The Chemistry of Life 2021 Martin Welch

The Chemistry of Life: Part 2

Martin Welch
Department of Biochemistry (mw240@cam.ac.uk)
PLEASE BRING THIS HANDOUT TO EACH LECTURE! It includes all the
complicated diagrams, and the text of slides from the lectures in prose. You will
not need to make detailed notes. However, I have left a wide margin for brief
notes. The slides will also be made available on moodle. The subject is covered
in most text books, with some of it following closely to the descriptions in
“Biochemistry and Molecular Biology” by Elliott & Elliott (OUP) and Stryer
(Biochemistry, 6th ed. Berg, Tymoczko & Stryer; Chapters 15-17). For those
wishing to read further I recommend “Metabolic Regulation: A Human
Perspective” by Frayn and “Biochemistry for the Medical Sciences” by
Newsholme & Leech. These latter books are both very readable (honest!).

Further reading and other resources: These are provided for the keen reader
who wants to read more on the subject or for supervisors for discussion points
in supervisions.

“Metabolic pathways in the post-genomic era” JA Papin et al., TIBS 28, 250-258 (2003).
“Multifaceted roles of glycolytic enzymes” J-W Kim & CV Dang, TIBS, 30, 142-150
(2005).
“The danger of metabolic pathways with turbo design” B Teusink, et al. TIBS, 23, 162-
169 (1998)
“Glycolysis, turbo design and the endocrine pancreatic β cell.” PB. Iynedjian, TIBS, 23,
467-468 (1998).
“Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron
transport.” AR Fernie et al., Curr Opin in Plant Biology, 7, 254-261 (2004).
“Beta-oxidation of unsaturated fatty acids: a revised pathway.” H Schulz & W-H Kunau.
TIBS, 12, 403-406 (1987).
“Mitochondrial DNA, aconitase ‘wraps’ it up.” GS Shadel. TIBS 30, 294-296 (2005).
“Rocking cell metabolism: revised functions of the key glycolytic regulator PKM2 in
cancer.” B. Chaneton & E. Gottlieb, TIBS, 37, 309-316 (2012)
“A guardian angel phosphatase for mainline carbon metabolism.” Trends in
Biochemical Sciences, TIBS, 41 (11), 893-4 (2016).

A video of Bart and Homer explaining glycolysis: https://www.youtube.com/watch?

v=TZE_JEaAOKI
More scholarly but less entertaining this is a nice animation of the citric acid cycle:
http://www.bing.com/videos/search?
q=youtube+citric+acid+cycle+animation&qpvt=youtube+citric+acid+cycle+animation&vi
ew=detail&mid=F35BD20D7E0CD424C4F9F35BD20D7E0CD424C4F9&FORM=VRDG
AR.
And for β-oxidation.
http://www.wiley.com/college/grosvenor/0470197587/animations/
Animation_Lipid_Metabolism/Energy/media/content/met/anima/met4a/frameset.htm.

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Lecture 4 – An introduction to Bioenergetics

Aims
 To understand the concepts of entropy and free energy.
 To describe the coupling of free energy from one process to another
o e.g. oxidation of food (e.g. via glycolysis and the citric acid cycle)
coupled to muscle contraction;
o Absorption of light to the synthesis of macromolecules as part of
photosynthesis.

Thermodynamics and the cell

In order to understand whether a reaction occurs in a given environment we
must consider a number of thermodynamic processes and in particular the
properties of energy, entropy and free energy. In this lecture we will introduce
these concepts with a view to understanding how one reaction can be coupled
to drive a thermodynamically unfavourable process.

Do the laws of chemistry apply to living cells?

For a long time scientists (the so-called ‘Vitalists’) believed there was something
magical about the cell (hence the distinction between organic and inorganic
chemistry). In particular cells appeared to violate the laws of thermodynamics by
creating complex macromolecules and maintaining a high degree of order within
the cell. However, the same laws of thermodynamics that determine the
direction of change in a steam engine also dictate whether a reaction will occur
in a cell. This is what we will be considering in this lecture.

The First Law of Thermodynamics

“Energy can neither be created nor destroyed in a process.”
This law can be summarised mathematically as the change in internal energy
(∆U ) of a system (i.e. the energy the system possesses within it) is equal to the
heat energy absorbed (∆Q ) less the work done by the system.
 ∆U = ∆Q - ∆W

An example of this might be a gas in a box. The gas has internal energy from
the pressure it exerts (U). If we supply heat to the system, either the internal
energy rises (as measured by the pressure) or the gas does work (it expands).
Note the sign change for work, as this is work done by the system.

This places an upper limit on the energy costs of a process if it is to occur. For
example, energy stored in carbohydrates from photosynthesis must be less than
or equal to the energy coming into the system from light. Thus, we could add up
the energy costs of a reaction to see whether it would go or not.

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What makes a gas expand?

The expansion of a gas is difficult to explain using the First Law exclusively.
Other examples of processes like this include dissolving NaCl in water (an
endothermic reaction) and the flow of heat down a temperature gradient. To
understand these processes we need to consider entropy and the second law of
thermodynamics.

Entropy and the Second Law of Thermodynamics

“In a spontaneous process the entropy of the Universe increases.”
Entropy can be thought of as the wasted energy (energy not able to do work)
from a system. It is also often termed the degree of disorder of a system,
although one can question as to how one might measure such disorder.
 dS= dq/T where S is entropy, q is heat in and T is the temperature

In a spontaneous process the entropy of the Universe increases.

∆S > 0

At equilibrium
∆S = 0

However, entropy is particularly difficult to measure experimentally.

Furthermore, the problem with entropy and cellular processes is how does the
cell maintain so much order inside itself but still obey the second law of
thermodynamics? We would expect entropy to increase for spontaneous
processes (those not at equilibrium) including many biochemical reactions, but
the cell seems highly ordered. We need to consider both the first and second
law to understand the thermodynamic constraints placed on a reaction. As a
result a third term is commonly used when considering thermodynamics.

Gibbs Free Energy (∆G)

This is defined as:
∆G = ∆H - T ∆S at constant temperature (T).

Enthalpy (∆H) is related to how much energy is released by a reaction. It is a

constant pressure version of ∆U (the internal energy of the system).
The entropy change of the reaction represents the lost energy of the system
(we are considering the second law here).

Consideration of spontaneous reactions shows that either the reaction is

exothermic (∆H<0), the reaction occurs with a large increase in entropy (∆S>0),
or there is a combination of both factors. This can be summarised in terms of
the Gibbs Free Energy changes as follows:
For a spontaneous reaction: ∆G < 0
For a reaction at equilibrium: ∆G = 0
∆G = ∆H - T ∆S

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So in a spontaneous reaction either H has to be negative, associated with a
large release of energy (an exothermic reaction), or there is a large increase in
entropy (∆S) (e.g. a gas is produced), or we have some combination of the two.

The definition of standard Gibbs free energy

The standard Gibbs Free Energy Change (∆G°) is defined as the Free Energy
change which accompanies a reaction in which the reactants and products are
in their standard state at a specified temperature (usually 298K) and 1 atm of
pressure.
For solutions the standard state is 1M and gases at 1 atm of pressure.
However, for an acid the standard state would be equivalent to pH(1M)=0, not
very useful if we are considering the cell. It is actually much more convenient to
consider changes at pH = 7.
The biochemical standard Gibbs Free Energy change (∆G°´or delta G nought
prime in words) is defined as the standard Gibbs energy change for a reaction
at pH = 7.

Measuring ∆G°´
Unlike entropy, we can potentially measure ∆G°´ easily by measuring the
equilibrium constant of a reaction.
We can show for a reaction: A ↔ B in the cell
∆G = ∆G°´ + RT ln {[B]/[A]}
At equilibrium we know ∆G =0
Hence ∆G°´ = -RT ln {[B]/[A]} = -RT lnKeq

Where Keq is the equilibrium constant for the reaction (this can be measured)
(note Keq=[B]/[A] for this example)

∆G is a state function
Gibbs Free Energy is a ‘state function’, meaning the change in Gibbs free
energy for a process will be the same regardless of the pathway taken (compare
Hess’s law which you may have met at A-Level when calculating lattice energies
in chemistry). This means we can calculate a ∆G°´ for a process that is
experimentally difficult to measure by using tables of pre-determined ∆G°´ for
other reactions.

We can also ‘couple reactions’ and begin to understand how the cell seemingly
cheats the second law. The idea of ‘coupling of reactions’ is an incredibly
important concept in biochemistry.

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Coupling reactions:

We can also illustrate this with a set of general reactions. If we consider the
reactions:
A→B+C ∆G°´= +21 kJmol-1
B→D ∆G°´= -33 kJmol-1
Total: A → C + D ∆G°´= -12 kJmol-1

Summary of thermodynamics:
We can combine the First and Second Laws of Thermodynamics into the
concept of ∆G
 ∆G < 0 for a spontaneous process

We can measure ∆G°´ via the position of equilibrium for a reaction

 We can calculate ∆ G°´ by adding up smaller reaction steps for a more
complicated reaction
A reaction that is thermodynamically unfavoured (∆G >0) can be coupled to one
that is (∆G <0) to drive the former reaction provided that the ∆Gtotal <0.

ATP is one of the main driving forces for reactions

ATP is often termed the universal currency of energy in the cell as so many
reactions are coupled to the hydrolysis of ATP to drive a thermodynamically
unfavourable reaction. Pi is a phosphate and PPi is pyrophosphate (diphosphate)

 ATP + H2O ↔ ADP +Pi +H+ ∆G°´ = -30.5 kJmol-1

 ATP + H2O ↔ AMP +PPi +H+ ∆G°´ = -45.6 kJmol-1
 PPi + H2O↔ Pi +Pi +H+ ∆G°´ = -19.2 kJmol-1

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ATP

But the energy also depends on the concentration of reactants and products,
the concentration of Mg2+ and Ca2+ and the concentration of water.

Under typical cellular conditions ∆G~-50 kJmol-1 for ATP hydrolysis

(concentration also plays a part as we see below).
∆G = ∆G°´ + RT lnKeq

∆G = -30.5 kJmol-1 + RT ln {[ADP][Pi][H+]/[ATP][H2O]}

The concentrations of ATP, Pi and ADP are in the range of mM or less (ask your
supervisor why ADP is much lower than expected), while we set [H+] = 1 by
definition under standard cellular conditions for ∆G°´ (effectively we have taken
care of the fact the reaction will occur at pH=7 by using ∆G°´ and can effectively
ignore the concentration of hydrogen ions). However, the concentration of water
is ~ 55M and so the term on the right is large and negative, further decreasing
∆G.

Why is this reaction so exothermic (strictly speaking exogeronic)?

i. Phosphate and ADP have more resonance stabilisation than ATP
(see below for phosphate).
ii. Electrostatic repulsion. ATP has ~4 negative charges at pH 7 which
are in close proximity and this weakens the bridging P-O-P bonds in
ATP.
iii. Stabilisation due to hydration. More water can bind to ADP and Pi
than ATP (this is driven by both enthalpy and entropy).

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The free energy of ATP hydrolysis is referred to as its phosphorylation potential

- it is intermediate between a number of biologically important phosphorylated
molecules.

METABOLITE ∆G°´ (KJMOL-1)

Phosphoenolpyruvate -62
1,3-bisphosphoglycerate -49
Phosphocreatine -42
ATP -30.5
Glucose 6-phosphate -14
3-phosphoglycerate -10

We can use this table to determine whether a compound will phosphorylate

another. Considering the standard Gibbs free energy changes, metabolites at
the top of the table have a higher phosphorylation potential than those at the
bottom of the table.

e.g. i. ATP + H2O → ADP + Pi ∆G°´ = -30.5 kJmol-1

ii. glucose 6-phosphate + H2O → glucose and Pi ∆G°´ = -14 kJmol-1
i.-ii ATP + glucose → glucose 6-phosphate + ADP
∆G°´ = -30.5 --14 kJmol-1 = -16.5 kJmol-1

Phosphoenolpyruvate will phosphorylate ADP.

Phosphoenolpyruvate + ADP → pyruvate + ATP

Phosphocreatine will phosphorylate ADP.

PCr + ADP → Cr + ATP

Phosphocreatine acts as a high energy store to safe guard the cellular reserves
of ATP.

Measuring ATP changes

ATP has a very high turnover in cells (0.5 kg/min in humans during exercise). In
many tissues in mammals ATP is buffered by phosphocreatine. Under high
energy demands or metabolic crisis (e.g. a lack of oxygen which impairs the
production of ATP) initially PCr decreases in concentration prior to a depletion of
ATP. We can measure this in a working tissue using 31P NMR spectroscopy.

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31
P NMR spectrum from
intact working heart

Examples of coupling ATP hydrolysis to chemical reactions in the cell

1. ATP is used to phosphorylate glucose to provide enough energy to prime
the molecule to be broken down to pyruvate
2. Peptides are unstable thermodynamically (∆G~+17 kJmol-1 for a
dipeptide) with respect to their component amino acids, but ATP can be
used to build peptides
3. To join two nucleic acids to start to synthesize DNA requires energy and
this is derived from the hydrolysis of ATP

We can look at coupling reactions by considering a simple reaction.

A↔B ∆G°´ = +16.7 kJmol-1
The equilibrium constant K is given by K = [B]/[A] = exp(-∆G°´/RT) = 0.00115
Now let’s couple the hydrolysis of ATP (∆G°´ = -30.5 kJmol-1) with this reaction
somehow.

A + ATP + H2O ↔ B + ADP + Pi

∆G°´ = +16.7 kJmol-1 + - 30.5 kJmol-1 = -13.8 kJmol-1

K = [B]/[A] * [ADP][Pi]/[ATP]= 267

This has changed the equilibrium constant by 105. Using the cellular value for
the hydrolysis of ATP this is 108!

Activated carriers
A recurring motif in metabolism is the activated carrier (e.g. ATP). A relatively
small set of metabolites drive reactions which would be thermodynamically
unfavourable otherwise. Some examples we will meet:

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Carrier molecule Group carried
ATP Phosphoryl-
NADH & NADPH Electrons
FADH2 & FMNH2 Electrons
Coenzyme A Acyl
Biotin CO2
Uridine diphosphate glucose glucose

Reduction and oxidation

In terms of metabolism an important set of reactions we must consider involve
oxidation/reduction.
Gain of hydrogen = reduction; Loss of hydrogen = oxidation
Gain of electrons = reduction; Loss of Electrons = Oxidation (LEO)

For energy production the main redox system is NAD+/NADH (nicotinamide

adenine dinucleotide).

During reduction, NAD+ receives a H+ and 2 electrons, equivalent to a hydride

ion (H-). If you are worried about how this is a reduction, remember as well as
adding hydrogen to a molecule, reduction can also involve the acceptance of
electrons. As well as NADH we will also examine the flavin adenine dinucleotide
redox system (FAD/FADH2) in Prof. Hibberd’s lectures.

The NADP+/NADPH redox system:

For biosynthesis NADP+/NADPH is used. The phosphate group acts as a tag to
allow the recognition of this redox system by biosynthetic enzymes. This is one
way the cell can achieve two different redox potentials: one for production of
ATP; one for the synthesis of other metabolites.

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Redox centre

Phosphate
tag

High energy sulphur bonds: Acetyl-CoA

Another important class of compounds contain reactive sulphur bonds.
Coenzyme A is required by many enzymes to catalyse acetylation, providing an
activated form of acetate.
The hydrolysis has ∆G°´ = -31.5 kJmol-1
Thus, in the same way ATP has an ‘activated’ phosphoryl group acetyl-CoA has
an ‘activated’ acetate group (‘activated acetate’)

Coenzyme A is a universal carrier of acyl groups:

As well as acetate groups CoASH will also react with acyl groups in general i.e.
RCOOH (a fatty acid).
CoASH + RCOOH → R-CO-S-CoA
These reactions are important during the activation of fatty acids in β-oxidation
and at the start of the citric acid cycle.

Activated co-enzymes
We have met three activated carriers/co-enzymes: ATP, NADH/NADPH and
acetyl-CoA. These molecules have a variety of roles in driving
thermodynamically unfavourable reactions. In each of the cases notice that an
adenine base is present. RNA is thought to predate DNA and proteins. Could
the adenine have been used as a recognition motif by early RNA catalysts
(ribozymes)?

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Key reactions are repeated across metabolism
There are just six types of reaction that we will meet in metabolism.
Type of Reaction Description
Oxidation-reduction Electron Transfer
Ligation requiring ATP Formation of carbon bonds
Isomerization Rearrangements of atoms
Group transfer Transfer of a functional group
Hydrolytic cleavage of bonds by the addition of
water
Addition/removal of functional groups addition of functional groups to double
bonds or their removal to form double
bonds

The take home messages……

For a spontaneous reaction to happen ∆G < 0.

We can use this to couple reactions that are thermodynamically unfavourable
with the hydrolysis of ATP to drive the first reaction.
As well as ATP there are a host of other activated metabolites including NADH,
NADPH and acetyl-CoA.

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Lecture 5
Starts
Regulation of metabolism – General principles
Here We need regulation:
 1. To avoid substrate (‘futile’) cycles.
 2. To link energy production to energy usage.
 3. To respond to physiological changes
 Physiological responses after feeding will not be the same as that
during fasting.
A futile cycle is shown left and
represents a crossing point between
glycolysis and gluconeogenesis at
phosphofructokinase-1 and fructose
1,6-bisphosphatase. NB these futile
cycles are useful in terms of rapidly
increasing a flux through a pathway,
but control is necessary to prevent
such cycles consuming too much
ATP. Because of their use in
regulating flux some prefer the term
substrate cycle.

How are enzyme activities controlled?

1. Change in the amount of enzyme.
The amount of an enzyme can be changed by the rate of synthesis or the rate of
destruction (t1/2 = hours to days). In mammals this is quite a slow response and
occurs as a result of long term changes.
For example
An increase in lipoprotein lipase in lactating mammary glands.
Changes in liver enzymes during the shift from the fed state to starvation.
Increases in drug-metabolising enzymes following the intake of foreign
compounds.

In bacteria the induction of enzymes can be rapid, e.g. when E. coli is exposed
to lactose the induction of b-galactosidase occurs rapidly.

2.Metabolic control of an enzyme

 This is a much more rapid response
 In a pathway: A  B  C  D  E
 where E is the product of the pathway, it is often that E inhibits the A  B
 feedback inhibition.
 This prevents intermediates accumulating.

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Mechanisms for controlling enzyme reaction rates

Allosteric regulation involves the binding of an allosteric effector, which changes
the affinity of the enzyme for its substrate(s). The effect can be positive, with an
increase in affinity, or negative, with a decrease, and represents a mechanism
of very fast response to changes in the cell.

Covalent modifications of an enzyme most commonly involve phosphorylation

by protein kinases. These enzymes transfer a phosphate group from ATP to
specific sites on proteins, causing a conformational change.

This produces two levels of control within the cell.

 Allosterically regulated enzymes are rapid
 Signals are usually intracellular

 Phosphorylation/dephosphorylation regulated by extracellular agents,

e.g. hormones.
 Allows coordination at the whole organism level.
 Glucagon, insulin and adrenaline are particularly important in
controlling fat and carbohydrate metabolism.
 Glucagon is produced in response to low blood glucose and
insulin in response to high levels.
 Adrenaline is released from the adrenal gland and stimulates the
release of food reserves.

Carbohydrate metabolism
Aims
 Understand the basis of glycolysis.
 Understand its interaction with other key metabolic pathways
(glycogenolysis, the pentose phosphate pathway and the citric acid
cycle).

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 Understand the importance of the ‘Cori cycle’.
 Understand the importance of ‘turbo design’ for glycolysis (and metabolic
pathways in general).

The Strategy for Carbohydrate Metabolism

Three pathways are required to completely oxidise glucose and produce ATP.
The overall reaction can be summarised as:

C6H12O6 + 6O2 → 6CO2 + 6H2O + energy as ATP

Initially glucose is broken down to produce pyruvate and a small amount of

energy as part of glycolysis. The pyruvate is then oxidised in the citric acid
cycle, producing CO2 and the reduced metabolites NADH and FADH2, as well
as GTP.

Finally the reduced NADH and FADH2 are oxidised during oxidative
phosphorylation, generating ATP and regenerating the NAD+ and FADH
required by both glycolysis and the citric acid cycle.

NB some textbooks
give different values
for the ATP produced
by these processes.
Ask your supervisor
why or look at Stryer
or Elliott & Elliott for
a discussion.

The influence of insulin on glucose metabolism in mammals

Although the hormone insulin is central to controlling metabolism across the
body a number of tissues take up glucose in an insulin independent manner.
This includes the brain, liver cells and erythrocytes. These tissues use insulin-
independent transporters such as GluT1 (Glucose Transporter 1), GluT2 and
GluT3.

However, entry of glucose into fat and muscle cells is controlled by GluT4, an
insulin dependent transporter. These tissues demonstrate how hormonal
signalling can control the metabolism of a cell. Prior to the cells being exposed
to insulin the GluT4 proteins are trapped in intracellular vesicles. Insulin recruits
these vesicles to the cell membrane, allowing transport of glucose.

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Glycolysis
Glucose (and also glycogen) is metabolised to:
pyruvate, 2 ATP; NAD+ is reduced to NADH
 Two fates for NADH
 Can be transported into the mitochondria for oxidation
 Can be used to reduce pyruvate to lactate, thus regenerating
NAD+

Glycolysis, as well as producing pyruvate for the citric acid cycle, can be used to
produce energy in the absence of oxygen. This is important in tissues lacking
mitochondria such as red blood cells and the lens in the eye, and for tissues
where a burst in activity is required, such as fast-twitch (white) muscle. This
oxygen debt is then repaid in mammals by increasing the citric acid cycle rate to
oxidise the lactate produced in the body.

Hyperlactaemia and lactic acidosis

Normally there is about 1 mM lactate in the blood.
 pKa = 3.86 so the acid is fully disassociated
If lactate increases above 5 mM the buffering capacity of the blood is
overpowered.
 pH drops from 7.35-7.45 to ~ 7
This usually arises from tissue hypoxia or decreased clearance of lactate
from blood to other organs

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The pathway of glycolysis

Glycolysis

We can consider the pathway to

be in two halves. The first part up
to glyceraldehyde 3-phosphate
involves the chemical priming
phase, where glucose is prepared
for the production of ATP (and
ATP is consumed in this half).
The second half involves the
energy generating stages (Figure
from Stryer).

We can also consider the pathway in

terms of Gibbs Free Energy changes
for the various pathways (figure by
Matthews & van Holde). Stars show
reactions controlled by allosteric
modifications (see Dr. Waller’s
lectures and these lectures for a
reminder) – these steps are
irreversible because of the large
change in Gibbs energy. Numbers in
brackets show typical concentration
in the liver in µM.

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The pathway in more detail
There are three stages in the overall pathway of glycolysis. Initially glucose is
prepared for lysis and then split into two three carbon monosaccharides. This
process consumes 2 ATP per glucose which are required to activate the
glucose (glucose→glucose 6-phosphate; fructose 6-phosphate→fructose 1,6-
phosphate). It ends with lysis of glucose to glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate.

The glyceraldehyde 3-phosphate produced is then oxidised to produce 2 ATP

and 2 NADH per glucose. This step represents oxidation of the three carbon
chain intermediates. The aldehyde in glyceraldehyde is converted to a
carboxylic acid.
The oxygen for the oxidation comes from water and the oxidising agent is NAD+
(forms NADH). We have gone from a C=O bond to an energetically favoured
COOH group (a high degree of resonance stabilisation).

A further 2 ATP are produced in the third stage consisting of rearrangement and
hydrolysis to produce pyruvate. Note in the final reaction (catalysed by pyruvate
kinase) the energy required to phosphorylate ADP to ATP comes from replacing
a C=C and C=O bonds with two C=O bonds.

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The total of these reactions

There are three stages – lysis, oxidation and rearrangement:
 Lysis consumes 2 ATP per glucose
 Oxidation produces 2 ATP and 2 NADH per glucose
 Rearrangement produces 2 ATP per glucose
The net reaction is:
Glucose + 2Pi +2ADP + 2NAD+→2pyruvate + 2ATP +2NADH + 2H+ + 2H2O
However, this reaction will soon cease if the NAD+ is not regenerated somehow
by oxidation of NADH. There are two alternatives to regenerate this NAD+ in
mammalian tissues. Firstly, the NADH can be oxidised in mitochondria. The
overall process is then referred to as aerobic glycolysis. Alternatively, the NADH
can be oxidised by lactate dehydrogenase during the conversion of pyruvate to
lactate. This occurs in anaerobic glycolysis. This lactate is either exported to the
blood stream (see the Cori cycle below) or is converted back to pyruvate for
oxidation of the carbon backbone in the citric acid cycle.

Moonlighting enzymes:
Many enzymes in metabolism (and in particular glycolytic enzymes) are
often termed house keeping, and thought not to vary under different
conditions. However, recently many of the enzymes of glycolysis have
been discovered to possess multi-faceted roles.

Enzyme Role
Hexokinase Transcriptional regulation, apoptosis, glucose homeostasis

Lactate dehydrogenase Transcriptional regulation

Glyceraldehyde-3-phosphate Transcriptional regulation, apoptosis.

dehydrogenase

Enolase 1 Transcriptional regulation, the degradosome

Glucose 6-phosphate isomerase Cell motility and invasion

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Metabolic control in glycolysis: The control of glycolysis will vary according to
tissue type and we will broadly consider two tissues here. In muscle, glycolysis
is used often to generate ATP as part of an explosive response (e.g. the so-
called fight or flight response). However, in the liver very little ATP is produced,
and instead the tissue is a net producer of glucose from gluconeogenesis and
glycogenolysis in the fasted state, while liver tissue also synthesises
triglycerides and glycogen in the fed state.

The control of glycolysis can be considered in key stages:

1. Transport of glucose into the cell
 Glucose transported into the cell via glucose transporters in most tissues
 GluT4 transporters in muscle cells and adipocytes
 Insulin causes the recruitment of GluT4 proteins from vesicles in the cell
to the plasma membrane (see previously)
 Liver uses GluT2 transporters
 This occurs in a non-insulin dependent manner to regulate the
body’s total glucose load

2. Phosphorylation of glucose

In liver glucokinase is present. This enzyme

has a Km(glucose) = 10 mM, while in muscle
hexokinase the Km(glucose) = 0.1 mM. Thus,
the liver can deal with high concentrations of
glucose, while muscle operates at Vmax/2
even under low glucose conditions. Also
prevents the liver from taking up low conc.
of glucose it releases during fasting.

Also hexokinase is inhibited by glucose 6-phosphate, but glucokinase is not.

Muscle tissue can conserve glucose and ‘shut off’ glycolysis when glucose 6-
phosphate builds up. Liver tissue can still produce glucose 6-phosphate for
glycogen or lipid synthesis.

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3. Phosphofructokinase-1

ATP is both a substrate and allosteric

inhibitor of PFK-1
This is potentiated by citrate
Thus, in the inactive state (high citrate
and ATP) glycolysis is inhibited in
muscle
 AMP overcomes this inhibition
(produced during exercise)

4. Fructose-2,6-bisphosphate:
A futile cycle exists between fructose 6-phosphate and fructose 1,6-
bisphosphate via PFK-1 and fructose 1,6-bisphosphatase. Left unchecked this
cycle would consume ATP. However, this cycle is used to control flux through
glycolysis and gluconeogenesis. In muscle control is maintained by AMP
(AMP↑, glycolysis ↑). In liver it is controlled by fructose-2, 6-bisphosphate
(fructose-2, 6-bisphosphate ↑, glycolysis ↑, gluconeogenesis ↓).

PFK-2 is under hormonal control

In cardiac muscle this is adrenaline. Fight or flight – stimulate glycolysis.
Adrenaline (also called epinephrine, esp in the USA) is released from the
adrenal gland. In liver glucagon causes a decrease in fructose 2, 6-
bisphosphate, stimulating gluconeogenesis. In skeletal muscle there is no
hormonal control and AMP determines the concentration of fructose 2, 6-
bisphosphate.

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 The Chemistry of Life 2021 Martin Welch

5. Pyruvate kinase
Fructose 1, 6-phosphate stimulates pyruvate kinase, and this reaction is an
example of ‘feed forward’ stimulation. This ensures that the start of the pathway
where glucose is ‘prepared’ for lysis can stimulate the end of the pathway where
ATP is generated.

Production of lactate

Lactate dehydrogenase

NADH NAD+

This process regenerates NAD+ for glycolysis. Lactate is either exported to the
liver or converted back to pyruvate for oxidation of NADH and pyruvate in
mitochondria.
Lecture
six The fate of pyruvate
Starts
roughly
here Glucose
Glycolysi
Gluconeogenesis
s
Pyruvat
e Ethano
l
Fermentation in yeast
Lactat
e
2 Acetyl- Anaerobic
CoA metabolism: lactate in
animals and some
microorganisms
BOTH REACTIONS
Citric acid REGENERATE
NAD+
cycle

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The Chemistry of Life 2021 Martin Welch
Gluconeogenesis
Lactate (via pyruvate of course!) has two fates in the body
 Oxidation in the citric acid cycle (see below)
 Or conversion back to glucose (gluconeogenesis)

 Some organs in the body have little oxidative capacity

 Red blood cells
 Fast-twitch, white muscle (c.f. red/slow-twitch muscle).
 We need to recover this lactate in some form that either other
organs can use, or re-cycle the carbon chain back to these
anaerobic organs as glucose.

As mentioned above, lactate produced as part of anaerobic glycolysis is

exported from muscle tissue where it is generated to the liver for subsequent
conversion back into glucose via the pathway gluconeogenesis.

Mechanism of glucose synthesis from pyruvate

Most of the reactions of

glycolysis are readily
(thermodynamically)
reversible but there are
3 which are not.
The phosphorylation of
glucose using ATP.
The phosphorylation of
fructose-6-phosphate,
again using ATP.
The step between
phosphoenolpyruvate
and pyruvate.

In the case of pyruvate kinase the forward reaction yields an ATP. However, it
requires two ATP to go backwards. The reaction occurs in two steps i.e.
pyruvate, ATP and bicarbonate react to form oxaloacetate. The ATP is
hydrolysed to ADP and Pi. The enzyme responsible is pyruvate carboxylase.
PEP carboxykinase (PEP-CK) catalyses the conversion of oxaloacetate to PEP,
using GTP.

The steps as far as fructose-1, 6-bisphosphate are reversible. Fructose-1, 6-

bisphosphate is converted back to fructose-6-phosphate by the hydrolysis of a

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The Chemistry of Life 2021 Martin Welch
phosphate group. The enzyme is fructose-1:6-bisphosphatase. In a similar way
glucose-6-phosphate to glucose is achieved by simple hydrolysis by glucose-6-
phosphatase.

The Cori Cycle between muscle and liver tissue

The interplay between anaerobic glycolysis in muscle tissue and
gluconeogenesis in liver tissue is described by the Cori Cycle (named after a
husband and wife team). Muscle tissue generates lactate during explosive
exercise and this would cause acidosis in the muscle tissue if it were not
exported into blood. This lactate still represents a large amount of potential
energy to the body, and so it is converted back into glucose via
gluconeogenesis. After exercise, this glucose is transported back to the muscle
tissue and stored as glycogen, ready for the next burst of explosive exercise.

Lactic acid
during
During exercise
exercise

Glucose after
exercise
The importance of gluconeogenesis
This process is also important for maintaining normal function in the brain.

Glucose is the primary fuel of the brain. When stores are depleted the first call
on the body is to convert lactate generated by other organs into glucose to fuel
the brain.

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The Chemistry of Life 2021 Martin Welch
Negligible quantities of glucose are produced by fat (however, the brain can use
ketone bodies). Hence, during long term starvation the body must convert
proteins into glucose via amino acids and the citric acid cycle (more in a
moment on this....).

Glycogen and glycogenolysis

Because of the central importance of glucose in the body, especially for the
normal function of the brain, the body needs to store glucose. It stores it in the
form of glycogen (c.f. starch in plants). This also reduces the osmotic potential
of glucose (which would otherwise damage cells in the body) and avoids the
non-enzymatic glycosylation (glycation) of proteins as occurs in diabetes.
Far left:
Glycogen
granules in
hepatocytes.
Near left:
the
structure of
glycogen.

The structure of glycogen

Glycogen is a polymer of glucose which is predominantly joined at a(1→4)
glucose, except at branch points, every 10 or so glucose units, where the
branching is a(1→6). Branch ~10 glucoses (see lectures on Macromolecules).
One end is joined to glycogenin (a protein).
 The size of these glycogen particles varies with the state of feeding
 ~10 nm between meals
 >40 nm after feeding

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The Chemistry of Life 2021 Martin Welch
The structure exposes a large number of glucose units to the surface.
Glycogenolysis (break down of glycogen) can occur very rapidly during ‘fight or
flight’ situations.

The synthesis and degradation of glycogen

During feeding citrate and ATP are produced by the citric acid cycle. Both of
these metabolites act as allosteric inhibitors of glycolysis. This prevents the
breakdown of glucose to pyruvate, and allows the conversion of glucose to
glycogen (see pathways below). 70 g of glycogen in liver; 200 g of glycogen in
muscle.

Turbo design of glycolysis

There are at least two reasons for the first stages of glycolysis consuming ATP.
Firstly, the conversion of glucose to glucose 6-phosphate prevents the sugar
from leaving the cell via the transporter that allows it to enter. Also, as we have
mentioned, the phosphorylation of glucose primes the molecule for degradation.
This turbo design is a general feature of many metabolic pathways, where the
hydrolysis of ATP is used to drive a thermodynamically ‘uphill’ process prior to
the subsequent release of energy.

A metabolic mystery has surrounded this turbo design for some time. Over a
century ago, Harden observed that glycolysis ceased if the supply of phosphate
is limited when yeast juice was exposed to high concentrations of glucose. This

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The Chemistry of Life 2021 Martin Welch
was the first observation of substrate accelerated death in a single cellular
organism.

Since then, several strains of yeast have been produced which are unable to
grow in high concentrations of glucose, despite all the evidence indicating the
rate of enzymes at the start of glycolysis were increased. The evidence
appeared to be at odds with one another, until it was discovered the mutations
were deletions in the trehalose 6-phosphate (Tre-6-P) synthase gene (Tps1p).
While the interaction between Tre-6-P and HK is still debated, the model shown
below replicates the metabolic control found in these yeast mutants, illustrating
a general problem for turbo designed pathways.

An explanation for substrate

accelerated death in mutants
of trehalose 6-phosphate
synthase gene.
Key: HMP Hexose
monophosphates (i.e.
glucose 6-phosphate and
fructose 6-phosphate). Tre-
6-P trehalose 6-phosphate.
The broken lines indicate the
suggested interactions
necessary to reproduce the
effects of substrate
accelerated death in the
mutant yeast strains.

Glucose sensors: Monitoring glucose use experimentally

In 1962 Clark described how to make an electrochemical sensor “more
intelligent" by adding "enzyme transducers as membrane enclosed
sandwiches". This was illustrated by an experiment in which glucose oxidase
was entrapped at a Clark oxygen electrode using a dialysis membrane. The
decrease in measured oxygen concentration was proportional to glucose
concentration. This was the first generation of a glucose monitor.
‘Glucose pens’ used to conveniently monitor blood glucose concentrations are
one of the most successful exports from the UK. These devices are commonly
used to monitor glucose metabolism in diabetic patients, indicating a market of
~7% of the Westernised populations.
A Recap
 Glycolysis converts glucose to pyruvate generating some ATP and some
NADH (see why this is not a lot in a moment!).
 Under anaerobic conditions NADH is used to reduce pyruvate to lactate.
 Under aerobic conditions pyruvate and NADH are oxidised by the citric
acid cycle and oxidative phosphorylation.
 Some cells in the mammalian body rely on glycolysis and this produces a
cycle of metabolites e.g. muscle and liver in the Cori cycle.

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The Chemistry of Life 2021 Martin Welch

Oxidative metabolism
Aims
Understand the key steps of the citric acid cycle.
Understand its interaction with other key metabolic pathways (glycolysis, amino
acid synthesis and b-oxidation).
Understand the key steps involved in b-oxidation and fatty acid synthesis.
Understand how we might measure rates in these pathways.

The citric acid cycle

This is also known as the Krebs cycle and the tricarboxylic acid cycle. The cycle
holds a central position in terms of many metabolic pathways and is important
for both the production of intermediates as well as the generation of energy.

Gluconeogenesis

The citric acid cycle involves overall oxidation and only occurs under oxidative
conditions, taking place in the mitochondria. The cycle generates 3 NADH and 1
FADH2 for each turn of the cycle. These are used as part of oxidative
phosphorylation to generate ATP. The cycle also generates GTP, which is
readily converted into ATP.
CO2 is also produced by the cycle (two for each acetyl group entering the cycle).

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The Chemistry of Life 2021 Martin Welch
Pyruvate, the end of glycolysis and the start of the Citric Acid Cycle
The conversion of pyruvate to acetyl-CoA is catalysed by pyruvate
dehydrogenase (PDH). This enzyme is in fact a complex of 3 enzymes and 5
co-enzymes! By co-localising these enzymes side reactions are minimised and
the overall rate is increased.
O CoASH + NAD+ H3C S
CoA + O C O + NADH
H3C
OH O

Pyruvate Acetyl-CoA

Each reaction has to be coupled to ensure the free energy released during the
loss of CO2 is coupled to the subsequent generation of acetyl-CoA and NADH.
To achieve this, a flexible arm (lipoamide) tethers the acetyl group to transfer it
between two active sites, as well as transferring reducing potential to a third site
In this manner a coupled reaction is achieved.

The Citric Acid Cycle

Unlike glycolysis, where there is both oxidation and reduction in the pathway,
the citric acid cycle involves overall oxidation and only occurs under oxidative
conditions. As a flip-side of this oxidation, NAD+ and FAD are reduced to NADH
and FADH2
 3 NADH and 1 FADH2
These reduced species are used to generate ATP as part of oxidative
phosphorylation. The cycle also generates GTP. CO2 is produced by the cycle
(two for each acetyl group entering the cycle).

We need not
concern ourselves
with the
asymmetric
metabolism of
citrate, but this
may be a good
question for your
supervisor!
Alternatively there
is a discussion in
Stryer.

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The Chemistry of Life 2021 Martin Welch

Mitochondria and the Citric Acid Cycle

The enzymes of glycolysis, pentose phosphate pathway (see the appendix) and
fatty acid synthesis are largely cytosolic.
The citric acid cycle, b-oxidation and the respiratory chain are in the
mitochondria. The matrix of the mitochondrion contains the enzymes of b-
oxidation and most of the citric acid cycle. An important exception is succinate
dehydrogenase (you will see why later in the lectures on oxidative
phosphorylation).

The overall stoichiometry of the cycle

If we take acetyl-CoA as the start of the cycle, two carbon atoms are introduced
into the cycle and condense with oxaloacetate to form citrate. The citrate is
oxidised across a series of steps and the carbon backbone loses two CO2
molecules to reform oxaloacetate. Thus, two carbons enter the cycle and two
carbons leave. This has very important implications in terms of
gluconeogenesis. Mammals cannot synthesize glucose from fat (at least the
fatty acid part). Gluconeogenesis requires CO2 fixation.

The cyclic nature of the citric acid cycle

 Acetyl-CoA + oxaloacetate → citrate + CoA
 High energy sulphur bond broken
 Condensation
 Citrate → cis-Aconitate → iso-citrate
 Rearrangement
 Iso-citrate → a-ketoglutarate + CO2
 Oxidation and decarboxylation
 Generate NADH
 a-ketoglutarate + CoASH → succinyl-CoA

 Succinyl-CoA + GDP → succinate + GTP + CoA-SH

 Generation of high energy phosphate
 GTP + ADP → GDP + ATP
 Succinate → fumarate
 Generation of FADH2
 Fumarate → malate
 hydration
 Malate → oxaloacetate
 Generate NADH
 Return to the start of the cycle

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The Chemistry of Life 2021 Martin Welch

Anaplerosis: One problem with such a cycle is that if we use the cycle to
generate new compounds we lose carbon from the cycle. So an anaplerotic
pathway is needed as well as the catabolic pathway just described.
E.g. Pyruvate carboxylase
Pyruvate + CO2 + ATP + H2O → oxaloacetate + ADP + Pi + 2H+

How many ATP molecules are generated from glucose?

We are now in a position to compare how much energy is generated from
glycolysis and the citric acid cycle.
Assume 1 NADH ≡ 2.5 ATP
And 1 FADH2 ≡ 1.5 ATP

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The Chemistry of Life 2021 Martin Welch
In oxidative glycolysis
For 1 mole of glucose we
Use 1 mole of ATP to form glucose-6-P
Use 1 mole of ATP to form fructose 1,6-bisphosphate
Generates 2 moles of NADH from glyceraldehyde 3-phosphate dehydrogenase
BUT we ‘lose energy’ getting NADH into the mitochondria (only get 1.5 ATP per
NADH)
2 mole of ATP from 1,3-bisphosphoglycerate
And 2 ATP from phosphoenolpyruvate
Total -1 + -1 + 2*1.5 + 2 + 2 = 5 ATP

N.B. Only get 2 ATP under anaerobic metabolism

In the citric acid cycle

Two pyruvates from one glucose.
2 NADH from pyruvate dehydrogenase (1 per pyruvate)
2 NADH from isocitrate dehydrogenase
2 NADH from a-ketoglutarate dehydrogenase
2 GTP equivalent to 2 ATP
2 FADH2 from succinate dehydrogenase
2 NADH from malate dehydrogenase
Total 8 NADH + 2 FADH2 + 2GTP = 8*2.5 + 2*1.5 + 2*1 = 25

Aerobic glycolysis + citric acid cycle = 30 ATP

Measuring the rate of the citric acid cycle

We can measure the citric acid cycle rate experimentally either making use of
the coupling between the citric acid cycle rate and oxygen consumption or
monitoring the rate of labelling of key citric acid cycle intermediates. One
important method for monitoring oxygen consumption involves the use of an
oxygen electrode. Alternatively, both 14C and 13C experiments can be performed
to ‘chase the label’ around the citric acid cycle.
13
C label is introduced to an
organ/organism. E.g. labelled butyrate (a
short fatty acid). Labelling of key
metabolites is observed in vivo using
NMR spectroscopy. In the example
shown we monitor the citric acid cycle
rate by following the rate of labelling of
glutamate, which is made via the citric
acid cycle (see Prof. Hibberd’s lectures
to come).

An alternative method to monitor oxidative metabolism in the brain involves

functional magnetic resonance imaging (fMRI). Magnetic resonance imaging

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 The Chemistry of Life 2021 Martin Welch
(MRI) relies on imaging the brain by detecting hydrogen nuclei largely in water.
Paramagnetic substances will modify this signal. Deoxyhaemoglobin is
paramagnetic, while haemoglobin is diamagnetic. We can use fMRI as a tool for
monitoring blood oxygenation through the paramagnetism of
deoxyhaemoglobin. This approach is used to monitor which regions of the brain
are stimulated by various stimuli including alcohol, drugs and even sex! It seems
some researchers are not restricted by what they can fit into clinical magnets!

Lecture 7 Metabolic control in the citric acid cycle

starts We have already seen the central importance of the citric acid cycle in terms of
approx. controlling flux into other pathways and the generation of energy by the cell. In
here particular, the body must conserve glucose metabolism for the brain and red
blood cells. Acetyl-CoA is the point of no return for glucose derived carbon as it
cannot be converted back into glucose after this. This is controlled by pyruvate
dehydrogenase.

1. Pyruvate dehydrogenase
PDH is a multienzyme complex which possesses two regulatory enzymes:
PDH kinase phosphorylates the complex and deactivates PDH
PDH phosphatase dephosphorylates the complex and activates PDH.

PDH kinase is inhibited by pyruvate

 Ensures PDH is ‘on’
when lots of pyruvate
PDH phosphatase is activated by Ca2+
in muscle, and insulin in adipocytes
 Stimulates PDH
during exercise (Ca2+)
and feeding (for lipid
synthesis)
 PDH is also regulated
by the ratio of
NADH/NAD+ and
acetyl CoA/CoA.
 Turn ‘off’ PDH if lots

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The Chemistry of Life 2021 Martin Welch

2. Citrate synthase: As mentioned previously this enzyme is allosterically

inhibited by ATP. Regulation at this point is important for gluconeogenesis
during ‘starvation.’ Oxaloacetate is used at the start of gluconeogenesis. If ATP
is in high concentration, rather than using oxaloacetate and acetyl-CoA to
produce citrate, inhibition of citrate synthase results in the diversion of
oxaloacetate to gluconeogenesis while acetyl-CoA is used to generate ketone
bodies, producing substrates for the rest of the body. Both pathways can be
used to fuel the brain for example.

3 + 4. Isocitrate dehydrogenase and a-ketoglutarate dehydrogenase

Isocitrate dehydrogenase
ICDH is inhibited by high
NADH/NAD+ ratio typical of the
fed state.
 Also stimulated by ADP,
and inhibited by ATP.

4. a-ketoglutarate dehydrogenase
 Inhibited by products
succinyl-CoA and NADH
 Stimulated by Ca2+

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The Chemistry of Life 2021 Martin Welch
5. The purine nucleotide cycle
When there are large quantities of acetyl-CoA present the concentration of
oxaloacetate may be rate limiting. The breakdown of ATP during strenuous
exercise can be used to generate fumarate (through several steps!) to prime the
cycle with more oxaloacetate. Patients with deficiencies in this pathway (the
purine nucleotide cycle) experience muscle cramps during exercise but apart
from this appear normal.

Neurotransmission and the Citric Acid Cycle: In brain tissue metabolism is

compartmentalised across cell types. In the majority of excitatory neuronal cells
glutamate is released as part of neurotransmission. In glial cells, this glutamate
is taken up and converted to glutamine to be returned to neuronal cells to
ensure the citric acid cycle is not depleted of carbon. In addition glial cells can
‘fix carbon’ using pyruvate carboxylase to generate more citric acid cycle
intermediates.

Fuels for long distance running: We obtain much more ATP from aerobic
metabolism compared with anaerobic. The body burns sugars initially but then
requires other substrates during prolonged exercise -‘fats’. This is especially
true in the heart (the ‘dustbin’ of the body in terms of substrate metabolism).

b-oxidation and fatty acid degradation

Fats represent the largest energy store in the body and are stored as
triacylglycerols (also called neutral fats or triglycerides), which are uncharged
esters of fatty acids with glycerol. Storage is in adipocytes and the liver, and
triacylglycerols are a particularly efficient way to store energy – requires less
water than glycogen and produces more energy following complete oxidation.

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The Chemistry of Life 2021 Martin Welch

Triglycerides are converted into glycerol and free fatty acids by a set of
enzymes called lipases. Oxidation of fats occurs in mitochondria via a process
called b-oxidation. This process converts an aliphatic fat into a set of activated
acetyl units (acetyl CoA) that can be processed by the citric acid cycle.

The steps are as follows: Fatty acids cleaved from their glycerol backbone are
activated using Co-Enzyme A to form acyl-CoA. The formation of a high energy
bond between CoA-SH and a fatty acid results in ATP being converted to AMP.
The overall reaction is made favourable because the PPi formed is hydrolysed
to Pi. This occurs outside the inner membrane of the mitochondria.There are
different enzymes for short, medium, and long chain fatty acids.

Activation occurs at the outer

mitochondrial membrane, catalysed by
carnitine acyltransferase I. The fatty acyl-
CoA cannot diffuse across the inner
membrane. The activated fatty acid is
carried across attached to carnitine. Once
inside the fatty acid is transferred back to
CoA-SH in a reaction catalysed by
carnitine acyltransferase II.

An activated fatty acid is oxidized to

introduce a double bond.
The double bond is hydrated (addition
of water) to introduce an oxygen; the
alcohol (from the water) is oxidized to
a ketone.
NB the similarity with succinate being
converted to oxaloacetate.
The four carbon fragment is cleaved by
coenzyme A to yield acetyl CoA and a
fatty acid chain two carbons shorter.
35 The process is repeated until the fatty
acid is completely converted into acetyl
CoA units.
The Chemistry of Life 2021 Martin Welch

The purpose of b-oxidation

As part of this process FADH2, NADH, and acetyl-CoA are all generated. Thus,
we can generate ATP by oxidation in the citric acid cycle (for acetyl-CoA) and
oxidative phosphorylation (for all the NADH and FADH2 we generate – see Prof.
Hibberd’s lectures). From palmitate ~106 ATP is produced according to the
reaction below.

 ~2.5 molecules of ATP for each of the 7 molecules of NADH.

 ~1.5 molecules of ATP for each of the 7 molecules of FADH2.
 oxidation of acetyl CoA by the citric acid cycle yields 10 molecules of
ATP (8 acetyl CoA)
 The total for palmitoyl CoA = 17.5 + 10.5 + 80 = 108 ATP.
 (2 molecules of ATP are consumed in the activation of palmitate so the
real total is 106 ATP)

Three metabolic facts of note about b-oxidation

Mammals cannot synthesize glucose from fat.
Acetyl-CoA produced for the citric acid cycle is completely oxidised to CO2. This
has implications for long term starvation – muscle break down to generate
glucose.
In starvation or diabetes the liver may produce more acetyl-CoA than can be
metabolised via the citric acid cycle. This is particularly likely when oxaloacetate
concentration decreases during gluconeogenesis. Under these conditions
ketone bodies, primarily acetoacetate and b-hydroxybutyrate, are formed in the
liver and released into the blood.

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The Chemistry of Life 2021 Martin Welch
The energy from fatty acid oxidation is more O2 intensive than that from glucose.
As a result fetal heart metabolism relies on glucose, but once there is a ready
supply of oxygen (and no longer such a danger of hypoxia (lack of oxygen in the
blood) associated with birth itself) the fetal heart switches to fatty acids soon
after birth.

The integration of metabolism

We have met the major metabolic pathways and are now in a position to
investigate how they interact in a physiological process such as running.

For exercise the metabolic changes are determined by energy reserves and
how long they last during a particular sporting event. Phosphocreatine (PCr) is
the first reserve used during sprinting. Explosive sports (sprinting, shot-put,
javelin) induce a 1000-fold increase in glycolysis. The phosphate produced by
breakdown of ATP is used by phosphorylase to produce glucose 1-P from
glycogen. This process is stimulated by Ca2+ (from muscle contraction) and
adrenaline.

During exercise AMP is formed by adenylate kinase. In turn AMP is then

deaminated to inosine monophosphate (IMP) which in turn stimulates
glycogenolysis. IMP is further degraded to adenosine which stimulates
vasodilation. Both of which allows us to continue using glycolysis during longer
bursts of anaerobic metabolism – e.g. 200-400 m sprint.

Mid-term and after exercise we begin to re-circulate lactate as glucose as part of

the Cori cycle.

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The Chemistry of Life 2021 Martin Welch

Long term exercise: During endurance races glycogen and fatty acids are
oxidized. The majority of fatty acids are released from adipose courtesy of
hormone sensitive lipase. In addition there is a small amount of fatty acid
released from the breakdown of muscle triglyceride. Fatty acids and glycogen
derived pyruvate both produce acetyl-CoA. This produces ATP via oxidative
phosphorylation and the citric acid cycle.

However, during exercise the supply of oxaloacetate can be outstripped by the

supply of acetyl-CoA (especially if we are metabolizing glucose and fat at the
same time). To produce more citric acid cycle metabolites the cell converts
isoleucine and valine to succinyl-CoA. Once glycogen is depleted we rely on fat
but this is a slower process for generating ATP. This is when the ‘Wall’ hits in
long distance running.

A Recap
The Citric Acid Cycle generates much larger amounts of ATP than glycolysis.
Acetyl-CoA introduces two carbons into the cycle, while two carbons leave as
CO2 each cycle.
The citric acid cycle is also central to integrating a number of metabolic
pathways including fatty acid metabolism, fatty acid synthesis, glycolysis,
gluconeogenesis and amino acid production.
The rate of the citric acid cycle can be monitored by a number of different
approaches including carbon labelling experiments, oxygen electrodes and
fMRI.

Appendix:
There are two major pathways which we did not have time to discuss. These are
mentioned here briefly for completion. This will not be directly examined as part
of Biology of Cells but may help you place the metabolic pathways we have met
in the lectures in context.

The pentose phosphate pathway: This pathway of glucose metabolism has three
special functions:

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The Chemistry of Life 2021 Martin Welch
1. It generates pentose sugars (ribose-5-phosphate) for nucleic acid synthesis.
2. It allows the reduction of NADP+ to NADPH for fat synthesis.
3. It is the route for the metabolic utilisation of pentose sugars.
This pathway occurs in tissues where there is a high demand for NADPH or pentoses
(e.g. liver, red blood cells, lactating mammary glands, and adrenal cortex in mammals).
This pathway should not be viewed as a pathway for the oxidation of glucose: it does
not produce ATP or oxidise glucose completely. Instead it balances the need of the cell
for NADPH or nucleotide synthesis.
The pathway consists of two parts (the oxidative and non-oxidative sections). By
balancing flux through either of these sections, tissues that only require NADPH (e.g.
adipose tissue synthesising fats) or pentose sugars (e.g. proliferative tissue producing
DNA) can focus on just this. The non-oxidative section is also important in the
metabolism of pentose sugars.

The Synthesis of Fatty acids: This is important for energy storage, lipid biosynthesis
and biosynthesis of compounds such as certain steroids. The steps are essentially
similar to b-oxidation, but in reverse. However, as we have seen before, we cannot just
simply reverse b-oxidation. Synthesis occurs outside mitochondria (in the cytosol). The
precursor for acetyl-CoA and ultimately activated malonyl group is citrate.

Fatty acid synthesis begins with acetyl-CoA. While acetyl-CoA is produced in

mitochondria it has to be transported into the cytosol as citrate (using citrate lyase to
reform the acetyl-CoA in the cytosol – this step also generates NADPH which is used in
fatty acid synthesis). To drive the reaction necessary to begin elongation of the fatty
acid chain, the acetyl-CoA is initially converted into malonyl-CoA (malonyl contains 3
carbons), by carboxylation.

A multienzyme complex called fatty acid synthase is used to produce fats. This enzyme
complex has two positions to accept units from acyl-CoA derivatives. At the start of the
cycle, one position receives an acetyl group from acetyl-CoA, while the other is
occupied by malonyl (from malonyl-CoA). During the donation reaction, CO2 is released

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The Chemistry of Life 2021 Martin Welch
from malonyl-CoA; with this mechanism ensuring that fatty acid synthesis is an
irreversible pathway.

The next step is reduction, which converts one C=O into a CH2 group. This step
requires two NADPH (one from the conversion of citrate to acetyl-CoA, and the other
from the pentose phosphate pathway). Following this another malonyl group is
introduced to the complex and the fat is then elongated by another C2 unit again. In this
manner saturated fatty acids are synthesized.

Understanding metabolic control (note this is the fourth year this material has
appeared in the appendix and is no longer considered core to the CoL lectures but
may help to understand the subject in more detail)
We have met a range of pathways in the chemistry of life lecture series so far
(and more is to come!). Traditionally these pathways have been taught in
isolation. However, the current drive in metabolic research is understanding how
they interact. The key question is how do we go from a pathway to a network of
metabolism?

Thermodynamics and metabolic networks:

We have seen that thermodynamics can help us understand whether a
particular reaction can go or not. We can extend this analysis to the complete
metabolic network, for example placing upper limits on concentrations within the
cell. This type of analysis has been applied to E. coli, yeast and even cancer in
humans.
The example shown in the lecture is from a recent publication in Nature
Chemical Biology (Bennet BD et al., (2009) Nature Chem Biol) who investigated
the relationship between metabolite concentrations and the Km values of their
consuming enzymes in E. coli. This demonstrated that for most metabolites their
concentration was very much greater than the corresponding Km value.
However, enzymes in central carbon metabolism (the citric acid cycle in
particular) are notable exceptions demonstrating their important role in
regulating global metabolism within the cell.

Metabolic control analysis (MCA):

Analysis of metabolic enzymes separately cannot generate a complete picture
of how the overall system is controlled. The concept of demand control has
been used to analyse the flow through pathways. An example of this is that
increasing the enzymes for ethanol production in yeast does not increase the
amount of ethanol produced but increasing the need for ATP does. Metabolic
control analysis attempts to predict the effect of varying one or more of the
enzymes of the pathway. The net flux of a pathway can be very different to the
flux through a given enzyme. In the below pathway enzymes A and C catalyse
reactions which are close to equilibrium. If the concentration of the enzyme
increases it will have little effect on the total flux. Enzyme B catalyses a reaction
far from equilibrium. This enzyme has the potential to control flux in the
pathway. This field of research is also referred to as metabolic flux analysis
(MFA) in some publications.

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The Chemistry of Life 2021 Martin Welch

For glycolysis: Although ‘a key control enzyme’ is phosphofructokinase-1, the

rate of lactate production is not simply controlled by this enzyme because of the
influence of all the other enzymes in the pathway. Feedback inhibition may
operate and, in addition, metabolites may flow into other pathways. MCA
demonstrates that a large number of parameters affect the rate of lactate
production from glucose-6-phosphate and there is no rate determining step!

Metabolomics:
Clearly, we need to consider how pathways interact but how can we measure a
range of metabolites? Metabolomics is the ‘quantitative measurement of
metabolic responses to pathophysiological stimuli or genetic modification.’ The
approach measures the small molecule concentrations through a global
approach and uses pattern recognition to define a metabolic phenotype
(metabotype) associated with the disease or genotype.

Global Profiling tools:

A variety of different analytical tools have been used to measure a wide range of
metabolites in a given cell, biofluid, tissue or even organism. In each case the
aim is to gain a snapshot of a large number of steady state concentrations in
order to describe the metabolic state the cell is in.
 NMR spectroscopy. This is used either in solution state or indeed as part
of in vivo NMR spectroscopy. It is high throughput and relatively robust,
although it has low sensitivity.
 Gas Chromatography (GC)- and Liquid Chromatography (LC)-Mass
spectrometry (MS). This is a more sensitive approach than NMR,
although because the mass spectrometers get dirty, reproducibility can
be an issue, especially in large datasets. LC-MS can look at larger
macromolecules like intact lipids.
 Fourier Transform-Mass Spectrometry – this is a very powerful form of
mass spectrometry for identifying unknowns. Often it is not performed
with chromatography (see LC-MS and GC-MS above) but samples are
injected as complex mixtures with no chromatographic separation.
 Coulombic arrays – this operates in conjunction with a liquid
chromatography and metabolites are detected by the virtue of the redox
properties. This makes this approach very suitable for certain types of
reactive metabolites.
 Fourier Transform – Infra Red (FT-IR) spectroscopy. While not terribly
discriminatory this is being used to image cancers in vivo.
 Thin Layer Chromatography – often used to separate out different lipid
species which can then be investigated by GC-MS, LC-MS or other
suitable approaches.

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The Chemistry of Life 2021 Martin Welch
 Metabolite arrays
 Use biochemical assays. This is being used to phenotype E. coli
using a 96-well plate format for the biochemical assays.
Currently GC-MS is being used to profile metabolites in plants. One of the
analytical challenges associated with this approach is the large number of
metabolites that are present in plant kingdom, with ~50,000 metabolites having
been identified with this number set to rise to ~200,000 (c.f. 20,000-50,000
genes in a typical plant genome). Fiehn et al. used GC-MS to quantify 326
distinct compounds in Arabidopsis thaliana leaf extracts (chemical structure to
half of these). Pattern recognition processes were then used to separate out
different mutants and eco-types (plants that grow in a particular ecological
niche). (Fiehn O et al. 2000 Nature Biotechnology, 18, 1157-1161).

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