Yuan Provido

Chem 2065 AY2023-24

17 Glycolysis
1. State the primary function of glycolysis

• In glycolysis, one molecule of glucose (a six-carbon compound) is converted to

fructose-1,6-bisphosphate (also a six-carbon compound), which eventually gives
rise to two molecules of pyruvate (a three-carbon compound). It uses 2 ATP while
producing 4 ATP and 2 NADH molecules.
• Glycolysis involves the breaking of sugar molecules to release energy that is
required for cellular metabolism.

2. Differentiate between glycolysis and anaerobic glycolysis

Glycolysis:
• In glycolysis, one molecule of glucose (a six-carbon compound) is converted to
fructose-1,6-bisphosphate (also a six-carbon compound), which eventually gives
rise to two molecules of pyruvate (a three-carbon compound)
Anaerobic Glycolysis:
• anaerobic glycolysis the pathway of conversion of glucose to lactate;
distinguished from glycolysis, which is the conversion of glucose to pyruvate

3. Write the net reaction of glycolysis (focusing on the carbon atoms of glucose)

4. Write the ten reactions of glycolysis including the enzymes involved

First Phase
Reaction 1 (Phosphorylation of glucose):
 • Glucose is primed to start the sequence that will produce ATP
• Enzymes: Hexokinase/Glucokinase

•
• Free energy change in cell is even more favorable than at standard state

•
• Glucose is kept in the cell; glucose-6-phosphate cannot diffuse out of the cell
anymore.
• Cellular concentration of glucose is low, hence glucose diffuses in the cell
• Equilibrium is not achieved, therefore makes it favorable site for regulation
Reaction 2 (Isomerization to Fructose-6-Phosphate):
• Isomerization of sugar, carbonyl is shifted from C1 to C2
• Enzyme: Phosphoglucoisomerase
• There is an ene-diol intermediate in the reaction.

•
o The reaction is equilibrium-controlled meaning if there's a high
concentration of glucose 6-phosphate then it gets converted to fructose 6-
phosphate by this enzyme or if there's now a high concentration of
fructose 6-phosphate then it goes back to the glucose 6-phosphate.

•
• Isomerization activates the C-3 for cleavage in aldoses reaction
Reaction 3 (Phosphorylation of Fructose-6-Phosphate):
• 2nd priming reaction that requires ATP and 2nd priming reaction of glycolysis
• Enzyme: Phosphofructokinase (PFK)
• The reaction itself is endergonic

•
•
• PFK is the committed step in glycolysis
• It is a committed and large step, -deltaG means PFK is highly regulated
 • ATP inhibits, while AMP reverses the inhibition
• Citrate is also an allosteric inhibitor
• Fructose-2, 6-bisphosphate is an allosteric activator
• PFK increases activity when energy status is low, but it decreases activity when
energy status is high
Reaction 4 (Cleavage of Fructose-1, 6-bisphosphate):
• Products are dihydroxyacetone phosphate and 3-Glyceraldehyde-3-phosphate
• Enzyme: Fructose biphosphate aldolase

•
o This observes equilibrium meaning, if there's high levels of fructose-1, 6-
bisphosphate then it gets converted into dihydroxyl acetone phosphate,
and in glyceraldehyde-3-phosphate, if there are high levels of this then
they get converted into fructose-1, 6-bisphosphate
Reaction 5: Isomerization of Dihydroxyacetone
• Product is glyceraldehyde-3-phophate
• Enzyme: Triose phosphate isomerase
• This approaches catalytic perfection
• At this stage we have 2 molecules of glyceraldehyde 3-phosphate.
o In the rate of diffusion molecules are floating around in the solution and as
soon as the dihydroxy acetone phosphate hits the active site of the
enzyme, it gets converted into glyceraldehyde-3-phophate, in other words
there is almost no lag in the conversion

•
o It still very close to 0, therefore it still can be equilibrium driven.
Second Phase
Reaction 6 (Oxidation of Glyceraldehyde-3-Phosphate):
• G-3-P + NAD+ + HPO24- ➔ 1,3- Bisphosphoglycerate + NADH + H+
• This involves a redox reaction.
• Aldehyde is converted into a carboxylate (mized anhydrive with phosphate)
• NAD+ is reduced to NADH
• Enzyme: glyceraldehyde -3-phosphate dehydrogenase

•
Reaction 7 (Transfer of Phosphate Group):
• This is described as the break-even reaction
• Enzyme: Phosphoglycerate kinase (PGK)
 • True substrate: MgADP2-
• Products are 3-Phophoglycerate + ATP
• Since there are 2 molecules of 1,3-BPG being catalyzed then 2 molecules of ATP
is produced
• The ATP synthesis is from a high-energy phosphate (1,3-BPG)
• When it now transfers the phosphate group into ADP, it is now referred as
substrate-level phosphorylation

•
Reaction 8 (Isomerization of 3-Phosphoglycerate):
• Product is 2- Phosphoglycerate
• This transfers the phosphoryl group from C-3 to C-2
• Enzyme: Phosphoglycerate mutase
• This reaction prepares the molecule for synthesis of second ATP equivalent

•
o Equilibrium driven
Reaction 9 (Formation of Phosphoenolpyruvate (PEP)):
• This reaction forms PEP
• Enzyme: Enolase
• This involves the removal of water (H2O)

•
o Almost equilibrium driven
Reaction 10 (Formation of Pyruvate):
• This reaction forms the pyruvate with the release of ATP
• This is the 2nd ATP forming reaction in glycolysis
• Pyruvate is a branch point in metabolism
• Enzyme: Pyruvate kinase

•
o This is highly negative, which means this is another site for regulation
• The 2 ATP (from one glucose) can be viewed as the payoff of glycolysis
o From the 2 ATP molecules it produced 4 ATP with a net of 2 ATP
• Allosterically activated by AMP, F-1, 6-bisP
• Allosterically inhibited by ATP and acetyl-CoA
• PEP to pyruvate is viewed in 2 steps: phosphoryl transfer followed by an enol-
keto tautomerization.
o The tautomerization is spontaneous and account for much of the free
energy change for PEP hydrolysis
 ▪
5. Explain factors that control whether the steps in glycolysis goes in the forward or
reverse direction

• One of the factors that control the direction of glycolysis is the PFK in reaction 3,
it is a committed step meaning it can only go in a forward direction.
• In the case of reaction 2, has a deltaG of , the value is close
to 0 meaning, if there's a high concentration of glucose 6-phosphate then it gets
converted to fructose 6-phosphate by this enzyme or if there's now a high
concentration of fructose 6-phosphate then it goes back to the glucose 6-
phosphate. This could cause a reverse direction.
• Another factor is the pyruvate kinase in reaction 10, the reaction is favored so
strongly in the forward direction that cells must do a ‘two-step’ around it in the
reverse direction when making glucose. To put it another way, in the process of
gluconeogenesis, two enzymes, two reactions, and two triphosphates are required.
When cells need to produce glucose, they cannot be diverted by pyruvate kinase
converting the PEP they produced during gluconeogenesis back to pyruvate.

6. Correlate the significance of the changes in the functional groups to the ‘logic’ of the
glycolytic pathway (Recall your organic chemistry. What are the significance of the
following functional groups: alcohol, phosphoester, aldol, aldehyde, ketone,
carboxylic acid, mixed anhydride, enol, phosphoenol?)

• Alcohol: breakdown of sugars by yeasts to form pyruvate molecules

• Phosphoester: Phosphorylates the glucose
• Aldol: Catalysis for reaction 4 in glycolysis
• Ketone: contribute to ATP production via oxidative phosphorylation only
• Aldehyde: The aldehyde of the substrate is oxidized to the level of a carboxylic
acid
• Carboxylic acid: Pyruvate, a three-carbon carboxylic acid, is the end product of
anaerobic glycolysis
• Mixed anhydride: This correlates to the isomerization reaction occurs in
glycolysis when glucose-6-phosphate is converted into fructose-6-phosphate
• Enol: phosphoryl transfer followed by an enol-keto tautomerization.
• Phosphoenol: Phosphoenolpyruvate acts as the second source of ATP in
glycolysis
The changes in the functional groups in the glycolytic pathway is important in
many ways, a functional group is a substituent or moiety in a molecule that causes the
molecule's characteristic chemical reactions, these chemical reactions are important as
 it serve help in the catalysis of the molecules as it changes, this help in producing the
necessary products of the glycolytic pathway.
7. Explain why reactions with significant standard free energy changes are the control
points in glycolysis (You do not need to memorize the exact values of ‘delta G’, just
whether they’re large or close to zero)

• This helps in identifying if a certain reaction’s deltaG is close to zero then it is

equilibrium driven
• When the deltaG of the reaction is far from zero then it is considered to be a
regulation site.

8. Explain the significance of substrate level phosphorylation (Note: the main ATP
production occurs in the mitochondria)

• The free energy of hydrolysis of a substrate is the energetic driving force in

substrate-level phosphorylation.
o An example of this is the conversion of glyceraldehyde-3-phosphate to
1,3-bisphosphoglycerate, this is part of reaction 6 and 7.
▪ In this step in glycolysis, a phosphate group is transferred from
1,3-bisphosphoglycerate to a molecule of ADP, producing ATP,
the first of two such reactions in the glycolytic pathway. 1,3-
bisphosphoglycerate can easily transfer a phosphate group to other
substances, 1,3-bisphosphoglycerate, has transferred a phosphate
group to ADP.
• substrate-level phosphorylation is a reaction in which the source of phosphorus is
inorganic phosphate ion, not ATP
• This is a reaction used in reactions 6,7, and 10.

9. Discuss the logic of converting pyruvate to lactic acid in mammals (lactate

dehydrogenase LDH, anaerobic glycolysis, dead-end in metabolism)

• In animal tissues the pyruvate is converted to lactate by the lactate dehydrogenase

LDH.
 •
• it's a one-step reaction to recycle the NADH into NAD+ so that again even if there
is a lack of oxygen this is still not in aerobic conditions, production of ATP is still
there.
• Lactate is described as dead-end in metabolism this is because our life forms has
not evolved to convert lactate into any other material, the only reaction that lactate
undergoes is to go back to being pyruvate.

10. Discuss how pyruvate is converted to ethanol during the fermentation process
(alcohol dehydrogenase, role of thiamine pyrophosphate TPP)

• In the first of the two reactions that lead to the production of ethanol, pyruvate is
decarboxylated (loses carbon dioxide) to produce acetaldehyde
o The enzyme that catalyzes this reaction is pyruvate decarboxylase.
▪ This enzyme requires Mg21 and a cofactor we have not seen
before, thiamine pyrophosphate (TPP).
• thiamine pyrophosphate a coenzyme involved in the transfer of two-carbon units
o In TPP the carbon atom between the nitrogen and the sulfur in the thiazole
ring is highly reactive.
 o It forms a carbanion (an ion with a negative charge on a carbon atom)
quite easily, and the carbanion, in turn, attacks the carbonyl group of
pyruvates to form an adduct.
o Carbon dioxide splits off, leaving a two-carbon fragment covalently
bonded to TPP.
o There is a shift of electrons, and the two-carbon fragment splits off,
producing acetaldehyde
o Acetaldehyde is then reduced to produce ethanol, and, at the same time,
one molecule of NADH is oxidized to NAD+ for each molecule of ethanol
produced.
• Alcohol dehydrogenase, the enzyme that catalyzes the conversion of acetaldehyde
to ethanol
o similar to lactate dehydrogenase in many ways
o most striking similarity is that both are NADH-linked dehydrogenases,
and both are tetramers

11. Calculate the energy production efficiency in glycolysis

• We can calculate DeltaG°' for the entire reaction by adding up the DeltaG°' values
from each of the steps.
• Note: all of the reactions from triose phosphate isomerase to pyruvate kinase are
doubled.
• This gives a final figure from glucose to two pyruvates of -74.0 kJ mol-1
• The energy released in the exergonic phases of the process drives the endergonic
reactions.
• The net reaction of glycolysis explicitly includes an important endergonic
process, that of phosphorylation of two molecules of ADP.
•
• The corresponding figure for the conversion of one mole of glucose to two moles
of lactate is -184.6 kJ mol-1 = -44.1 kcal mol-1 .
• The energy required to produce the two molecules of ATP for each molecule of
glucose can be recovered by the organism when the ATP is hydrolyzed in some
metabolic process.
• The percentage of the energy released by the breakdown of glucose to lactate that
is “captured” by the organism when ADP is phosphorylated to ATP is the
efficiency of energy use in glycolysis; it is (61.0/184.6) x 100, or about 33%.
o This comes from calculating the energy used to phosphorylate two moles
of ATP as a percentage of the energy released by the conversion of one
mole of glucose to two moles of lactate.
• The net release of energy in glycolysis, 123.6 kJ (29.5 kcal) for each mole of
glucose converted to lactate, is dissipated as heat by the organism.