Biochemistry of Metabolism

Biochemical Energetics

Copyright © 1999-2004 by Joyce J.

Diwan.
All rights reserved.
 Free energy of a reaction
The free energy change (G) of a reaction determines
its spontaneity. A reaction is spontaneous if G is
negative (if the free energy of products is less than that
of reactants).

For a reaction A + B  C + D

o [C] [D]
G = G ' + RT ln
[A] [B]

Go' = standard free energy change (at pH 7, 1M

reactants & products); R = gas constant; T = temp.
 For a reaction A + B  C + D
[C] [D]
G = Gº' + RT ln
[A] [B]

Go' of a reaction may be positive, & G negative,

depending on cellular concentrations of reactants and
products.
Many reactions for which Go' is positive are
spontaneous because other reactions cause depletion of
products or maintenance of high substrate concentration.
 [ C] [ D]
G = Gº' + RT ln
At equilibrium [A] [B]
G = 0.
[C] [D]
K'eq, the ratio [C][D]/[A]  = Gº' + RT ln [A] [B]
[B] at equilibrium, is the
equilibrium constant. [C] [D]
Gº' = - RTln
[A] [B]
An equilibrium constant
(K'eq) greater than one [ C] [ D]
defining K'eq =
indicates a spontaneous [A] [B]
reaction (negative G').
Gº' = - RT ln K'eq
Go' = RT ln K'eq
Variation of equilibrium constant with Go‘ (25 oC)

K'eq G º' Starting with 1 M reactants &

kJ/mol products, the reaction:
4
10 - 23 proceeds forward (spontaneous)
2
10 - 11 proceeds forward (spontaneous)
0
10 = 1 0 is at equilibrium
-2
10 + 11 reverses to form “reactants”
-4
10 + 23 reverses to form “reactants”
 Energy coupling
 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 + H2O  ADP + Pi Go' = 31 kJ/mol
Pi + glucose  glucose-6-P + H2O Go' = +14 kJ/mol
Coupled reaction:
ATP + glucose  ADP + glucose-6-P Go' = 17 kJ/mol

The structure of the enzyme active site, from which H2O

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 PPi:
Enzyme 1:
A + ATP  B + AMP + PPi Go' = + 15 kJ/mol
Enzyme 2:
PPi + H2O  2 Pi Go' = – 33 kJ/mol
Overall spontaneous reaction:
A + ATP + H2O  B + AMP + 2 Pi Go' = – 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.
 Energy coupling in ion transport

Ion Transport may be

coupled to a chemical
reaction, e.g., hydrolysis or ADP + Pi
synthesis of ATP.
S1 S2
In this diagram & below,
water is not shown. It should ATP
be recalled that the ATP
Side 1 Side 2
hydrolysis/synthesis reaction
is: ATP + H2O  ADP + Pi.
 S1 S2

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:

[S]2
G = R T ln + Z F 
[S]1

R = gas constant, T = temperature, Z = charge on the ion,

F = Faraday constant,  = voltage.
Since free energy changes ADP + Pi
are additive, the
spontaneous direction S1 S2
for the coupled reaction
will depend on relative ATP
magnitudes of: Side 1 Side 2

 G for ion flux - varies with ion gradient & voltage.

 G for chemical reaction - negative Go' for ATP
hydrolysis; G depends also on [ATP], [ADP], [Pi].
 ADP + Pi ADP + Pi

S1 S2 H +
1 H+2
active ATP
transport ATP synthesis ATP

Two examples:
Active Transport: Spontaneous ATP hydrolysis
(negative G) is coupled to (drives) ion flux against a
gradient (positive G).
ATP synthesis: Spontaneous H+ flux (negative G) is
coupled to (drives) ATP synthesis (positive G).
 “High energy” bonds
NH 2
ATP
adenosine triphosphate N
N

O O O N N

-O P O P O P O CH 2
adenine
O
O- O- O- H H
H H
phosphoanhydride OH OH
bonds (~) ribose

Phosphoanhydride bonds (formed by splitting out H2O

between 2 phosphoric acids or between carboxylic &
phosphoric acids) have a large negative G of hydrolysis.
 NH 2
ATP adenine
adenosine triphosphate N
N

O O O N N

-O P O P O P O CH 2
O
O- O- O- H H ribose
phosphoanhydride H H
bonds (~) OH OH

Phosphoanhydride linkages are said to be "high energy"

bonds. Bond energy is not high, just G of hydrolysis.
"High energy" bonds are represented by the "~" symbol.
~P represents a phosphate group with a large negative G
of hydrolysis.
 “High energy” bonds

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 on glycerol.
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)
 NH 2
Artificial ATP N
analogs have N

been designed O H O O N N
that are resistant -O P N P O P O CH 2
to cleavage of H
O
H
O- O- O-
the terminal H H
OH OH
phosphate by
hydrolysis. A M PP N P (A D PN P) A T P analog

Example: AMPPNP.
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.
 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 G
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.
Phosphocreatine (creatine O CH3
O
phosphate), another 
O P
H
N C N CH2 C
compound with a "high O 
 +
energy" phosphate linkage, O NH2

is used in nerve & muscle p h o s p h o c re a tin e

for storage of ~P bonds.
Creatine Kinase catalyzes the reversible reaction:  
Phosphocreatine + ADP  ATP + creatine
Phosphocreatine is produced when ATP levels are high.
During exercise in muscle, phosphate is transferred from
phosphocreatine to ADP, to replenish ATP.
Phosphocreatine may also be used to transport ~P from
one compartment of a cell to another.
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.
 O O O O O O
C C C
ADP ATP
2
C O PO 3 C OH C O

CH2 H+ CH2 CH3

PEP en o lp yru v a te p yru v a te

Phosphoenolpyruvate (PEP), involved in ATP synthesis

in Glycolysis, has a very high G of Pi hydrolysis.
Removal of Pi from ester linkage in PEP is spontaneous
because the enol spontaneously converts to a ketone.
The ester linkage in PEP is an exception.
 NH2

N
N

ester linkage
O O O N N
adenine
-O P O P O P O CH2
O
O- O- O- H H
ribose
H H
ATP (adenosine triphosphate) OH OH

Generally phosphate esters (formed by splitting out

water between a phosphoric acid and an OH group) have
a low but negative G of hydrolysis. Examples:
 the linkage between the first phosphate of ATP & the
ribose hydroxyl
 O
6 CH O P OH
2
CH2 OH
OH
5 O
H H HO CH O
H
4 H 1
OH
CH2 O P O
OH OH
3 2
O
H OH
glucose-6-phosphate glycerol-3-phosphate

Other examples of phosphate esters with low but

negative G of hydrolysis:
 the linkage between phosphate & a hydroxyl group
in glucose-6-phosphate or glycerol-3-phosphate.
ATP has special roles in energy coupling & Pi transfer.
G of phosphate hydrolysis from ATP is intermediate
among examples below.
ATP can thus act as a Pi donor, & ATP can be synthesized
by Pi transfer, e.g., from PEP.
Go' of phosphate
Compound hydrolysis, kJ/mol
Phosphoenolpyruvate (PEP) 
Phosphocreatine 
Pyrophosphate 
ATP (to ADP) 
Glucose-6-phosphate 
Glycerol-3-phosphate 
 O
Some other
“high energy” Coenzyme A-SH + HO C R
bonds:

Coenzyme A-S C R + H2O

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 G of hydrolysis.
 O

Coenzym e A -S H + HO C CH 3
acetic acid

C oenzym e A -S C CH 3 + H 2 O
acetyl-CoA

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

CH2

Coenzyme A includes -mercaptoethylamine

CH2

-mercaptoethylamine, NH

in amide linkage to the C O

carboxyl group of the B CH2

vitamin pantothenate. CH2

pantothenate

The hydroxyl of NH
NH2

pantothenate is in ester C O
N
N
linkage to a phosphate HO C H

of ADP-3'-phosphate. H3C C CH3 O O N N

H2C O P O P O CH2
The functional group is H
O
H
O O
the thiol (SH) of H H
-mercaptoethylamine. ADP-3'-phosphate

O OH

O P O

Coenzyme A O
 cAMP NH2
3',5'-Cyclic AMP (cAMP), is used by
cells as a transient signal. N
N
Adenylate Cyclase catalyzes cAMP
synthesis: ATP  cAMP + PPi. N N

The reaction is highly spontaneous due H2 O

to the production of PPi, which 5' C 4'
H H 1'
spontaneously hydrolyzes. O
H 3' 2' H
Phosphodiesterase catalyzes P O OH
hydrolytic cleavage of one Pi ester O
O-
(red), converting cAMP  5'-AMP.
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, PPi, polyphosphate, phosphocreatine

 Group transfer
ATP, Coenzyme A

 Transient signal
cyclic AMP
 Kinetics vs Thermodynamics

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 metabolic roles.
Phosphate is removed from ATP only when the reaction
is coupled to some other reaction useful to the cell, such
as transport of ions or phosphorylation of glucose.
 Oxidation & reduction
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. Oxidation of C is spontaneous.

H H O O O

H C H H C OH C C C
H H H OH
H H O

In c r e a s in g o x i d a t i o n o f c a r b o n
NAD+, Nicotinamide
Adenine Dinucleotide, Nicotinamide H O
Adenine C
is an electronacceptor Dinucleotide NH2
in catabolic pathways. O +
N
The nicotinamide ring,

O P O CH2
O
nicotinamide
derived from the H H
H H
vitamin niacin, accepts OH OH
2 e- & 1 H+ (a hydride) O NH2
in going to the reduced N
N
state, NADH.
NADP+/NADPH is 
O P O CH2
N N adenine
O
similar except for Pi. O H H
NADPH is e donor in H H esterified to
OH OH Pi in NADP+
synthetic pathways.
 NAD+/NADH
H O O
H H
C C
NH 2 NH 2

+
N  2 e  + H+ N

R R
NAD + NADH

The electron transfer reaction may be summarized as :

NAD+ + 2e + H+  NADH.
It may also be written as:
NAD+ + 2e + 2H+  NADH + H+
dimethylisoalloxazineO O
H H H
C N C  + C N C
H3C C C C NH 2e +2H H C C C C NH
3

H3C C C C C O H3C C C C C O
C N N C N N
H H H
CH2 CH2

HC OH HC OH

HC OH HC OH
FAD Adenine
FADH2
HC OHO O Adenine
HC OHO O

H2C O P O P O Ribose H2C O P O P O Ribose

O- O- O- O-

FAD (Flavin Adenine Dinucleotide), derived from the

vitamin riboflavin, functions as an e acceptor. The
dimethylisoalloxazine ring undergoes reduction/oxidation.
FAD accepts 2 e- + 2 H+ in going to its reduced state:
FAD + 2 e- + 2 H+  FADH2
 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.