatine (creatine phosphate), another
O CH
3 compound with a "high
-
O P
H
N C N CH
2 energy" phosphate linkage,
O
-
NH
2
+ is used in nerve & muscle for storage of ~P bonds . phosphocreatine
Creatine Kinase catalyzes:
Phosphocreatine + ADP

ATP + creatine
C
O
O
-
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 .

O O
-
O O
-
O O
-
C C C
ADP ATP
C O PO
3
2
-
C OH C O
CH
2
H
+
CH
2
CH
3
PEP enolpyruvate pyruvate
Phosphoenolpyruvate (PEP), involved in ATP synthesis in Glycolysis, has a very high
D
G of P i hydrolysis.
Removal of P i from ester linkage in PEP is spontaneous because the enol spontaneously converts to a ketone.
The ester linkage in PEP is an exception.

NH
2
N
N
O O ester linkage
O
N
N adenine
-O P O P O P O CH
2
O
OOO-
H
ATP (adenosine triphosphate)
H
OH
H
OH
H ribose
Generally phosphate esters , formed by splitting out water between a phosphoric acid and an OH group, have a low but negative
D
G of hydrolysis . Examples:
 the linkage between the first phosphate and the ribose hydroxyl of ATP.

O
6
CH
2
O P OH
5
OH
O
H H
4
H
OH H
1
OH
2
OH
3
H OH glucose-6-phosphate
CH
2
OH
HO CH O
CH
2
O P O
-
O
glycerol-3-phosphate
Other examples of phosphate esters with low but negative
D
G of hydrolysis:
 the linkage between phosphate & a hydroxyl group in glucose-6-phosphate or glycerol-3-phosphate .

Protein Kinase
Protein OH + ATP
O
Protein O P
O
-
P i
H
2
O
Protein Phosphatase
O
-
+ ADP
 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.

ATP has special roles in energy coupling & P i transfer.
D
G of phosphate hydrolysis from ATP is intermediate among examples below.
ATP can thus act as a P i by P i donor, & ATP can be synthesized transfer, e.g., from PEP.
Compound
Phosphoenolpyruvate (PEP)
Phosphocreatine
Pyrophosphate
ATP (to ADP)
Glucose-6-phosphate
Glycerol-3-phosphate
D
G o
' of phosphate hydrolysis, kJ/mol
-
-
-
-
-
-

Some other
“high energy” bonds:
O
Coenzyme A-S H + HO C R
O
Coenzyme A-S C R + H
2
O
A thioester forms between a carboxylic acid & a thiol
(SH), e.g., the thiol of coenzyme A (abbreviated CoA-SH).
Thioesters are ~ linkages. In contrast to phosphate esters, thioesters have a large negative
D
G of hydrolysis.

O
Coenzyme A-S H + HO C CH
3 acetic acid
O
Coenzyme A-S C acetyl-CoA
CH
3
+ H
2
O
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, CoA has a high group transfer potential.

SH
CH
2
Coenzyme A includes b
-mercaptoethylamine , in amide linkage to the carboxyl group of the B vitamin pantothenate .
CH
2
NH
C
CH
2
O b
-mercaptoethylamine pantothenate
CH
2
The hydroxyl of pantothenate is in ester linkage to a phosphate of ADP-3'-phosphate .
HO
H
3
C
NH
C
C
C
O
H
CH
3
O O
N
H
2
C O P O P O CH
2 The functional group is the thiol (SH) of b
-mercaptoethylamine.
O
-
O
H
ADP-3'-phosphate
-
O
H
O
P
Coenzyme A
O
NH
2
N
O
O
-
H
OH
H
N
N

3',5'-Cyclic AMP ( cAMP ), is used by cells as a transient signal. cAMP NH
2
N
N
Adenylate Cyclase catalyzes cAMP synthesis: ATP
 cAMP + PP i
.
The reaction is highly spontaneous due to the production of PP spontaneously hydrolyzes. i
, which
Phosphodiesterase catalyzes hydrolytic cleavage of one P i ester
(red), converting cAMP

5'-AMP .
N
N
O
5'
H
2
C 4'
H
P
H
O
3'
O
O-
O
H
2'
H
OH
1'
This is a highly spontaneous reaction, because cAMP is sterically constrained by having a phosphate with ester links to 2 hydroxyls of the same ribose. The lability of cAMP to hydrolysis makes it an excellent transient signal .

List compounds exemplifying the following roles of "high energy" bonds:

Energy transfer or storage
ATP, PP i
, polyphosphate, phosphocreatine

Group transfer
ATP, Coenzyme A

Transient signal cyclic AMP

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.

Oxidation & reduction will be covered in more detail later.
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 an acceptor): Fe ++ (reduced)

Fe +++ (oxidized) + e -
Since electrons in a C-O bond are associated more with O, increased oxidation of a C atom means increased number of C-O bonds.
H H O
H C H H C OH
H
C
H H
Increasing oxidation of carbon
H H
O
C
OH
O
C
O
Oxidation of carbon is spontaneous ( energy-yielding ).
Two important e
carriers in metabolism: NAD + & FAD .

NAD + , N icotinamide
A denine D inucleotide, is an electron acceptor in catabolic pathways.
The nicotinamide ring, derived from the vitamin niacin , accepts 2 e & 1
H + (a hydride) in going to the reduced state,
NADH .
N icotinamide
A denine
D inucleotide
O
-
O P O CH
2
O
H
H
OH
N
O
NH
2
H
H
OH
H
N
O
C
NH
2
+
N nicotinamide
-
O P O
N adenine
NADP + /NADPH is similar except for P i
.
NADPH is e
donor in synthetic pathways.
O
CH
2
H
H
OH
N
O
H
OH
H esterified to
P i
in NADP
+

H
+
N
R
NAD
+
+
O
C
NH
2

2 e
-
+ H
+
H
N
R
NADH
H
O
C
NH
2
The electron transfer reaction may be summarized as :
NAD + + 2e
-
+ H +

NADH.
It may also be written as:
NAD + + 2e
-
+ 2H +

NADH + H +

dimethylisoalloxazine
O
H
C N C
H
3
C C C C
H
3
C C C C
C
H
N N
CH
2
HC OH
NH
C O
2 e
-
+ 2 H
+
H
3
C
H
3
C
FAD
HC
HC
H
2
C
OH
OH O O
O P O P O
OO-
Adenine
Ribose
O
H
C
H
N C
C C C NH
C
C
H
C C
N
CH
2
HC OH
N
H
C O
FADH
2
HC
HC
OH
OH O O
H
2
C O P O P O
OO-
Adenine
Ribose
FAD ( F lavin A denine D inucleotide), derived from the vitamin riboflavin , functions as an e
acceptor.
The dimethylisoalloxazine ring undergoes reduction/oxidation.
FAD accepts 2 e
-
FAD + 2 e
-
+ 2 H + in going to its reduced state:
+ 2 H +

FADH
2


NAD + is a coenzyme , that reversibly binds to enzymes.

FAD is a prosthetic group , that remains tightly bound at the active site of an enzyme.