Lecture 2: Thermodynamics review

You should have read chapter 1 in Lehninger by now

It would be a good idea to review chapters 2,3,4, and 5

Suggested reading: chapter 13 – sections 13.1 and 13.2

Answer the following questions at the end of chapter 13:

1, 4,6, 8, 10
Again, a reminder that while the
answers to these questions are found at
the back of your textbook, for exam
purposes, you need to be able to show
you can solve these problems
* MBB 321 1
Bioenergetics

A subset of thermodyamics (the science of energy transformations)

The quantitative analysis of how organisms gain and use energy

Cells require energy for all of their

functions
• Living
• Growing
• Reproducing
• Movement
• And anything else that might not
be covered by the above

Maintaining life requires the

constant investment of energy

* MBB 321 2
Organisms transform energy and matter from their surroundings

What is the system and what is the surroundings?

a system is something that we define
• Biochemical reaction
• Metabolic pathway
• An individual cell
• An organism

The surroundings are everything else in the universe

system + surroundings = universe

Living organisms are generally open systems

Open system: energy (heat) exchange and/or matter exchange may occur
Living organisms exchange both energy and matter with their surroundings
* MBB 321 3
First law of thermodynamics

The total amount of energy in the universe remains constant:

(the form of energy can change, but energy cannot be created or destroyed)

ΔE total (universe) = ΔE system + ΔE surroundings

Cells interconvert energy

Kinetic energy: the energy of movement

• thermal energy
• radiant energy Stretch a rubber band and you
• mechanical energy give it potential energy
• electric energy Release the stretched rubber band
and the potential energy is
converted to kinetic energy

Potential energy: stored energy

• chemical potential energy (bond energy)
• concentration gradients
• electric potential (charge separation)

* MBB 321 4
 First law of thermodynamics

The total energy of a system is a constant: energy is conserved

(can be gained or lost (and changed) but not created or destroyed)

E = internal energy of a system:

all the energies that might be exchanged in physical or chemical processes
(including the energy stored in covalent and noncovalent bonds)

note that internal energy depends only on the present state of a

system and not how the system got there (independent of path)
= State Function

How does E change?

If energy flows into or out of the system in the form of heat or work
For a process that converts one state into another state (∆E)
∆E = E2 – E1 = q + w
q is the heat absorbed by the system from the surroundings; w is the work done on
the system by the surroundings (q - w = work done by the system)

* MBB 321 5
first law cont - Enthalpy

∆E = E2 – E1 = q + w

If volume is constant then ∆E is the heat exchanged at constant volume: ∆E = q

biochemical processes are usually carried out at constant pressure

and not constant volume (and in constant pressure reactions
∆E is not necessarily equal to the heat transfer)

A function was created for constant pressure processes = Enthalpy (H)

∆E = heat transferred in a constant volume process

∆H = heat transfer in a constant pressure process

Enthalpy (H): can be measured and reflects the number and kinds of
chemical bonds in reactants and products

Exothermic: chemical reaction that releases heat: ∆H is negative (-)

Endothermic: chemical reaction that takes up heat: ∆H is positive (+)
The First Law of Thermodynamics keeps track of energy changes
(bookkeeping), but it does not tell us if a reaction is favorable/spontaneous
* 6
MBB 321
First law of thermodynamics

The total energy of a system is a

constant: energy is conserved

Energy can be gained or lost - and

changed - but not created or
destroyed

The First Law of Thermodynamics

keeps track of energy changes
(bookkeeping), but it does not tell us if
a reaction is favorable/spontaneous

* MBB 321 7
Second law of thermodynamics

Indicates which processes are spontaneous (favorable)

Example: If you layer water on top of sucrose in a beaker what will

happen over time?

Will the reverse ever happen?

What is this telling us?

Systems of molecules have a natural tendency to randomize

* MBB 321 8
 Entropy (S): a measure of a systems disorder or randomness

Reversing the tendency

toward disorder requires
an intentional effort and
an input of energy
It is not spontaneous

If entropy is a measurement
of disorder (with more
disorder equaling greater
entropy) which room has the
greatest entropy?

Entropy (S): measurement of disorder (randomness) of a system or

the surroundings
low-entropy state – is an organized or ordered state
high-entropy state – is a disordered state 9
* MBB 321
Second law of thermodynamics
systems tend to proceed from ordered to disordered states (and the
entropy of the universe increases)

Does the formation of highly ordered biological structures violate the 2 nd

law of thermodynamics? Why or why not?

No: a cell/organism is not an isolated system

While it takes in energy to create order within itself, in the course of the
chemical reactions that generate order, part of the energy used is released
back into the system so the disorder of the surroundings is increased 10
* MBB 321
Gibbs free energy:

Enthalpy will tell us the heat content (energy) of a system

Entropy will tell us whether a process is favorable (spontaneous)

BUT: We still need to know the direction of a thermodynamically

favorable process

In a biological system where energy can be exchanged with the

surroundings, both enthalpy and entropy are important in
determining the direction of a thermodynamically favorable process

Changes in enthalpy (∆H) and changes in entropy (∆S) are related by the

Gibbs free energy: A combination of the first and second laws of thermodynamics

For any process (A B) at constant pressure and temperature

The free energy change is defined as:

∆G = ∆H – T∆S (T = absolute temperature)

* 11
MBB 321
 Gibbs free energy: summary
A reaction can occur spontaneously (is favored) only if ∆G is negative
If: ∆G is negative (-)
• the process is exergonic
• the reaction proceeds with the release of free energy
• the reaction will be thermodynamically favorable in the direction written
A reaction cannot occur spontaneously (is not favored) if ∆G is positive
If: ∆G is positive (+)
• the process is endergonic
• an input of free energy is required to drive the reaction
• the reaction will be thermodynamically unfavored
(reverse process is favored)

A system is at equilibrium and no net change can take place if ∆G is zero

If: ∆G is zero (0)
• The process is at equilibrium
• No net flow in either the forward or the reverse direction
• Neither process is favored
• Enthalpy and entropy changes are exactly balanced

12
note that Gibbs free energy does not describe the rate of a reaction
 What is going on biochemically at equilibrium?

* 13
MBB 321
What is going on biochemically at equilibrium?
Concentration of products A B
K’eq=
Concentration of reactants
20 units of B = ?
K’eq=
20 units of A
2 units of B = ?
K’eq=
2 units of A

Therefore a K’eq of ___ means what?

There are equal ________ of A and B at equilibrium

K’eq=
20 units of B = ?
2 units of A
Therefore a K’e of ____ means what?
There is more of
q
________________ at equilibrium so the
biochemical reaction was proceeding in the ______ direction
and ∆Go’ is __________________

K’eq=
2 units of B = less than 1
20 units of A
There is more of A at equilibrium so the
biochemical reaction was not proceeding in the
forward direction and ∆Go’ is positive 14
* MBB 321
Equilibrium and steady state
Equilibrium: the state of a system in which no further net
change is occurring (∆G is zero (0)

Steady state: a “non-equilibrium” state of a

system through which matter is flowing and in
which all components remain at constant
concentrations

Equilibrium and steady state are often

used interchangeably but there is a
MBB 321
difference and equilibrium is a
15
* “special kind” of steady state
Steady state vs. equilibrium

The intracellular and extracellular

concentrations of sodium in your
body remain constant over time

Constant over time = steady state

But not necessarily equilibrium

there is a substantial net flux

of Na+ into the cell through
Na + channels in the cell
membrane, and there is an
opposite but equal net flux
of Na+ extruded from the cell
by the action of the
Na+K+ATPase

Is the ∆G zero (0)?

Thermodynamic equilibrium is achieved only when the cell is dead

(and this is technically a “special case” of a steady state) 16
 Dynamic Steady State and Homeostasis
Equilibrium: ΔG=0
Steady state: a (non-equilibrium) state of a system through which matter is
flowing and in which all components remain at constant concentration

Because equilibrium is a special case of a steady state a better

term for the above definition would be dynamic steady state
(to indicate a steady state that is not at equilibrium

Homeostasis: the maintenance

of dynamic steady state by
regulatory mechanisms that
compensate for external
changes

Homeostasis is NOT equilibrium

* MBB 321 To maintain (dynamic) steady state = regulation 17

How do we determine the Gibbs free energy for a reaction?

∆G = free energy change under

intracellular conditions
(non-standard )

∆Go’ : standard state free energy

change (or just standard free
energy change)
Reference value whereby the
intrinsic free energy changes in
different reactions can be
R = gas constant compared under equivalent (or
T = absolute temperature standard) circumstances

Standard conditions: when each of the

reactants is present at a concentration of:

1 M, 1 atm pressure, pH of 7.0

* MBB 321 18
 Standard free energy change and relationship to equilibrium constant (cont)

equation # 1

At equilibrium: ∆G = 0 so equation #1 becomes

[C]c [D]d [C]c [D]d

0= ∆Go’ + RT ln and rearranging ∆Go’ = - RT ln
[A]a [B]b [A]a [B]b
equation #2

The equilibrium constant under standard conditions is defined as

K’eq= [C]c [D]d equation # 3 The equilibrium constant can be measured

[A]a [B]b experimentally

How is the equilibrium constant measured experimentally?

• Mix 1 M concentrations of all reactants and products under standard conditions
25 oC, 1 atm pressure, pH 7.0

• Allow the reaction to come to equilibrium

• Measure the concentrations, at equilibrium of A,B,C and D
• Calculate K’eq using those concentrations
19
MBB 321
Standard free energy change and relationship to equilibrium constant (cont)

[C]c [D]d K’eq= [C]c [D]d

∆Go’ = - RT ln equation #2 equation # 3
[A]a [B]b [A]a [B]b

Substituting equation #3 into equation #2 gives

so now we have a direct (simple)
∆Go’ = -2.303 RT log K’ eq relationship between the
equation #4 equilibrium constant and the
OR
standard free energy change

(the standard free energy

change of a system is simply an
alternative mathematical way of
expressing its equilibrium
constant)

This relationship allows the standard-state free energy

change for any process to be determined if the equilibrium
constant is known and the equilibrium constant can be
determined experimentally
20
MBB 321
 Summary: relationship between equilibrium
constant and change in standard free energy

Note: the presence of a catalyst (enzyme) does NOT affect K’ eq

* MBB 321 21
Applying a free energy calculation to a biochemical process

glucose 6-phosphate fructose 6-phosphate this is the second step in glycolysis

• 1 M concentrations of each reagent are mixed under standard conditions

(with an enzyme to catalyze the reaction)

• When the reaction reaches equilibrium the concentrations are measured

K’eq = [fructose 6-phosphate] = 0.67M = 0.504

[glucose 6-phosphate] 1.33M

Now use equation #4 to calculate the standard free energy change

∆Go’ = -2.303 RT log K’ eq

∆Go’ = (-2.303) (8.315 J/mol) (298K) log 0.5

∆Go’ = + 1718 J/mol or + 1.718 kJ/mol

MBB 321 22
 What does the ∆Go’ we just calculated tell us about the reaction?

glucose 6-phosphate fructose 6-phosphate ∆Go’ = + 1.718 kJ/mol

It tells us that
• under standard conditions the reaction is endergonic

• that the system is not at equilibrium when G6P and F6P are both at 1 M

• in fact, the reverse reaction is favored under these conditions

because ∆Go’ is positive

• therefore the equilibrium must lie to the left, with a higher concentration of G6P
than F6P
The concentrations of products and reactants are
fractions of the total amount of product and reactant

at equilibrium 1.33 x 100 = 66.5%

But does this reflect what 2
is happening in a cell? G6P F6P
0.67 x 100 = 33.5%
(66.5%) (33.5%) 2

Using standard conditions is a

great way to compare things
but it does not necessarily
reflect reality
MBB 321 23
In the cell, conditions are not standard

glucose 6-phosphate fructose 6-phosphate ∆Go’ = + 1.718 kJ/mol

In a living cell the F6P is “pulled” to the right by the next reaction and
the Keq (in a cell type that the concentrations of G6P and F6P were
determined) is actually 0.2
Keq = [FGP] = 0.2
[G6P]

We can now calculate the ∆G of this reaction (as opposed to the ∆Go’ )

∆G = ∆Go’ + RT ln 0.2
= 1,718 J/mol + (8.315 J/mol) (310K) (-1.609)
= - 2,498 J/mol or –2.5 kJ/mol

so even though the ∆Go’ is unfavorable for this reaction

the ∆G is actually exergonic (negative) and in the cell is therefore favorable

Every metabolic pathway must be, overall, thermodynamically favorable

By siphoning off a product for another reaction, the products and reactants of
the first reaction are at concentrations that do not reach equilibrium, therefore
forcing the first reaction in the direction of product

these two reactions are coupled MBB 321 24

Many biochemical reactions are unfavorable under standard conditions

K’eq= less than 1

∆Go’ is positive

Are conditions in a cell standard?

By siphoning off a product for another reaction, the products and reactants
of the first reaction are at concentrations that do not reach equilibrium,
therefore forcing the first reaction in the direction of product

these two reactions are coupled

The free energy changes of coupled reactions are additive

Rx1 A B+X ΔG = + 5 kJ/mol Note ΔG and not ΔGo’

Rx2 X Y+Z ΔG = -10 kJ/mol

A B+Z ΔG = -5 kJ/mol
* MBB 321 25
Coupling of biochemical reactions through the use of phosphate
containing compounds
Example: the synthesis of glucose 6-phosphate
( the first step in glucose utilization by many organisms)

glucose + Pi glucose 6-phosphate ∆Go’ = + 13.8 kJ/mol

What does the positive ∆Go’ indicate?

ATP + H20 ADP + Pi ∆Go’ = -30.5 kJ/mol

What does the negative ∆Go’ indicate?

Do these two reactions

share any intermediates?

MBB 321 26
*
Coupling of biochemical reactions through the use of phosphate
containing compounds - continued

These two reactions share an

intermediate (Pi) and may be
expressed as sequential
(coupled) reactions

glucose + Pi glucose 6 -phosphate ∆Go’ = + 13.8 kJ/mol

ATP + H20 ADP + Pi ∆Go’ = - 30.5 kJ/mol
ATP + glucose ADP + glucose 6-phosphate ∆Go’ = -16.7 kJ/mol

The energy stored in the bonds of ATP is used to drive the synthesis of glucose
6-phosphate even though its formation from glucose and phosphate is endergonic

In thermodynamic calculations, all that matters is the state of the system at the
beginning of the process and its state at the end 27
MBB 321
Coupling of biochemical reactions: summary

* MBB 321 28
The free energy change for the hydrolysis of ATP is large and negative
This equilibrium lies so far to the right
that ATP hydrolysis can be considered
essentially irreversible
compounds that can undergo reactions
with a resulting large negative free
energy change (like ATP) are used as
shuttles of free energy in the cell
(the bonds are said to contain
potential transfer energy)

MBB 321 29
Note the range of
potential transfer
energy of these
compounds (-60 to –10
∆Go’ kJ/mol )

This indicates that some

of these phosphate
hydrolysis reactions are
very high energy
processes while others
are not

Q: WHY do some
compounds have a
higher free energy of
hydrolysis?

It is not the phosphate

bonds themselves, but
rather some property of
both the reactants and
products in the reactions
that contribute to the
differences in free
energy

30
MBB 321
 Chemical basis for the large free-energy change associated with ATP hydrolysis

1. The hydrolytic cleavage of the

terminal phosphoric acid anhydride
(phosphoanhydride) bond in ATP
separates one of the three
negatively charged phosphates
Water is acting as which relieves some of the
a nucleophile electrostatic repulsion in ATP
(bond strain is relieved)

2. The inorganic phosphate released

by hydrolysis is stabilized by
formation of a resonance hybrid, in
which each of the four P-O bonds
has the same degree of
double-bond character and the
hydrogen ion is not permanently
associated with any one of the
oxygens

3. ADP immediately ionizes, releasing

H+ into the medium

4. Greater degree of solvation of products

(Pi and ADP) further stabilizes products
* MBB 321
relative to reactants 31
Chemical basis for the large free-energy change associated with the
hydrolysis of phosphoenolpyruvate (PEP) to pyruvate

The greater the

potential transfer
energy (the more
negative the ∆Go’ )
the greater the
tendency to
transfer a
phosphoryl group

PEP contains a phosphate ester bond that can undergo hydrolysis to yield
the enol form of pyruvate and this direct product of hydrolysis can
immediately tautomerize to the more stable keto form of pyruvate

Because the reactant (PEP) has only one form (enol) and the product (pyruvate) has two
possible forms, the product is stabilized relative to the reactant (and this is the greatest
contributing factor to the high std free energy of hydrolysis of PEP)

* MBB 321 32
 Chemical basis for the large free-energy change associated with the hydrolysis of
1,3-bisphosphoglycerate to 3-phosphoglycerate

1,3-bisphosphoglycerate has an
anhydride bond between C-1 and
phosphoric acid

The large negative standard free

energy change is again explained
in terms of structure of products
and reactants

When H2O is added across the

anhydride bond, one of the direct
products, 3-phosphoglyceric acid
can immediately lose a proton to
yield a carboxylate ion,
3-phosphoglycerate which has two
equally probably resonance forms

Removal of the direct product

(3-phosphoglyceric acid) and
formation of the
resonance-stabilized ion favor the
Note that in these three phosphate releasing reactions, forward reaction
the several resonance forms available to Pi stabilize this
product relative to the reactant, contributing to an
already negative free-energy change MBB 321 33
 Because the free-energy changes of sequential reactions are additive;
Any phosphorylated compound can be synthesized by coupling the synthesis
to the breakdown of another phosphorylated compound with a more
negative free energy of hydrolysis

Example: cleavage of Pi from phosphoenolpyruvate (PEP) releases more free energy than is
needed to drive the condensation of Pi with ADP, so the direct donation of a phosphoryl group
from PEP to ADP is thermodynamically feasible

(1) Hydrolysis of PEP: PEP pyruvate + Pi ∆G o’ = - 62 kJ/mol

(2) Phosphorylation of ADP: ADP + Pi ATP ∆G o’ = + 31 kJ/mol

Coupled phosphorylation of ADP by PEP: PEP + ADP pyruvate + ATP ∆G o’ = -31 kJ/mol

Remember – even though the term “high-energy phosphate bond” is often used
• when we talk about high energy bonds, it is not the bonds themselves that
contain the energy

• the free energy released by hydrolysis of phosphate containing compounds

results from the products of the reaction having a smaller free energy content
than the reactants (are more stable)

* MBB 321 34
 Chemical basis for the large free-energy change associated with the hydrolysis of
acetyl-coenzyme A

Thioesters, in which a sulfur atom replaces the oxygen

atom in the ester bond, also have large, negative,
standard free energies of hydrolysis
Acetyl Co-A is an important thioester in metabolism
The acyl group is activated for transacylation,
condensation or oxidation-reduction reactions

Thioesters have a higher free energy content than oxygen

esters because orbital overlap between the oxygen and
carbon atoms allows resonance stabilization in oxygen
esters while orbital overlap between S and C atoms is
poorer and provides little resonance stabilization

MBB 321
* 35
 ∆Go’ values for ATP hydrolysis do not represent the actual the ∆G values
under biological conditions

What are the reasons for this difference?

1) ∆G depends on temperature and different organisms live at different temperatures
2) ∆Go’ is defined at pH 7.0, but the actual pH may vary from 6.5 to 8.0 in different
organisms, tissues, cells and parts of a cell
3) Divalent ions like Mg2+ are present in significant quantities in all cells and the
reactants and products in the hydrolysis of ATP have varying affinities for these ions

most ATP in cells for example, exists in this form

Binding of Mg2+ introduces strain into

the ATP molecule that favors hydrolysis

Varying levels of magnesium will change ∆G in

complicated ways, depending on the relative
affinities of reactants and products for the
magnesium ion

4) The actual concentrations of ATP, ADP and Pi in the cell are not at the 1M
standard state concentration

Note: the same distinction between ∆Go’ and ∆G applies to all reactions, therefore the
phosphate transfer potentials (on slide 2.30) are only a rough guide to what may happen in the cell
because cellular concentrations of reactants and products can vary greatly, depending on the state of
the cells metabolism
36
MBB 321
 ATP provides energy by group transfers, not by simple hydrolysis

The contribution of ATP to a reaction is often shown as a

single step (a) which suggests that water displaces either
Pi (orthophosphate) or PPi (pyrophosphate)

But ATP hydrolysis alone would accomplish only the release

of heat (which would not drive reactions in isothermal
systems

The single arrow indicated in an ATP reaction generally

represents a two-step process

1) Part of the ATP (a phosphoryl or pyrophosphoryl or

AMP) is first transferred to a substrate molecule or to
an amino acid residue in the enzyme, becoming
covalently attached to the substrate or the enzyme and
raising its free-energy content

2) The phosphate containing moiety of step one is

displaced (in this example by NH3)
generating Pi, (in this example) PPi, or AMP

Note: some processes DO involve direct hydrolysis of ATP (or GTP)

• Noncovalent binding of ATP (GTP) followed by hydrolysis to ADP (GDP) and Pi can provide the
energy to cycle some proteins between two conformations, producing mechanical motion
(muscle contraction, movement of enzymes along DNA, movement of ribosomes along mRNA)
• GTP-binding proteins that participate in signaling pathways directly hydrolyze GTP to drive
conformational changes 37
MBB 321
The formation of high energy phosphate compounds is the means of activating a
wide variety of compounds for further chemical transformation

Example: phosphorylation of glucose coupled to ATP hydrolysis

(1) Hydrolysis of ATP: ATP ADP + Pi ∆Go’ = -31 kJ/mol

(2) Phosphorylation of glucose: glucose + Pi ADP + G6P ∆G o’ = +14
kJ/mol

Coupled phosphorylation of glucose by ATP: ATP + glucose ADP + G6P ∆G o’ = - 17

kJ/mol

Glucose by itself is relatively inert, but by phosphorylating it, glucose is now “activated” and
a more chemically reactive compound that can be further catabolized
( remember that phosphates are good “leaving groups”)

ATP is thermodynamically unstable, but is kinetically stable

Kinetically stable: if ATP is placed in an aqueous solution buffered at pH 7 the half-time for
its hydrolysis is many hours without a catalyst (the activation energy of an uncatalyzed
cleavage of its phosphoanhydride bonds is 200 to 400 kJ/mol)

ATP does not spontaneously donate phosphoryl groups to water or to the hundreds of other
potential acceptors in the cell.

Only when specific enzymes are present to lower the energy of activation does phosphoryl
group transfer from ATP occur
38
* MBB 321
 At the end of this section you should be able to:

• Discuss what information the change in the Gibbs free energy

tells us about a reaction

• Describe how an equilibrium constant is measured

experimentally

• Be able to calculate an equilibrium constant

• Discuss the relationship between the Gibbs free energy and

the equilibrium constant
Additional terms to be
• Be able to couple two reactions
familiar with:
•Potential energy
• Understand the relationship between equilibrium,
•Enthalpy
steady state and homeostasis
•Entropy
•Exergonic
• Understand why phosphate transfer potentials differ
•Endergonic
among compounds

• Understand what contributes to the large free-energy change

associated with ATP hydrolysis
39
MBB 321