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[2023]

Metabolic Biochemistry

Biotechnology [2A]
Miguel Baños García

ETSIAMN (UPV) | B2

Metabolic Biochemistry | Biotechnology [2A]

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UNIT I: PRINCIPLES OF BIOENERGETICS

LESSON 1: INTRODUCTION TO
BIOENERGETICS
Without bioenergetics (thermodynamics), metabolism is just a series of recipes to
learn by heart. Thermodynamics gives sense to biochemistry.

01. UNIFORMITY OF LIVING BEINGS. WHAT IS LIFE?

From a scientific point of view, life is almost impossible to define. Several non-
consensus definitions attempt to define what life is, but no clear definition exists.
Every living organism shares the same chemistry, made and maintained by a
collection of processes named metabolism:
 Aqueous solution
 Same chemical elements: C H O N P S
 Same chemistry, molecules and covalent nature
 METABOLISM to make and maintain their components.
The boundary between living and non-living beings is in viruses: they share the
first three characteristics. Despite not having their own metabolism, they still need
one to survive (parasites).

01.1. Are we so different from inert objects?

We appear to be unique (and we are). If we compare the main properties of
biosphere and lithosphere:
Biosphere (living beings) Lithosphere (inert beings)
Complex molecules Simple molecules
Chemically reduced compounds: Oxidised compounds
difficult to understand since we live in a
highly oxidising environment
Unstable, highly reactive molecules. Stable (low reactivity)
They have the tendency to react and
degrade, but still, they do not do.
Order, structure and information Tendency to reach chaos and decay.
This complexity is self-created and
maintained.
Homeostasis: tendency to stay the Tendency to reach equilibrium (death)
same (keep the same composition,
living through a steady state).

Miguel Baños García | ETSIAMN (UPV)

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01.1.1. Equilibrium vs. steady state

Inert beings tend to reach an equilibrium where there is no change, no movement.
These systems are dead:
 Forward processes equal backward processes
 The concentration of reactants and products is constant, does not vary
(closed system)
 The equilibrium is self achieved
 The system is closed: there is stable and with no net variation of properties. It
is self-achieved.
Living beings live in a steady state (estado estacionario), where:
 There is a continuous flow in one direction (no way back).
 The concentrations of intermediates are constant (open system with matter
and energy exchange)
 The concentrations of reactants and products do change.
 The system is open: there is no net variation of properties, but a continuous
supply of matter and energy from outside is required.

LESSON 2: BIOCHEMICAL
THERMODYNAMICS
According to the First Principle of Thermodynamics, the energy of the whole
universe is constant. Assuming this idea, we can consider that if a given system
consumes energy, it must take it from another part of the universe.
There is a natural tendency for things to go in just one sense. This idea is reflected
in the concept of entropy. Regarding the Second Law of TD, the universe's entropy
always increases. In practical terms, entropy is the driving force leading systems to
equilibrium.
Living beings are open systems in a steady state. They are thermal machines
working at constant pressure, volume and temperature. (Since they do not have
their own metabolism, viruses are the exception to the rule).
By burning paper (made of cellulose, a glucose polymer), we are oxidising glucose:
Glc +O2 → H 2 O+C O 2+Q+W
However, we cannot take advantage of the energy produced. Still, our primary
energy source is glucose oxidation, but carried out in increasingly less violent
reactions.
The constant of free energy or Gibbs' energy was introduced to describe the
changes in energy in highly restricted systems (such as living beings).
∆ G=∆ H−T ∆ S
We can apply this expression to living beings, taking the change in Gibbs’ free
energy as the usable energy obtained in a process.

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The question is: how can we, living beings, produce order when the natural
tendency of things is to chaos and decay? The answer is simple: by producing a
larger amount of chaos in our surroundings.
∆ Gº=−RT ln K eq
The standard free energy change (ΔGº) is a real, constant value that can be
calculated experimentally on an easy way. This value is defined as the energy that
a system produces going to equilibrium when these three conditions are
accomplished:
 Initial concentrations equal 1M
 The temperature is 25ºC or 298.15K
 The pressure is of 1atm
In biochemistry, there is no problem in working at standard conditions and even
with 1M concentrations, with an exception: protons. Protons are incredibly
important in biochemistry, and since a concentration of 1M will lead to a pH of 0,
these conditions cannot be accomplished.
Therefore, a new value named ΔGº’, considering a pH of 7, is created to work in
Biochemistry.
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∆ G º =−RT ln K ' eq
When a process’ ΔG is lower than 0, a process is spontaneous or exergonic.
However, spontaneous does not mean quick or fast, but with a natural tendency to
happen. Thermodynamics do not consider time.
If a process’ ΔG is higher than zero, the process
is not spontaneous or endergonic. However, this
does not mean that a process is “forbidden” by
thermodynamics. This process does not happen
by itself, but can happen with an external source
of energy.
Spontaneity has just to do with tendency, not
with something forbidden or permitted.
We can get some processes that go against our
interest. For them to occur, living beings can
couple some endergonic processes to other
exergonic ones in order to get our product of
interest. This is intimately related to the use of
ATP.
Since the free energy change is a state function (and therefore, additive), living
beings’ metabolism can promote non-spontaneous processes by coupling them to
other exergonic ones.
Most of the metabolic processes are chained. In a two-step process ABC, the
first step can be non-spontaneous but if, the second step is very spontaneous (and
therefore the global process will be spontaneous as well), the chain will naturally
happen.

Miguel Baños García | ETSIAMN (UPV)

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Sometimes we can find processes coupled to others:

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1 ¿ A ⇌ B , ∆ Gº =+3 kcal/mol
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2 ¿ C ⇌ D , ∆ G º =−7 kcal /mol
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A+C ⇌ B+ D , ∆ Gº =−4 kcal/mol
By combining these processes, we can make a non-spontaneous process to
naturally occur. Moreover, we can reconvert D into C, applying an external source of
energy, for it to keep producing B. C/D are usually the same compound in different
states, named energy intermediate or energy shuttle molecule.

LESSON 3: ATP: THE UNIVERSAL

“CURRENCY” OF FREE ENERGY
Since for nature it would have been very difficult to develop a specific pathway for
each non spontaneous process, it has developed a universal intermediate named
ATP (adenosyn tri-phosphate).
Some natural processes are very exergonic. These are mainly:
 Catabolic pathways (degradation or destruction of biomolecules)
 Light absorption in photosynthesis
These reactions produce a lot of energy, which is seized by our organism to
synthesize the proteins we are made of. These reactions are very endergonic:
 Biosynthesis
 Movement (not only of the whole organism, but also at a cell level).
 Osmotic processes
These processes need a supply of
energy.
ATP can be found (mainly) in 2 forms:
ADP and ATP. The first one is
dephosphorylated with respect to
ATP:
ATP ⇌ ADP+ Pi
The structure of ATP is very simple:
This is an energy rich compound, since the bonds between the phosphate groups
are very energetic. The phosphates are named α, β and γ respectively. According to
the classical approach, the two ester bonds accumulate a large amount of energy,
so hydrolising the γ and β phosphates we could obtain a lot of energy. This is not
completely true, though.
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ATP+ H 2 O ⇌ ADP + Pi , ∆G º =−7.3 kcal /mol

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The value was calculated under cell conditions (though the calculus is very difficult
and inaccurate). This value is translated to an equilibrium constant of around 10 7.
At standard conditions, the value of the free energy change would be -7.3 kcal/mol,
and the constant would move from 1 to 5 millions.
After the γ phosphate has been hydrolized, the process may continue with the
hydrolization of the β phosphate.
ADP+ H 2 O ⇌ AMP + Pi
Going one step further, if we hydrolize the α phosphate, we will find that the
process is spontaneous but not as much as the rest. In this case, the remaining
compound will be adenosine. The free energy change would be -3.4 kcal/mol.
In some biochemical process, ATP is hydrolized losing two phosphate groups:
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ATP+ H 2 O ⇌ AMP+ P Pi ( pyrophosphate ) , ∆ G º =−7.7 kcal /mol
Moreover, pyrophosphate is highly unstable in the cell:
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Pyrophosphate+ H 2 O ⇌2 Pi , ∆ Gº =−6.9 kcal/mol
One ATP molecule has 4 negative charges in a relatively low space. This creates a
high electrostatic repulsion which implies a tension for the molecule. After the
release of a phosphate, this will be expelled far away from the resulting ADP and
kept away.
By applying the same amount of energy (7.3 kcal/mol) we can reassemple a
phosphate group into an ADP molecule. This same behaviours can be applied to
ADP/AMP equilibrium.
There is a second reason why ATP is so energetically rich: hydration. If the molecule
is surrounded by water molecules, it will gain stability. The ADP+Pi complex will be
surrounded by more water molecules than just ATP, so it will be a more stable
complex. Moreover, when water moves to surround the complex, a tiny amount of
energy is released.
The last reason for this is resonance (explained by organic chemistry). This
phenomenon can be applied to the phosphates: the inorganic P by itself, once it has
been released from the ATP, contains 3 negative charges non-statically. As a result,
the pi electrons (double bond) will be shared by the P and the 4 O atoms, and their
bonds will be described neither as single nor as double, but as an intermediate.
According to the classic model, the energy was stored in the ester bonds. However,
it has been demonstrated that it is the whole molecule which stores the energy: the
molecule is a strong tension, and this stress is released by breaking the bond. The
energy is stored in the whole system, not only in the ester bond.
Still, there is a question to answer: How can ATP’s accumulated tension promote
non spontaneous processes?
Living beings manage by making the ATP hydrolisis an inevitable part of the process
to couple the this to an endergonic reaction. Then, the whole process will be a
single process and the free energy change will be overall negative. We can produce
these conditions using very specific catalysts: enzymes. There are many ways to

Miguel Baños García | ETSIAMN (UPV)

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couple ATP to non-spontaneous processes: For example, if ATP places in the correct
place of the enzyme, the energy can be transferred in form of conformational
changes which forces the appeareance of an activated complex which will then give
the product.
ATP is the link between exergonic (or spontaneous) and endergonic (or non-
spontaneous) reactions. It is, in other words, the free-energy intermediate.

WHY ATP?
ATP is not the only energy-rich compound in metabolism.
There are others which contain even more energy. This is
the case of PEP (phosphoenolpyruvate), the most energy-
rich biomolecule. It can be hydrolized according to the
following reaction:
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PEP+ H 2 O → Pyr + Pi , ∆ Gº =−14.8 kcal/mol
Due to the high energy release, the enzyme pyruvate
kinase is able to directly synthesize ATP.
However, due to its unique properties, evolution has selected ATP as the main free-
energy intermediate in metabolic processes:
1. Average energy-rich value: As we have seen before, ATP is able to directly
receive phosphate groups from other energy-rich compounds (such as PEP) as
well as from other exergonic processes such as respiration.
2. Adjusted perfectly to most metabolic needs: ATP is powerful enough for
its functions. Additionally, using more powerful molecules may result in a
waste of energy in not very endergonic processes.
3. Kinetic stability: Despite being thermodynamically unstable (due to its
tensioned structure) in an aqueous solution, ATP will never self-hydrolize. It is
time stable. This is because the activation energy of ATP hydrolization is
very high (around 10 times higher than the free-energy released).

Metabolic Biochemistry | Biotechnology [2A]