13

Abdullah Al-Rawashdeh

Dua’ Al-Shrouf
Diala Abu Hassan
 Glycolysis
Synthesis of 2,3-bisphophoglycerate in RBCs
▪ This process doesn’t need oxygen and it occurs in the cytoplasm
so even RBCs (that don’t have mitochondria in them) mainly
depends on glycolysis to synthesize its energy, although
glycolysis creates little energy compared to aerobic but it’s
enough for the RBC because it has one function.
▪ Sometimes in the step where 1,3-bisphophoglycerate is
converted into 3-phosphoglyecrate, an RBC that reached the
site for gas exchange (peripheral tissues where it needs to
release the oxygen attached to it ) another pathway is used,
instead of one direct conversion it becomes a two steps
pathway using 2 enzymes (same product)
▪ In the first step the mutase enzyme transport the phosphate from C1 to C2 and the
product is 2,3-bisphosphoglycerate, then by using an H2O molecule the phosphatase
enzyme removes the phosphate on C2 producing the same product (3-
Phosphoglycerate). It releases it as an inorganic
phosphate instead of the usual ATP. This pathway
(glycolysis in this case) net ATP is zero (2ATP are used and
2 are produced) but we must ask why a cell would waste
2 ATP molecules to synthesize 2,3-bisphosphoglycerate?
▪ The molecule 2,3-BPG is important because it binds to
hemoglobin after it releases its oxygens (T state), to
reduce the affinity of the hemoglobin to rebind oxygen (since the oxygen is a gas it
diffuses but it can rebind, so it alternates the hemoglobin btw the R and T states so to
prevent this, this molecule binds to the hemoglobin)

Energy Need and Production

▪ We can see each step and it’s energy usage or
Production, the net ATP is 2, we don’t consider the -ATP
NADHs as a part of the energy production because they -ATP
are produced in the cytoplasm and they may not enter the 2NADH
mitochondria because they don’t have a carrier in the 2ATP
membrane so we cannot guarantee that they are going to
be used in the ETC and participate in energy production 2ATP
▪ In glycolysis NADH is no energy molecule, but
it’s an electron carrying molecule, so it has nothing
to do with creating energy in anaerobic
respiration (so the main role of NADH is to carry electrons in redox rxns even though they
have the high energy phosphate bond but sometimes I can extract energy from them)

Pyruvate fate
▪ We know that pyruvate loses one carbon and becomes acetyl CoA to enter krebs cycle
▪ But it could gain CO2 and become oxaloacetate to either supply TCA
cycle or to create amino acids (aspartate).
▪ Under anaerobic conditions it can be converted to lactate.
▪ In non-human cells it can also be converted into acetaldehyde by
decarboxylation and then into ethanol.
▪ The conversion of pyruvate to ethanol reaction happens in the
yeast, so the decarboxylation of pyruvate removes a CO2 and it
becomes acetaldehyde, then it will be reduced to ethanol by
alcohol dehydrogenase (NADH is oxidized), so when we use the
yeast for baking we put some sugar and hot water with it so it
feeds on these sugars and glycolysis will take place, which
results in the production of pyruvate and this process happens,
and because CO2 is a gas it will try to diffuse and move away
which makes the food fluffy.
▪ In anaerobic conditions the pyruvate is reduced to lactate by
accepting a proton(a carbonyl -ketone- is converted to a hydroxyl
group -secondary alcohol-) using NADH as a coenzyme and the
enzyme lactate dehydrogenase, this enzyme can reconvert lactate
to pyruvate so it works in both directions based on equilibrium and
concentrations, it oxidizes NADH in the forward rxn but reduces it
in the backward rxn, the NADH and NAD+ are the same but
it alternates between these two states (they supply the same pool) but there’s a ratio
between these two states depending on the oxidative status.
▪ The point of the forward being is to regenerate the NAD+ to use it again in glycolysis
under anaerobic conditions. And remember this is a reversible reaction.

When is lactate produced?

1)To cope with increased energy demands like in a rigorously muscles activation
(exercising), when a muscle is being activated for too long the amount of oxygen
reaching it will become less and less, that causes the cells to rely on anaerobic
respiration for energy instead of aerobic even though it produces a much less energy, so
after a while lactate accumulates and results on fatigue(lactate level is increased 5 to 10
folds).
 2) Low energy demanding cells like RBCs also contribute to lactate production because
it doesn’t have a mitochondrion and depends on glycolysis (there’s no fatigue here it
occurs only in exercising muscles because muscle cells need much higher energy, so
they produce more lactate)
3) During hypoxia (wether it’s generalized or localized) to survive brief episodes of
hypoxia, the lactate production also increases.
▪ Climbing high mountains causes generalized hypoxia, also during corona viruse there
was a big problem in the respiratory system, so a low amount of oxygen entered the
body and the whole body suffered from hypoxia
▪ The body will try to recover and survive by depending more and more on the
anaerobic respiration, so more lactate production
▪ While localized hypoxia occurs in different types of thrombosis, which results in
partial or complete loss of blood supply to a certain region in your body which reduces
the amount of oxygen that reaches this region, this causes the death of the cells in this
region especially if these cells are very active (heart, brain and stomach cells) which
need a high amount of energy and cannot live without oxygen and blood supply for a
long period of time

Clinical Hint:Lactic Acidosis

▪ High amounts of lactic acid may cause lactic acidosis, and it contains a hydroxyl group
and a carboxylic group which are acidic groups, and it leads to:
1)low pH of plasma, it may be caused by high production of lactate or low breaking
down of it.
2)it is the most common cause of metabolic acidosis, because this lactate shouldn’t stay
in the cells, the body can reduce its concentration by using it to reproduce pyruvate
using lactate dehydrogenase, but if it stays it will cause metabolic acidosis by:
➢ ↑Production of lactic acid
➢ ↓Utilization of lactic acid
Pyruvate + NADH ↔Lactate + NAD+
▪ Most common cause of this acidosis: Impairment of oxidative metabolism due to
collapse of circulatory system
▪ Other causes:
- Impaired O2 transport (if someone has a blood disease that affects the structure of
hemoglobin, and it interferes with its ability to transport oxygen)
- Respiratory failure
- Uncontrolled hemorrhage (losing a big amount of blood leads to the loss of the RBCs
and the oxygen attached to them, so the transport of oxygen is reduced leading to more
dependence on the anaerobic respiration)
 - Direct inhibition of oxidative phosphorylation (even if Krebs cycle is still working, I
cannot produce energy so there will be more shifting towards the anaerobic respiration)
- Hypoxia in any tissue (generalized or localized)
- Alcohol intoxication (high NADH/ NAD+) (in people who drinks large quantities of
alcohol, the metabolism of this alcohol increases the ratio of NADH/NAD+, so most of
the available coenzyme in the previous rxn will be NADH, which results in the inhibition
of the Krebs cycle because it needs NAD+, so even if we have enough concentrations of
the reactant, product and enzyme the rxn won’t move forward, more shifting towards
anaerobic respiration)
-↓ gluconeogenesis (it’s the pathway in which glucose is produced from non
carbohydrate precursors, this occurs in the case of long periods of fasting, oxaloacetate
is used in this process so if it was activated at low levels this means there is less
oxaloacetate and more pyruvate, so more production of lactate)
-↓ pyruvate dehydrogenase (this enzyme converts the pyruvate to Acytel-CoA, so if this
conversion decreases, we won’t be able to supply enough Acyrel-CoA to the Krebs cycle,
so again the cells will depend more on the anaerobic respiration)
-↓ TCA cycle activity (if any enzyme in the TCA cycle has a mutation or a problem it will
be reduced, and the cells will depend more on anaerobic respiration)
-↓ pyruvate carboxylase (this enzyme converts the pyruvate to oxaloacetate, so again
less supple to the Krebs cycle or we can say that we will have more pyruvate that will
undergo anaerobic respiration instead of aerobic because it is limited by the oxygen
level and the availability of the coenzyme NAD+)
Regulation of Glycolysis
▪ Regulation usually occurs at the irreversible steps; it happens on 3 steps.
Regulators of PFK and PK
▪ On the left side of this picture, we can see the
regulatory molecules of glycolysis, while we can see
the regulators of gluconeogenesis on the right side
(which is the opposite rxn of glycolysis)
▪ Glycolysis is regulated at the hexokinase,
phosphofructo-kinase and pyruvate kinase steps
▪ The first regulation happens at the
phosphofructokinase, where it can be activated by
fructose 2,6-bisphosphofructose (not fructose 1,6-
bisphosphate which is the product of this enzyme)
which is a regulatory molecule that acts as a switch because it activates the glycolysis
and inhibits the gluconeogenesis to make sure that the two opposite pathways aren’t
on simultaneously (because if they worked together it’ll be a waste of energy)
▪ We can also activate this enzyme using the AMP, because this molecule indicates a low
energy state in the cells
▪ While the ATP is an inhibitor because it indicates a high energy state in the cells , so
there’s no need to break down glucose
▪ The citrate also works as an inhibitor, which is an intermediate of the Krebs cycle so if
we have a high concentration of citrate this indicates that the Krebs cycle is working
enough so there’s production of energy so there’s no need to break down the glucose
▪ The proton inhibits this enzyme, because the proton is a byproduct of the ETC so if we
have a high concentration of H+ this means we have a high concentration of ATP
because the ETC is working
▪ We can also regulate glycolysis through the pyruvate kinase which is the last enzyme in
this process, which is activated by fructose 1,6-
All of these regulators are allosteric
phosphate which is ྀྀྀྀྀྀthe product of the
previous regulated step, this is called feed regulators, so they have binding sites on
forward because a product from one step
the enzyme, so they either convert it to
activates the enzyme of a step that’s far ahead
in the cycle (this activator is produced in step the active or the inactive form
no.3 and activates the enzyme of step no.10)
▪ This enzyme is inhibited by the ATP, because it indicates a high energy state in the cells,
so there’s no need to break down glucose
▪ Alanine amino acid also inhibits this enzyme, because if you removed the amine group
from its structure it will become pyruvate, so it’s a source of pyruvate, so if it’s in high
concentration there’s no need to synthesize pyruvate out of this step

Glucokinase and hexokinase activity

▪ On the X axis we can see the substrate
concentration, while on the Y axis the enzyme
activity is shown
▪ Notice how the curve of the hexokinase is all
down compared to the glucokinase, so the
Vmax of the hexokinase is much less than that
of glucokinase
▪ The Vmax of the hexokinase can be reached at a
very low concentration of glucose, whereas the
Vmax of glucokinase needs a really high
concentration of glucose to be reached
▪ The orange region represents the concentration
of glucose at fasting conditions, it takes 2 hours for the levels of the sugar in the blood
 to go down to fasting blood sugar which is the orange region (in diabetic patients the
sugar level doesn’t go down even if they’re fasting)
▪ The sugar level shouldn’t go lower than the orange zone because the person will lose
consciousness and that’s because the brain is exclusively dependent on glucose as a
source of energy, so while the brain is consuming glucose, we have to supply it with
enough glucose instead of the consumed concentration
▪ Notice that at the orange concentration the hexokinase is at the Vmax even though
we’re fasting, however the glucokinase is working at 20% or less of its total activity at
fasting conditions

Glucokinase regulation
▪ Glucokinase as a molecule is found in the nucleus when
it’s inactive, the Glucokinase regulatory protein (GKRP) is
attached to it, so they form a complex together that is
present inside the nucleus, this attachment makes the
glucokinase inactive and keeps it in the nucleus
▪ Once glucose levels go up after a meal, insulin is
secreted, so more expression of GLUTs especially GLUT 4,
and as a result more uptake of the glucose into the cells
▪ A high concentration of glucose inside the cells is going
to activate the enzymes (hexokinase and glucokinase)
▪ This happens because the glucose activated the dissociation of glucokinase away from
this regulatory protein, when the glucokinase dissociates it can be transported from
the nucleus to the cytoplasm where it can act on the glucose
▪ If the fructose 6-phosphate (one of the intermediates) increases a lot, this indicates
that we’re breaking down a lot of the glucose,
Sequestration: you move the molecule
so it’s going to activate the return of the
glucokinase back to the nucleus, this is called away from where it can function
sequestration, when it’s in the nucleus it’s
going to bind to the regulatory protein again so it gets inactivated
Regulation of ATP and AMP
▪ As we said ATP and AMP indicate different energy
states, and their levels are different between rest
and exercise (ATP levels are low at the exercise
state because it’s being consumed because we
need energy, whereas the ADP and AMP levels go
up while exercising)
End of sheet 13