Biochemical Energetics

What is energy ?
The capacity for doing work.

Laws of Thermodynamics = laws of energy

Zeroth law
If two thermodynamic systems are each in thermal equilibrium with a
third, then they are in thermal equilibrium with each other

First law
Energy can neither be created nor destroyed. It can only change forms.
In any process in an isolated system, the total energy remains the
same.

Second law
Energy systems have a tendency to increase their entropy rather than
decrease it.
Enthalpy (H) - equals Energy plus Work at constant pressure -
which is the same as the change in heat at constant pressure
ΔH – mean variation of entalpy in a system and can be used in
biochemistry to see if a reaction it is thermodynamic posible.

Entropy (S) – means the rate of disorder in a system

ΔS – show how a system can be transformed in a more ordered or
disordered one.

Temperature T – is a measure of thermal agitation. It is expresed

only in Kelvin (K). The minimum T can not be lower than 0 K (-
273,16 Celsius).
ΔT – variation of temperature in a system.

ΔG – means free energy. This variation of energy in a biochemical

system predict the direction of reactions.
 Free energy of a reaction
The free energy change (DG) of a reaction determines
its spontaneity. A reaction is spontaneous if DG is
negative (if the free energy of products is less than that
of reactants).

For a reaction A + B  C + D
[C] [D]
DG = DG ' + RT ln
o
[A] [B]

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

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

DGo' of a reaction may be positive, & 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 concentration.
 [C] [D]
DG = DGº' + RT ln
At equilibrium [A] [B]
DG = 0.
[C] [D]
K'eq, the ratio  = DGº' + RT ln
[A] [B]
[C][D]/[A][B] at
equilibrium, is the [C] [D]
DGº' = - RTln
equilibrium constant. [A] [B]
An equilibrium constant [C] [D]
(K'eq) greater than one defining K'eq =
[A] [B]
indicates a spontaneous
reaction (negative DG'). DGº' = - RT ln K'eq
DGo' = - RT ln K'eq
Variation of equilibrium constant with DGo‘ (25 oC)

K'eq DG º' Starting with 1 M reactants &

kJ/mol products, the reaction:
4
10 - 23 proceeds forward (spontaneous)
2
10 - 11 proceeds forward (spontaneous)
100 = 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 DGo' = -31 kJ/mol
Pi + glucose  glucose-6-P + H2O DGo' = +14 kJ/mol
Coupled reaction:
ATP + glucose  ADP + glucose-6-P DGo' = -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 DGo' = + 15 kJ/mol
Enzyme 2:
PPi + H2O  2 Pi DGo' = – 33 kJ/mol
Overall spontaneous reaction:
A + ATP + H2O  B + AMP + 2 Pi 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.
 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
DG = R T ln + Z F DY
[S]1

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

F = Faraday constant, DY = 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

 DG for ion flux - varies with ion gradient & voltage.

 DG for chemical reaction - negative DGo' for ATP
hydrolysis; DG 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 DG) is coupled to (drives) ion flux against a
gradient (positive DG).
ATP synthesis: Spontaneous H+ flux (negative DG) is
coupled to (drives) ATP synthesis (positive DG).
 “High energy” bonds
NH 2
ATP
adenosine triphosphate N
N

O O O N N

-O P O P O P O CH2 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 DG 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 DG of hydrolysis.
"High energy" bonds are represented by the "~" symbol.
~P represents a phosphate group with a large negative DG
of hydrolysis. Mangesium ions are key factors.
 “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 group on glucose).
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
(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 N
N
analogs have
been designed O H O O N N
that are resistant -O P N P O P O CH 2
O
to cleavage of H H
O- O- O-
the terminal H H
OH OH
phosphate by
hydrolysis. AMPPNP (ADPNP) ATP 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.
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.
 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 DG
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
phosphate), another H
O
-
compound with a "high O P N C N CH2 C
energy" phosphate linkage, - O-
O NH2+
is used in nerve & muscle
for storage of ~P bonds. phosphocreatine

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.
 O O- O O- O O-
C C C
ADP ATP
C OPO32- C OH C O

CH2 H+ CH2 CH3

PEP enolpyruvate pyruvate

Phosphoenolpyruvate (PEP), involved in ATP synthesis

in Glycolysis, has a very high DG 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 DG of hydrolysis. Examples:
 the linkage between the first phosphate and the ribose
hydroxyl of ATP.
 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 DG of hydrolysis:
 the linkage between phosphate & a hydroxyl group
in glucose-6-phosphate or glycerol-3-phosphate.
 Protein Kinase O

Protein OH + ATP Protein O P O- + ADP

O-
Pi H2O
Protein Phosphatase

 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 & Pi transfer.
DG 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.
DGo' 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 DG of hydrolysis.
 O

Coenzyme A-SH + HO C CH3

acetic acid

Coenzyme A-S C CH3 + H2O

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 b-mercaptoethylamine

CH2

b-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 O
O- O- H H
the thiol (SH) of H H
b-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 H2 O
due to the production of PPi, which 5' C 4'
H H 1'
spontaneously hydrolyzes. O
H 3' 2' H
Phosphodiesterase catalyzes P O OH
O
hydrolytic cleavage of one Pi ester 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 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
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 O O

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

Increasing oxidation of carbon

Oxidation of carbon is spontaneous (energy-yielding).

Two important e- carriers in metabolism: NAD+ & FAD.
NAD+, Nicotinamide
Adenine Dinucleotide, Nicotinamide H O

is an electron acceptor Adenine C

Dinucleotide NH 2
in catabolic pathways. O +
-
N
The nicotinamide ring, O P O CH 2
O
nicotinamide
derived from the vitamin H H
H H
niacin, accepts 2 e- & 1 OH OH
H+ (a hydride) in going O NH 2
to the reduced state, N
N
NADH.
- N N adenine
NADP+/NADPH is O P O CH 2
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
NH2 NH2

+
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+
dimethylisoalloxazine O O
H H H
C N C - + C N C
H3C C C C NH 2e +2H H3C C C C NH

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 Adenine
HC OH O O HC OH O 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.