Module 1 - Basic Concepts and Design of Metabolism

15.1 Energy is Required to Meet 3 Fundamental Needs

Living organisms require a continual input of free energy for 3 major purposes:

(1) Performance of mechanical work in muscle contraction and cellular movements

(2) Active transport of molecules and ions
(3) Synthesis of macromolecules and other biomolecules from simple building blocks

15.2 Metabolism is Composed of Many Interconnecting Reactions

Metabolism = the enzyme catalyzed chemical reactions that occur in a cell to either
breakdown biomolecules to obtain energy (ATP), or reactions that synthesize biomolecules
- Metabolic pathways process a biomolecule from a starting point to an end point
- E.g. Glycolysis, starts w/ glucose, and converts it into carbon dioxide, water,
and energy
- The metabolic pathway of glycolysis starts w/ glucose and through a series of
reactions is converted to pyruvate
- Metabolic pathways can be linear, branched, or circular

Some metabolic pathways occur simultaneously within a cell

- Important: metabolic pathways can be interconnected; an intermediate of one
pathway can also be an intermediate in one or more pathways, which connects them
- Two or more pathways that are connected can still be regulated independently by
intricate and sensitive mechanisms

A given reaction within a pathway can be reversible or irreversible, depending on the reaction
- Important: the first committed step, which is often the first reaction in the pathway, is
usually a point of regulation for that pathway
- Reactions that are regulated in a pathway are often rate-limiting steps (i.e. slow
reactions that can be increased by a variety of mechanisms)

Some pathways can function anabolically AND catabolically, depending on the energy
conditions of the cell
- These are called amphibolic pathways
- E.g. The citric acid cycle is amphibolic

There are many metabolic pathways, but there are a limited number of types of reactions and
certain intermediates common to many pathways
Metabolism consists of energy yielding reactions and energy requiring reactions

We can divide metabolic pathways into two classes:

1) Those that convert energy into useful forms such as ATP or ion gradients
2) Those that require the input of energy to proceed

- Those that transform fuel into cellular energy are called catabolic reaction, or
catabolism
- E.g. Fuel (carbohydrates, fats) → (catabolism) CO2 + H2O + energy

- Those reactions that require energy such as the synthesis of glucose, fats, or DNA, are
called anabolic reactions, or anabolism
- Energy produced in catabolism are used in anabolism to generate complex structures
from building blocks or to create energy-rich states from energy-poor ones
- E.g. Useful energy + simple precursor → (anabolism) complex molecules

!! Metabolism is the sum total of Catabolism and Anabolism !!

The primary functions of metabolism are:

1. To obtain chemical energy in the form of ATP from the degradation of energy rich
nutrients from the environment (food) or from captured solar energy (light).
a. E.g. Food that you eat is used to move your muscles or use your brain, both of
which require energy
2. To convert molecules that we ingest as food into the building blocks (amino acids,
nucleotides, etc) of larger molecules needed in the body
3. To assemble the small building block molecules (amino acids, nucleotides, etc) into
larger molecules such as proteins, nucleic acids, lipids, polysaccharides
4. To synthesize and degrade biomolecules that have specialized functions in cells

An important general principle of metabolism: although biosynthetic (catabolic) and

degradative (anabolic) pathways often have reactions in common (i.e. common
intermediates), the regulated, irreversible reactions of each pathway are almost always
distinct from one another

Thermodynamically Unfavourable Reactions Can be Driven by a Favourable Reaction:

Important: enzymes only speed up the rate of a chemical reaction, but do not change the
equilibrium constant for reactions and thus cannot make a thermodynamically unfavourable
reaction proceed
- I.e. enzymes do not affect the equilibrium constant for a given reaction, they only
speed up the rate at which the reaction reaches equilibrium
A good analogy of this is the reaction is a bowling ball:
- Going downhill, it can occur on its own w/o any energy input (i.e. spontaneously)
although an enzyme greatly speeds up the rate at which the bowling ball travels down
the decline
- However, no bowling ball can spontaneously (on its own) move up an incline, and no
enzyme can ‘push’ it up

Thermodynamics in terms of metabolism are approached in terms of free energy (𝚫G)

- A reaction can spontaneously take place (w/o the input of energy) if 𝚫G is negative!
- These reactions release energy and are exergonic ( = energy releasing)
- Reactions that have a positive 𝚫G cannot proceed unless there is an input of energy
- This often involves the cleavage of ATP
- These reactions are endergonic ( = energy requiring)
- When a system is at equilibrium, there is no net change in the concentrations of
products and reactants, and 𝚫G is 0
- The 𝚫G is of a reaction is independent of the path where reactants are converted to
products
- The 𝚫G provides NO information about the rate of a reaction - only whether the
reaction will/will not occur spontaneously

The overall free-energy change for a series of reactions (called coupled reactions) is equal to
the free energy changes of the individual steps . . . For example:

A⇄B+C 𝚫G = +21 kJ mol -1

B ⇄ D 𝚫G = -34 kJ mol -1
—------------------------
A ⇄ C + D 𝚫G = - 13 kJ mol-1

- Under normal conditions, A cannot be spontaneously converted into B and C bc 𝚫G

is positive
- Under normal conditions, B can be spontaneously converted into D bc 𝚫G is negative
- However, free-energy changes are additive, and the conversion of A into C and D has
a 𝚫G of -13 kJ mol-1, which means it occurs spontaneously!!
- Thus, thermodynamically unfavourable reactions (like step 1), can be driven by a
thermodynamically favourable reaction (step 2) to which it is coupled
- In this example, the reactions are coupled by sharing the intermediate B
Determining 𝚫G:
To determining 𝚫G, we need to take into account the nature of the reactants and the products,
as well as their concentrations. Consider:

A+B ⇄C+D

The 𝚫G of the forward reaction is given by: 𝚫G = 𝚫G° + RT ln [C] [D] / [A] [B]
𝚫G° is the standard free energy change
R is the gas constant
T is the absolute temperature
[ A, B, C, D ] are the molar concentrations of the reactants and products

How is 𝚫G° calculated?

- A simple way is to measure the concentrations of reactants and products when the
reaction has reached equilibrium, and thus 𝚫G is zero.
- Depends on the nature of the reactants (𝚫G°) and on their concentrations (2nd term)
- 0 = 𝚫G° + RT ln [C] [D] / [A] [B] → so, subtracting 𝚫G° we have:
- 𝚫G° = - RT ln [C] [D] / [A] [B] → since Keq (equilibrium constant) = [C][D] / [A]
[B]
- 𝚫G° = -RT ln Keq → also written as 𝚫G° = -2.3RTlog Keq

Therefore:
When Keq = 1 𝚫G° = 0 at equilibrium
Keq = < 1 𝚫G° = a positive value, does not favour the forward reaction but favours the
reverse reaction
Keq = > 1 𝚫G° = a negative value, favours the forward reaction (spontaneous reaction)

15.3 ATP is the Universal Currency of Free Energy:

ATP Hydrolysis is Exergonic:

- ATP is a nucleotide consisting of adenine, a ribose, and a triphosphate unit
- ATP is an energy rich molecule bc its triphosphate unit contains 2
phosphoanhydride linkages
- Phosphoanhydride linkages (or anhydride bonds) are formed between 2 phosphoryl
groups, as well as the loss of a water molecule
- Large amounts of energy are released when ATP is:
- 1) Hydrolyzed into adenosine diphosphate (ADP) and orthophosphate (Pi)
- ATP + H2O ⇄ ADP + Pi 𝚫G = -30.5 kJ mol-1
- 2) Hydrolyzed into adenosine monophosphate (AMP) and pyrophosphate (PPi)
- ATP + H2O ⇄ AMP + PPi 𝚫G = -45.5 kJmol-1
 - The free energy released from ATP hydrolysis is used to drive reactions that require
an input of free energy such as muscle contraction
- In turn, ATP is formed from ADP and Pi when fuel molecules are oxidized in
chemotrophs (heterotrophs) and light energy in phototrophs (photosynthesis)

ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactions:

- Unfavourable reactions (endergonic → energy requiring) can be made possible by
coupling ATP hydrolysis
- In the cell, the hydrolysis of an ATP molecule in a coupled reaction changes the
equilibrium ratio of products to reactants by a large factor

The High Phosphoryl-Transfer Potential of ATP Results from Structural Differences

Between ATP and its Hydrolysis Products:

What makes ATP an efficient energy currency?

- ATP has a high phosphoryl-transfer potential, meaning it readily transfers its
phosphate group to water
- The standard free energy from the hydrolysis of ATP, where the terminal
phosphate group is cleaved to yield ADP and inorganic phosphate (Pi) is very
negative ( -30.5 kJ / mol)
- ATP + H2O ⇄ ADP + Pi 𝚫G° = =30.5 J/mol

- We need to compare the tendency of various organic compounds w/ phosphoryl

groups that transfer phosphoryl groups to an acceptor molecule
- A common means of comparison is to determine the amount of energy released when
the phosphorylated compound transfers the phosphoryl group to water
- The energy released from this reaction is called the standard free energy of
hydrolysis

- Looking at 𝚫G of glycerol-3-phosphate (-9.2 kJ mol-1) compared to 𝚫G of ATP (-

30.5 kJ mol-1) it is much smaller
- This means the tendency of ATP to transfer its phosphoryl group to water is much
stronger than glycerol-3-phosphate, therefore it has a larger phosphoryl-transfer
potential
This large standard free energy change is due to large differences in stability of the
products compared to the reactants; there are 4 reasons:

1) Electrostatic Repulsion - ATP carries 4 negative charges, which repel one another
- When ATP is hydrolyzed, the electrostatic repulsion is reduced
2) Resonance Stabilization - Pi (product of ATP hydrolysis) has greater resonance
stability than other phosphates in ATP
3) Increase in Entropy - the entropy of the products is greater (bc there are now 2
molecules, rather than just a single ATP molecule)
4) Stabilization Due to Hydration - Water binds to ADP and Pi , stabilizing these
molecules
- Makes the reverse reaction less favourable

Sometimes, ATP is hydrolyzed to release pyrophosphate, which is 2 phosphate groups joined

together, resulting in AMP as a product. This results in large amounts of energy being
released, since it is also an acid anhydride bond that gets cleaved !!

Phosphoryl-Transfer Potential is an Important Form of Cellular Energy

Transformation:
- ATP is not the only compound w/ a high phosphoryl-transfer potential, and some are
even higher
- Phosphoenolpyruvate (PEP, 1,3-bisphosphoglycerate (1,3-BPG), and creatine
phosphate have high phosphoryl-transfer potential
- PEP can transfer its phosphoryl group to ADP to form ATP!

Phosphates Play a Prominent Role in Biochemical Processes:

Phosphate plays a prominent role bc . . .
- 1) Phosphate esters are thermodynamically unstable while being kinetically stable -
thus phosphate esters’s energy release can be manipulated by enzymes
- 2) Phosphate ester’s stability is from the negative charges that make them resistant to
hydrolysis in the absence of enzymes
- 3) Phosphate esters are kinetically stable, and they can be added by kinases and
removed only by phosphatases
- No other ions have the chemical characteristics of phosphate

15.4 The Oxidation of Carbon Fuels is an Important Source of Cellular Energy:

- ATP serves as an immediate free energy source, and not as long term storage
- ATP molecules are immediately consumed within a minute of its formation
 - There needs to be a mechanism for regenerating ATP
- The generation of ATP from ADP is one of the primary roles of catabolism
Carbon Oxidation is Paired w/ a Reduction:
- The carbon in ‘fuel molecules’ - such as glucose and fats - is oxidized to CO2 and the
energy released is used to regenerate ATP from ADP and Pi
- All oxidation reactions include the loss of electrons from the molecule being oxidized
(carbon fuels in this case) and the gaining of those electrons by another molecule
- This process is called reduction
- In some cases, the loss or gain of electrons can be accompanied by the loss or
gain of protons
- These reactions are called oxidation-reduction or redox reactions
- The more reduced a carbon is to begin w/, the more free energy is released by its
oxidation
- In aerobic organisms, the electron acceptor in the oxidation of carbon is O2, and the
oxidation product is CO2

- When a fuel is oxidized, the oxidation takes place one carbon at a time
- Carbon-oxidation energy is used to create a compound w/ high phosphoryl-transfer
potential as well as to create an ion gradient
- In either case, the end point is to create ATP

Two major sources of fuel that we use are glucose and fatty acids!
- Fatty acids are far more reduced than glucose
- i.e. have far FEWER oxygens than glucose and far MORE C-H bonds that are
a source of electrons
- This is why the degradation of fatty acids yield more energy than glucose

Compounds w/ High Phosphoryl-Transfer Potential Can Couple Carbon Oxidation to

ATP Synthesis:
How is energy released in the oxidation of a carbon compound converted into ATP?
Example: glyceraldehyde-3-phosphate
- The C1 atom of an aldehyde is capable of further oxidation, and oxidation of the
aldehyde to an acid will release energy!
- However, oxidation does not take place directly. Instead, the carbon oxidation
generates an acyl phosphate, 1,3-BPG - which releases electrons
- The released electrons are captured by NAD+
- 1,3 BPG has a high phosphoryl-transfer potential, meaning its cleavage can be
coupled to the synthesis of ATP (like PEP!)
- Thus, the energy of oxidation is initially trapped as a high-phosphoryl-transfer
potential compound, and then used to form ATP
- The oxidation energy of the carbon atom is transformed into phosphoryl
transfer potential, first as 1,3 BPG and then as ATP
The oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate occurs in 2 steps:
- In the first step, the oxidation of glyceraldehyde-3-P results in electrons released
captured by NAD+ to form NADH
- NADH donates its electrons in a process called oxidative phosphorylation that will
result in ATP synthesis
- The other product, 1,3-BPG transfers a phosphate group to ADP to form ATP
- This redox reaction illustrates how the energy of oxidation is initially trapped as a
high phosphoryl-transfer potential compound and then used to form ATP
- ATP synthesis that results from the transfer of a phosphate group from one
high phosphoryl transfer potential compound to ADP is termed substrate
level phosphorylation

15.5 Metabolic Pathways Contain Many Recurring Motifs:

Activated Carriers Exemplify the Modular Design and Economy of Metabolism:

- Many phosphoryl transfers can be used to drive thermodynamically unfavourable
reactions, can alter the energy or conformation of a protein, and can signal to alter the
activity of a protein
- The phosphoryl group donor in all the above reactions in protein, meaning ATP is the
activated carrier of phosphoryl groups bc phosphoryl transfer from ATP is an
energetically favourable, or exergonic, process
- The use of activated carriers is a recurring motif in biochemistry
- Many function as coenzymes (cofactors of enzymes)

Activated Carriers of Electrons for Fuel Oxidation:

- In aerobic organisms, the electron acceptor is O2
- However, electrons are not transferred directly to O2, instead fuel molecules (glucose,
fats) reduce or transfer electrons to special carriers: pyridine nucleotides or flavins
- The reduced forms of these special carriers then transfer the electrons to
oxygen
- These carriers have higher affinity for electrons than carbon fuels, but a lower affinity
for electrons than O2
- Electrons than flow from unstable configuration (low affinity) to a stable one
(high affinity)
- The ability of electrons to flow to a more stable configuration is what accounts
for them being ‘activated’

- Nicotinamide adenine dinucleotide (NAD+), a pyridine nucleotide, is a major

electron carrier
- The reactive part of NAD+ is the nicotinamide ring - in the oxidation of a substrate,
the nicotinamide ring accepts a hydrogen ion and two electrons, which is equivalent to
a hydride ion (H+)
 - The reduced form of this carrier is NADH

- Flavin adenine dinucleotide (FAD) is the other major electron carrier in the
oxidation of fuel molecules, and is a coenzyme
- The oxidized and reduced forms of this carrier are FAD (electron acceptor) and
FADH2
- The reactive part of FAD is the isoalloxazine ring, which can accept two electrons and
protons

Activated Carriers of Electrons for the Synthesis of Biomolecules:

- High potential electrons are required for anabolic (breakdown) reactions
- Precursors (building blocks) are more oxidized than the final products, meaning
reducing power (or a need for electrons) is needed in addition to ATP
- This process is called reductive biosynthesis

The primary electron donor used in biosynthetic processes is NADPH. It is similar in

structure to NADH except that it has a phosphate group as well!
- NADPH is the electron donor, NADP+ is the oxidized form
- Important: NADPH is used almost exclusively for reductive biosyntheses, whereas
NADH is used primarily for the generation of ATP
- However both are providers of electrons
- The extra phosphoryl group on NADPH is a tag that allows enzymes to tell between
high potential electrons to be used in anabolism vs those to be used in catabolism

Activated Carriers of Two-Carbon Fragments:

- Coenzyme A, a central molecule in metabolism, is a carrier of 2-carbon units called
acyl groups
- It carries a pantothenic acid moiety, which is a B vitamin
- Acyl groups are important in . . .
- Catabolism - oxidation of fatty acids
- Anabolism - synthesis of membrane lipids
- Acetyl groups are attached to CoA through the reactive sulfhydryl group to form the
molecule acetyl CoA

- The hydrolysis of acetyl CoA (which releases acetyl groups) has a very large negative
standard free energy change and is thermodynamically favourable
- Consequently, acetyl CoA has a high acetyl-group-transfer potential bc transfer of
the acetyl group is exergonic
- I.e. Acetyl CoA carries an activated acetyl group, just as ATP carries an activated
phosphoryl group

Note: Most activated carriers that act as coenzymes are derived from vitamins, particularly
B vitamins
15.6 Metabolic Processes are Regulated in 3 Principal Ways:

Metabolism is regulated through control of (1) the amounts of enzymes, (2) their catalytic
activities, (3) the accessibility of substrates

The amounts of enzymes are controlled:

- The amount or abundance of an enzyme that catalyzes a regulated step in the pathway
can be regulated.
- Since enzymes are proteins, and thus coded for genes, the amount of an enzyme in a
cell can be controlled by affecting the expression of the gene that codes for it

Catalytic activity is regulated:

- Catalytic activity of the enzyme can be regulated in two ways:
- 1) Allosteric regulation - involves the binding of inhibitors or activators to the enzyme
that will either decrease or increase the activity of the enzyme
- 2) Covalently modified - occurs in response to hormones and other signals that can
lead to a change in the activity of an enzyme
- E.g. The addition of a phosphate group to a specific amino acid residue of an
enzyme will alter its activity

The Accessibility of Substrates is Regulated:

- Controlling the availability of substrates is another means of regulating a pathway
- E.g. Fatty acid breakdown occurs in the mitochondria, which means that fatty acids
that are present in the cytosol need to be transported into the mitochondria before they
can be degraded. Thus, the rate of fatty acid breakdown is controlled in part by
regulating the rate at which fatty acids are transported into the mitochondria
 Module 2 - Glycolysis:

16.1 Glycolysis is an Energy Conversion Pathway

- The glycolytic pathway is found in nearly all prokaryotic and eukaryotic cells
- In eukaryotic cells, glycolysis takes place in the cytoplasm/cytosol. This means as
soon as glucose is transported into the cell, enzymes of glycolysis can immediately act
on it.
- Glycolysis is the sequence of reactions that converts one molecule of glucose to two
molecules of pyruvate
- Glycolysis serves 2 functions:
- 1) It generates ATP
- 2) A number of intermediates of this pathway serve as building blocks for the
biosynthesis of other biomolecules, such as amino acids and fatty acids

The glycolytic pathway is central as it is involved in metabolizing glucose, a fuel used by

almost all organisms
- It takes glucose, a 6-carbon molecule, and through a series of reactions converts it to a
3-carbon molecule of pyruvate
- During this conversion, 2 ATP are consumed in the first stage, and in the second
stage, there are a total of 4 ATP produced per molecule of glucose
- 2 ATP / 3-carbon molecule of pyruvate (produces 2 pyruvate → 4 ATP)
- Thus, glycolysis generates a net of 2 ATP / molecule of glucose

Where do we get glucose?

- We eat very little free glucose
- Most dietary CHO is in the form of starch, a polymer of glucose
- We also get glucose from the digestion of lactose, a disaccharide found in milk, from
the degradation of sucrose found in table sugar, and from the breakdown of maltose (a
product of starch breakdown) and rich in foods that ferment by yeast

How does glucose get into the cell?

- Glucose doesn’t just diffuse across the cell - there are specific transporters that assist
- In mammals, there are several glucose transporters that have different functions and
are present in different tissues
- GLUT1 (all tissue), GLUT 2 (liver and pancreatic B cells), GLUT3 (all tissue),
GLUT4 (muscle and fat cells), GLUT5 (small intestine)
Glycolysis - 10 Step Reaction!

1. Kinase reaction (hexokinase): adds a phosphate onto glucose, forming glucose-6-

phosphate
a. This is one of two energy consumption reactions (ATP using) and is
irreversible

2. Isomerase reaction (phosphoglucose isomerase): converts glucose-6-phosphate into

fructose-6-phosphate by rearranging covalent bonds

3. Kinase reaction (Phosphofructokinase): removes a phosphate from ATP and gives it

to fructose-6-phosphate, to form fructose-1,6-bisphosphate
a. This is the second energy consumption reaction (ATP using) and is
irreversible

4. Lyase reaction (fructose bisphosphate aldolase): splits the 6-carbon sugar (fructose-
1,6-bisphosphate) into two 3-carbon sugars dihydroxyacetone phosphate and
glyceraldehyde-3-phosphate

5. Isomerase reaction (triosephosphate isomerase): dihydroxyacetone phosphate is

rearranged by another isomerase to form a second glyceraldehyde-3-phosphate

- At this point, glucose has been metabolized into 2 glyceraldehyde-3-phosphate

molecules, and 2 ATP have been consumed -

6. Dehydrogenase reaction (glyceraldehyde phosphate dehydrogenase): both

glyceraldehyde-3-phosphate molecules are oxidized to 1-3-bisphosphoglycerate by a
dehydrogenase
a. This produces one NADH for each oxidized glyceraldehyde-3-phosphate for a
total of 2 NADH
b. These NADH are used later to produce more ATP

7. Kinase reaction (phosphoglycerate kinase): a kinase transfers a phosphate from 1-3-

bisphosphoglycerate to ADP to form ATP and 3-phosphoglycerate
a. This step is reversible

8. Mutase reaction (phosphoglycerate mutase): moves a phosphate on the 3rd carbon of

3-phosphoglycerate to the 2nd carbon position to form 2-phosphoglycerate

9. Lyase reaction (enolase): removes water from 2-phosphoglycerate to form

phosphoenolpyruvate
 10. Kinase reaction (pyruvate kinase): removes the phosphate group from
phosphoenolpyruvate and donates it to ADP to form ATP and pyruvate
a. This step is irreversible

- At this point, 2 pyruvate molecules, 4 ATP’s and 2 NADH’s are formed / glucose
molecule -

Glycolysis is divided into two stages

Stage 1: trapping and preparation stage (no ATP generated)
- Begins w/ the conversion of glucose into fructose 1-6-bisphosphate
- Occurs in 3 steps: phosphorylation, isomerization, a second phosphorylation
- The strategy is to trap glucose into the cell and to form a compound that can easily be
cleaved into phosphorylated 3-carbon units
- The end of this stage is the cleavage of fructose 1-6,bisphosphate into two
phosphorylated 3-carbon units

Stage 2: ATP is harvested when the 3-carbon fragments are oxidized to pyruvate

Stage 1 - Step 1 - Phosphorylation of glucose

Glucose enters the cell by transport proteins, where it is rapidly phosphorylated by
hexokinase to form glucose-6-phosphate
- This step is important - phosphorylation of glucose traps it in the cell and prevents it
from being transported out
- Kinases = enzymes that phosphorylate molecules using ATP as the phosphate donor
- I.e. enzymes that transfer a phosphoryl group from ATP to an acceptor
- Hexokinases catalyze the transfer of a phosphoryl group from ATP to a 6-carbon
sugar, called hexoses, such as glucose and mannose
- Humans have over 500 different kinases that perform various phosphorylations
- This reaction is irreversible

Stage 1 - Step 2 - Isomerization

Glucose-6-phosphate is converted to fructose-6-phosphate
- This is an isomerization reaction, meaning there are no atoms lost but a
rearrangement of the atoms occur
- Remember: Glucose has an aldehyde group at carbon 1, and fructose has a ketose
group at carbon 2. Thus, the isomerization of glucose-6-phosphate into fructose 6-
phosphate is the conversion of an aldose into a ketose
- Note: converted into fructose 6 phosphate, not fructose 1-6-bisphosphate
- This reaction is catalyzed by phosphoglucose isomerase
- Isomerization is crucial bc only 3-carbon molecules are metabolized in later stages of
glycolysis. Glucose-6-phosphate is not easily cleaved into two 3-carbon molecules,
but fructose-6-phosphate is!!
 - This reaction is reversible

Stage 1 - Step 3 - Phosphorylation of Fructose-6-Phosphate

Fructose-6-Phosphate is phosphorylated at the second carbon to form fructose-1,6-
bisphosphate
- This reaction is catalyzed by phosphofructokinase (PFK) which is an allosteric
enzyme
- This reaction is irreversible

Stage 2 - Step 4 - Cleavage of fructose-1,6,-bisphosphate

Cleavage of fructose-1,6-bisphosphate into two 3-carbon molecules: dihydroxyacetone
phosphate and glyceraldehyde-3-phosphate
- This reaction is catalyzed by aldolase
- The products of the remaining steps in glycolysis consist of 3 carbon units rather than
6 carbon units
- Glyceraldehyde-3-phosphate can move directly onward in the glycolytic pathway
- Dihydroxyacetone phosphate cannot move forward directly, but is not wasted or lost,
as it can be readily converted to glyceraldehyde-3-phosphate by an isomerase in a
reversible reaction

Stage 2 - Step 5 -
- Isomerase reaction (triosephosphate isomerase): dihydroxyacetone phosphate is
rearranged by another isomerase to form a second glyceraldehyde-3-phosphate

Stage 2 - Step 6 - Oxidation of glyceraldehyde-3-phosphate

Oxidation of glyceraldehyde-3-phosphate powers the formation of 1,3-bisphosphoglycerate
which has a high phosphoryl-transfer potential
- This is catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase
- Dehydrogenases = enzymes that catalyze oxidation reduction reactions
- The net result of this reaction is that the phosphate group is attached to a carboxylic
acid group via a mixed acid anhydride bond. This bond has a high phosphoryl-
transfer potential

Stage 2 - Step 7 - Phosphoryl transfer -first energy producing step-

Phosphoryl transfer from 1,3-bisphosphoglycerate to ADP to form ATP and 3-
phosphoglycerate
- It can do this bc of the high phosphoryl-transfer potential of the mixed acid anhydride
bond
- This is the first energy producing step in glycolysis
- This reaction is catalyzed by phosphoglycerate kinase
 - ATP formed this way is called substrate level phosphorylation, since the donor of the
phosphate group is a substrate in the reaction
- **Remember, there are two ATP’s produced at this step for every molecule of
glucose, since one glucose generates two 3-carbon molecules (glyceraldehyde-3-P)
Stage 2 - Step 8 - Mutase reaction
- 3-phosphoglycerate kinase is converted to 2 phosphoglycerate by phosphoglycerate
mutase
- Mutases = isomerases that reposition phosphate groups within a molecule

Stage 2 - Step 9
- 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP) by the enzyme
enolase
- PEP is a high phosphoryl-transfer molecule

Stage 2 - Step 10
- Phosphoenolpyruvate (PEP) transfers its phosphate group to ADP to form ATP and
Pyruvate
- This reaction is catalyzed by pyruvate kinase
- This is another example of substrate level phosphorylation

Two ATP molecules are formed in the conversion of glucose into pyruvate
The net reaction of glucose into pyruvate is:
Glucose + 2Pi + 2ADP + 2NAD+ → 2 Pyruvate + 2 ATP + 2NADH + 2H+ + 2H2O

- The overall free energy change is about -22 kcal/mol

- This means that although individual steps may be reversible, glycolysis as a pathway
flows in one direction (the forward direction) towards the production of pyruvate

The pathway from glucose to pyruvate has no requirement for oxygen, which is why it is
referred to as anaerobic glycolysis

16.2 NAD+ is Regenerated from the Metabolism of Pyruvate:

- The conversion of glucose into 2 molecules of pyruvate results in the net synthesis of
ATP
- However, an energy converting pathway that stops at pyruvate will not proceed for
long, bc the redox balance has not been maintained
- This imbalance is caused by glyceraldehyde-3 phosphate dehydrogenase, which leads
to the reduction of NAD+ to NADH when glyceraldehyde 3 phosphate is oxidized.
- In the cell there are limited amounts of NAD+ (which is derived from the vitamin
niacin). In the reaction catalyzed by glyceraldehyde-3-P dehydrogenase, a molecule of
NAD+ is consumed which is not generated anywhere else in the pathway
 - Consequently, NAD+ needs to be regenerated for glycolysis to proceed
- The metabolism of pyruvate achieves this, although HOW this occurs is variable
depending on the organism and whether oxygen is available

There are three possible fates of pyruvate:

- The fate of pyruvate is variable, three reactions of pyruvate are of primary
importance: conversion into ethanol, lactate, or carbon dioxide and water
- The first two reactions (conversion to ethanol and lactate) are fermenations that take
place in the absence of oxygen
- Fermentation = ATP generating processes in which organic compounds act as both
donors and acceptors of electrons
- In most multicellular organisms and many unicellular organisms, the most common
situation is the conversion of pyruvate to carbon dioxide and water through the citric
acid cycle and the electron transport chain
- Oxygen accepts electrons and protons to form water

Fate #1 - Ethanol
- Pyruvate can be converted to ethanol: this pathway occurs in some microorganisms
and in yeast
- Most importantly, this reaction regenerates NAD+ is the second step of this
conversion
- The conversion of pyruvate to ethanol consists of a decarboxylation (loss of CO2)
reaction to form acetaldehyde and a redox reaction where acetaldehyde becomes
reduced to ethanol from electrons donated from NADH
- This reaction regenerated NAD+

- The conversion of glucose into ethanol is an example of alcoholic fermentation

- There is no net oxidation-reduction since the NAD+ used in glycolysis is regenerated
by the production of ethanol (i.e. there is so ‘profit’)
- The conversion of glucose into ethanol is an example of alcoholic fermentation
- This reaction is anaerobic

Fate #2 - Lactate
- Lactate is formed from pyruvate in a variety of microorganisms
- Pyruvate can be directly converted to lactate: in this pathway, which consists of only
one reaction, NADH is used to reduce pyruvate to lactate, in the process of
regenerating NAD+
- The process is called lactic acid fermentation
- The conversion of glucose to lactate is also redox neutral (i.e. no net oxidation
reduction / / no ‘profit’ )
- This reaction is anaerobic
Pyruvate to lactate also occurs in muscles when oxygen supply becomes limiting, forcing the
muscles to function anaerobically for short periods of time. As lactate builds up in the
muscle, muscle fatigue occurs. The lactate lowers the pH of the muscle as low as 6.3, which
inhibits phosphofructokinase, the main regulatory point of glycolysis. This slows the ability
of muscles to metabolize glucose for energy purposes, and fatigue sets in.
Fate #3 - Acetyl CoA
- This is the pathway where most energy is obtained from pyruvate, since acetyl CoA is
the entry point into the citric acid cycle and the electron transport chain
- This oxidizes glucose all the way to CO2 and H2O in aerobic manners
- NAD+ is NOT regenerated in this reaction, but rather more is consumed. Instead,
NAD+ is regenerated by the electron transport chain

16.3 Fructose and Galactose are Converted into Glycolytic Intermediates:

- Although glucose is the sugar commonly used for energy sources, others are also
important fuels
- Fructose and galactose are other important fuels
- Fructose is a component of sucrose, or table sugar, as well as in corn syrup
- Galactose is a component of lactose, known as mlk sugar
- Both sugars are metabolized by glycolysis, but enter the pathway at different points

Fructose is converted into glycolytic intermediates by Fructokinase

Fructose can take one of two pathways to enter the glycolytic pathway
- While there are two entry points for fructose into glycolysis, the vast majority of it is
metabolized by the liver
- In the liver, fructose is first phosphorylated to fructose-1-phosphate by fructokinase
- This is then split into two 3-carbon sugars, dihydroxyacetone phosphate and
glyceraldehyde by a special aldolase
- While dihydroxyacetone phosphate is already an intermediate of glycolysis
and can directly enter the pathway, glyceraldehyde has to be phosphorylated to
glyceraldehyde-3-phosphate in order for it to enter glycolysis
- This is carried out by triose kinase using ATP as the phosphate donor

Galactose is Converted into Glucose-6-Phosphate

- Galactose is converted into glucose-6-phosphate in four steps
- 1) The reactions involve the phosphorylation of galactose
- 2) the transfer of galactose to an activated carrier (UDP)
- 3) then the rearrangement of one hydroxyl group on galactose by an epimerase
enzyme, which is an isomerase that repositions hydroxyl groups
- 4) The product, Glucose-1-Phosphate, is then converted to Glucose-6-Phosphate by an
isomerase called phosphoglucomutase
16.4 The Glycolytic Pathway is Tightly Controlled:
The glycolytic pathway has a dual role: it degrades glucose to generate ATP, and it provides
building blocks for the formation of fatty acids and amino acids
- For these reasons, it is important that the rate of glycolysis is tightly controlled
- In metabolic pathways, enzymes catalyzing irreversible reactions are potential sites
of control
- In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and
pyruvate kinase are irreversible, and each serve as a control site
- Each of these enzymes has their catalytic activity increased or decreased by
the binding of allosteric effectors or through covalent modification

1. Muscle
In this tissue, glycolysis provides ATP that is used for muscle contraction.
- Glycolysis is primarily controlled by the energy state of the cell, which is represented
by the ATP:AMP ratio
- The higher the ratio, the greater the energy state and the less need for
glycolysis
- The lower the ratio, the lower the energy state, and the increased need for
glycolysis

Phosphofructokinase (PFK)
- Phosphofructokinase is the most important control site in the glycolytic
pathway!
- PFK is regulated allosterically
- ATP binds to a site on PFK, and inhibits its catalytic activity by decreasing PFK’s
affinity for one of its substrates, fructose-6-phosphate.
- AMP competes w/ ATP for the same site, but when AMP is bound, it does not inhibit
the enzyme
- Thus, the ratio of ATP:AMP controls the rate of glycolysis at this step

- As well, a drop in pH of the muscles also inhibits PFK by enhancing the effect of
ATP
- This makes sense physiologically. A drop in pH in the muscle is usually due to
excess lactate production, which occurs when the muscle is functioning
anaerobically.
- Excess lactate can damage the muscle, thus slowing glycolysis when lactate is
on the rise serves to protect the muscle

Hexokinase
 - Hexokinase plays a lesser role, however, the product of the reaction it catalyzes
(glucose-6-phosphate) inhibits hexokinase by an allosteric mechanism
- This makes sense physiologically. If glucose-6-P levels get too high, it means
that there is less flux through glycolysis occurring and thus a signal that there
is sufficient ATP available.
- It makes sense to slow the first step of glycolysis, which will lead to glucose
build up in the muscle, slowing the uptake of glucose into the muscle
Hexokinase continued . . .
Why is hexokinase not the most important point of control since it is the first reaction of
glycolysis? (often, the first step in a pathway is the one that is controlled)
- Phosphorylation of glucose to glucose-6-P, while being the first step, is NOT the
committed step of glycolysis
- This is bc glucose-6-P can enter other pathways, including the pentose
phosphate pathway and glycogen synthesis

Pyruvate Kinase
- Pyruvate kinase, the enzyme catalyzing the last irreversible step in glycolysis, is the
last enzyme regulated in muscle glycolysis
- It is allosterically inhibited by ATP and activated by fructose-1,6-bisphosphate
- The inhibition of ATP makes sense from an energy state perspective
- Why is pyruvate kinase activated by fructose-1,6-bisphosphate?
- This metabolite is an intermediate in glycolysis, and the product of
phosphofructokinase, the major rate limiting enzyme
- A rise in F-1,6-BisP is a clear indication of an increased flux through the
pathway, and thus makes sense that pyruvate kinase activity would be
increased to handle the increased flux

2. Liver
The liver doesn’t need energy for contraction, and obtains most of its energy needs
from the breakdown of fatty acids. So what is the purpose of glycolysis in this tissue?
- One of the major functions of the liver is to maintain glucose levels in the blood
- The glucose that it takes up is either
- (1) stored as glycogen (a polymer of glucose)
- (2) used to generate reducing power in the form of NADPH which is used for
biosynthesis (done in the pentose phosphate pathway)
- (3) converted via glycolysis to molecules that serve as building blocks for the
synthesis of other biomolecules
- Thus, the regulation of glycolysis is different than in muscle
Phosphofructokinase (PFK)
- Liver PFK is still the major point of regulation similar to muscle, however the
specifics of its regulation are different
- ATP and pH, the major regulators of PFK in muscle, play less of a role bc the liver
doesn’t have the sudden intense need (as in contracting muscle) and no lactate is
formed in the liver
- Bc glycolysis in the liver is primarily used to provide building block molecules,
signals indicating whether these building blocks are abundant or scarce are the
primary signals
- Citrate is a major inhibitor of PFK, since citrate is formed from acetyl CoA which in
turn is a product of pyruvate metabolism
- Thus a high level of citrate is an indicator that biosynthetic precursors are at
sufficient levels, and metabolizing more glucose through glycolysis can be
slowed

Glycolysis in the liver is able to respond to high blood sugar in a unique way
- When glucose rises in the blood after a meal, the lever takes up much of this, as a
result, there is more flux through glycolysis which leads to build up of fructose-6-P
levels.
- The enzyme PFK needs to be activated to handle this increase in the metabolite
- One way this is achieved is that some of fructose-6-P is converted to fructose-2,6-P
- Fructose-2,6-P is an allosteric activator of PFK, which it does by increasing its
affinity for one of its substrates and by blunting the inhibitory effect of ATP
- In this way, glycolysis is accelerated when glucose is abundant
- This is called feedforward stimulation

Hexokinase
- Hexokinase is also regulated in a special way in the liver
- The liver has a unique isoform of hexokinase, called glucokinase
- Glucokinase has a 50-fold higher Km for glucose than hexokinase, which
means that glucose-6-P is formed only when glucose is abundant, such as after
a meal
- Glucokinase also gives the brain and muscle ‘first dibs’ at the glucose, but
allows liver to take up what isn’t needed by the other cells and either store it as
glycogen or metabolize it to other compounds such as fatty acids and amino
acids
- Another unique feature is that glucokinase is not inhibited by its product, glucose-6-P
- This allows it to facilitate the liver to be able to use the excess glucose that it
takes up from the blood so it is not wasted

- Glucokinase phosphorylates glucose only when glucose is abundant, such as

after a meal