Glycolysis

Definition: pathway in which glucose is broken down through multiple steps into two
molecules of pyruvate with the release of energy in the form of ATP and NADH.
NADH will later be taken to electron transfer chain to form ATP
Pyruvate will enter Krebs Cycle (citric acid cycle) if mitochondria and oxygen are available, if
not it will go to make lactic acid.
RBCs have no mitochondria and so despite having loads of O2 they have to undergo
anaerobic respiration producing lactic acids.
Glycolysis occurs in every cell.
The enzymes used in the glycolytic pathway are mainly in the cytosol so this is where the
reactions occur.

How does the cell get glucose

First it is ingested
Enters portal circulation Portal circulation
Liver hepatocytes they are specialised at taking up large amounts of glucose, galactose
and fructose and convert it into glucose and store it allowing only small amounts to enter
the general circulation, thus preventing immediate hyperglycaemia. When the plasma
glucose levels drop, the liver releases the glucose into the plasma.
Glucose can easily diffuse across capillary membranes so the concentration is the same in
the blood as in the interstitial fluid.
Glucose is transported into cells by two methods:
o Facilitated diffusion
o Glucose-sodium cotransporter

1. Facilitated diffusion
Glucose Transporter (GLUT) - A transmembrane protein that, when bound to glucose, will alter its
shape pushing glucose into the cell. They do not use energy, rather they just make the membrane
more permeable to glucose (i.e. if more transporters then the membrane is more permeable to
glucose). So it relies on glucose moving from high concentration to low concentration (hence
diffusion). It is called Facilitated because it is using these transporters
Types (about 14 types found):

GLUT 1 + 3 present on most tissues (especially CNS, and RBCs). Their purpose is basal
uptake of glucose (meaning glucose movement into cell when the levels are low). They have
high affinity for glucose so they bind tightly with glucose even when the blood glucose is
low. Neurons have very high concentrations in neurons. Because it is diffusion, if the
neurons increase their metabolic activity then the intracellular level of glucose drops thus
creating an increased gradient and so increases the rate of diffusion.

GLUT 2 Two way glucose transporters they allow glucose to move in either direction.
These are present in the hepatocytes (so that when glucose in blood is high then it moves
into the hepatocytes, but when the glucose in blood is low, the glucose can move from the
hepatocytes into the blood). These transporters are present in the kidney, liver and
pancreas. They are present in the pancreas because these cells have to equilibrate their
glucose with the blood so they can produce the right amount of insulin.
GLUT 4 Present on skeletal muscles and adipose tissue. Their concentration on the
membrane is regulated by insulin. Insulin travels in blood to muscles and adipose tissue and
results in cellular production of proteins that pull vesicles containing Glut 4 receptors to
move to the cell membrane, thus inserting GLUT-4 receptors into the cell membrane. GLUT4 receptors can now uptake the excess glucose. Muscles and fat can store glucose in
different forms.
GLUT 5 = fructose transporter. This is present in GIT cells (to absorb fructose), testicles (they
like to run their metabolism on fructose) and sperm (uses fructose for their metabolism).
GLUT 7 Present in the membranes of smooth endoplasmic reticulum as it is involved in
gluconeogenesis (which occurs in the liver mainly and partly in the kidney)

Handling of glucose by liver

Liver cells take up glucose and form a polymer of glucose called glycogen (glyconeogenesis). When
glucose is low in blood then glycogenolysis (break down of glucose) occurs to bring the blood
glucose back up.
Gluconeogenesis (glucose formation from new sources (i.e. fatty acids or amino acids provide the
carbon skeleton). All organs that can do gluconeogenesis must have a GLUT-2 receptor.
Gluconeogenesis occurs in Liver and Kidneys.
2. Sodium-glucose co-transport System
Present in kidney and in intestine
They use active transport as it is against the concentration gradient
The image below shows the mechanism of their action in the proximal convoluted tubular cells of
kidney:

On the basolateral side Na+/K+ ATPases actively pump sodium out and potassium into cell.
This creates a low concentration in the cell.
On the luminal membrane there is a sodium-glucose transporter that binds sodium and
glucose. The concentration of sodium in the lumen is higher than intracellular so when
sodium binds to this pump it moves into cell down its concentration gradient. When this
happens the transporter flips and takes the glucose in as well. If either Na+ or glucose arent
present then the receptor will not flip.
This movement of sodium down its concentration gradient into the cell does not require
energy and is called facilitated diffusion. This Na+ transporter also carries glucose with it into
the cell. Once most of the glucose has been taken into the cell there is a low concentration
in the lumen and a high concentration in the cell, so glucose is moving against its
concentration gradient and so requires active transport. The energy is created, for this
process, by sodium moving down its concentration gradient, so this is called secondary

active transport as the actual energy (i.e. na+ concentration gradient) was created by the
Na+/K+ ATPase pump. This can be proven because giving a toxin that blocks the Na+/K+
ATPase pump then the Na+-glucose cotransporter also stops working.
On the basolateral side there are glucose channels that allow glucose to move via facilitated
diffusion freely back and forth between the cell and interstitum, however due to high
concentration the glucose in the cell, it moves out into the interstitium and then capillaries.

Proximal tubule cells have lots of mitochondria - These cells require a lot of Energy at the
Basolateral membrane for the Na+/K+ ATPase so there are lots of mitochondria to provide the
energy which are found near the basolateral membrane.

Primary active transport = when molecules being transported against concentration gradient and
energy is being directly used on that receptor
Secondary active transport = molecules are being moved across a membrane against their
concentration gradient but energy for this is being used in another location.

Glycolysis
Now that glucose has entered the cell via the mechanisms described above the following occurs.
1. Glucose enters cell

2. Kinase Enzymes (hexokinase and glucokinases) phosphorylate glucose forming Glucose 6

phosphate (meaning that the 6th (last) carbon in glucose is phosphorylated. This enzyme
works in one direction (i.e. cannot reverse the reaction) so they need to be very well
regulated.

a. Hexokinase reaction increases with small increases in glucose but quickly a plateu
is reached. This means that this enzyme will work on even little amounts of glucose,
and is most active when small concentrations of glucose are present. Most cells are
rich in these enzymes, because these cells just need to use the glucose that comes
to them for energy production. Regulation of activity: G6P inhibits Hexokinase
b. Glucokinase doesnt work much at low concentrations but as the glucose
concentration increases then the effect of the enzyme increases. This enzyme works
more effectively at higher concentrations of glucose and so is most active when
large amounts of glucose injested. Liver comes across large amounts of glucose and
so has a lot of this enzyme, allowing the liver to process and so store large amounts
of glucose. Regulation of activity: glucokinases are stored in the nucleus of
hepatocytes and are bound to glucokinase regulating protein. When plasma glucose
concentration is high it enters the nucleus and causes the glucokinase to be released
from this protein allowing glucokinase to become active. F6P binds to glucokinase
regulating proteins causing them to bind the enzyme again thus inhibiting them
when the F6P has reached a high concentration.

Amount of glucose

Hexokinase
Works on small amounts then
plateus

Works on
Found in
Regulation

Glucose, fructose and galactose

All cells
G6P inhibits its action

Glucokinase
Works in a linear fashion, i.e.
more glucose the better it
works
Hepatocytes
Bound to glucokinase
regulating proteins, glucose
reduces this and F6P increases
it.

Km = concentration of a substrate (glucose) at which half of the enzyme (hexokinase or glucokinase)

is saturated. So Km is low for hexokinase and high for glucokinase.
Vmax = the concentration of glucose when the enzyme has reached its maximum rate of activity. So
hexokinase has low Vmax and Glucokinase has a high Vmax.

3. G6P is then converted into Fructose 6 Phosphate by an enzyme. This enzyme also reverses
this reaction, depending on the concentration of G6P or F6P. This enzyme is called Phosphoglucose-isomerase.

4. Another enzyme (phospho-fructo-kinase 1 (PFK-1)) ads a phosphate to the 1st carbon so

now it is called fructose 1,6 bisphosphate.

5. A very small number of F6P molecules are converted to Fructose 2,6-Bisphosphonate by the
Kinase component of the enzyme PhosphofructoKinase 2. This enzyme also has a phosphate
component that can reverse this reaction.

6.

Regulation of PFK-1 PFK-1 has binding site for ATP, AMP, Citrate and F 2,6 BP. ATP and
Citrate are negative regulators meaning that if they are present the enzyme is less effective.
AMP and F 2,6 BP are positive regulators thus increase enzymes activity.

7. Regulation of PFK-2 Phosphate has dual action on PFK-2. It stimulates the Phosphatase
component and inhibits the Kinase component thus favouring the conversion of F 2,6 BP
back to F6P.

8. Phosphate levels are regulated by Insulin and Glucagon. Image below is self explanatory.

Insulin also has some other effects:

Increased gene expression of:

o Glucokinase
o Hexokinase
o PFK-1
o Pyruvate Kinase discussed later
Production of Protein phosphatases which dephosphorylate PFK-2

9. Fructose 1,6 BP is then broken down into Dihydroacetone Phosphate (DHAP) and Glyceral
Aldehyde 3-Phosphate (GA3P) by the enzyme aldolase

10. DHAP can then be reversibly converted also into GA3P by Triose Phosphate Isomerase. So
now we have two molecules of GA3P being produced from one glucose molecule. From now
on we will only talk about one molecule but it is important to remember that there are two
of every reaction occurring.

11. An enzyme (Glyceral Aldehyde 3-phosphate Dehydrogenase) converts GA3P into 1,3Bisphosphoglycerate. It does this by having several binding domain:
a. GA3P domain
b. Inorganic Phosphate (Pi) domain
c. NAD+ domain
12. GA3P dehydrogenase enzyme holds these molecules together and removes a Hydrogen ion
from GA3P and inserts it onto NAD creating NADH (high energy bond). NADH will later enter
the electron transport chain and results in production of 3 ATP molecules. This Enzyme also
adds the inorganic Pi to the molecule thus creating a high energy bond.

13. Next another enzyme (phosphoglycerate Kinase) removes the phosphate on carbon 1 and
adds it to ADP forming ATP. This type of phosphorylation is termed Substrate level
phosphorylation.

Arsenic Poisoning
Arsenic is a chemical element that when injested is converted to arsenate. Arsenate is able to bind
to the inorganic phosphate binding site on Glyceral Aldehyde 3-Phosphate Dehydrogenase and thus
stop Pi binding there. So now arsenic is inserted instead of Pi onto carbon 1 of the molecule. This
prevents NAD from attaching to NADH and so it cannot enter the electron transfer chain and so no
ATP will be produced.
Also, because there is no Pi on the molecule (not called 1,3 Bisphosphoglycerate now as Arsenic
molecule there instead) then Phosphoglycerate Kinase cannot convert ADP to ATP.
So the effect of Arsenic poisoning is lack of production of ATP.

RBCs
RBCs have another pathway here that most cells do not. They have an enzyme called 1,3
Bisphosphoglycerate Mutase which is able to move the phosphate from carbon 1 to carbon 2. This
then Forms 2,3 Bisphosphoglycerate (also called 2,3Diphosphoglycerate (2,3 DPG). 2,3 DPG is a
component of haemoglobin that when present hold the beta chains tightly together allowing the
haemoglobin to release their oxygen more easily.
So this pathway is used more when the tissues require more oxygen so that haemoglobin will be
more effeciient at letting go of it when it is at the tissues.
There is another enzyme (Phosphatase) which reverses this processes, This is used if RBC require
more energy. This also provdes a possible shunt (alternative route) to get from 1,3 BPG to 3
Phosphoglycerate.
14. Next the phosphate on 3 phosphoglycerate is moved from carbon 3 to carbon 2 by the
enzyme phosphoglycerate mutas, thus forming 2-phosphoglycerate.
15. Next Enolase converts 2-phosphoglycerate into phosphophenolpyruvate by removing H20
(in the form of H+ from on carbon and OH from another)

16. Finally the last phosphate is removed and added to ADP forming Pyruvate via the action of
Pyrivate Kinase enzyme.