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, ifnot it will go to make lactic acid.
RBCs have no mitochondria and so despite having loads of O2 they
have to undergoanaerobic 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 thereactions occur.

How does the cell get glucose

First it is ingested
Enters portal circulation Portal circulation
Liver hepatocytes they are specialized 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:
Facilitated diffusion
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 thehepatocytes 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 GLUT4 receptors into the cell membrane. GLUT-4 receptors can now
uptake the excess glucose. Muscles and fat can store glucose
indifferent 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
glycogenglyconeogenesis ). 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 isnt 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 theNa+/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 facilitateddiffusion freely back and forth between the cell
and interstitum, however due to highconcentration 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 theBasolateral membrane for the Na+/K+ ATPase so there are
lots of mitochondria to provide theenergy which are found near the
basolateral membrane

Primary active transport = when molecules being transported against

concentration gradient andenergy is being directly used on that receptor

Secondary active transport = molecules are being moved across a

membrane against theirconcentration 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 6phosphate (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

Glucokinase
Works in a linear fashion,
i.e.more glucose the better it
works

Works on

Glucose, fructose and

galactose

Found in

All cells

Hepatocytes

Regulation

G6P inhibits its action

Bound to glucokinaseregulating
proteins, glucosereduces this
and F6P increases i t.

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. Sohexokinase 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 Phospho-glucose-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 2,6Bisphosphonate 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 favoring 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 GlyceralAldehyde 3-Phosphate (GA3P) by the enzyme
aldolase

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

11. An enzyme (Glyceral Aldehyde 3-phosphate Dehydrogenase) converts

GA3P into 1,3-Bisphosphoglycerate. It does this by having several
binding domain:a.

a. GA3P domain
b. Inorganic Phosphate (Pi) domainc.
c. NAD+ domain

12. GA3P dehydrogenase enzyme holds these molecules together and

removes a Hydrogen ionfrom GA3P and inserts it onto NAD creating
NADH (high energy bond). NADH will later enterthe electron transport
chain and results in production of 3 ATP molecules. This Enzyme
alsoadds 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 3Phosphate 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
1,3Bisphosphoglycerate 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 acomponent 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 efficient 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 requiremore energy. This also provides a possible shunt (alternative
route) to get from 1,3 BPG to 3Phosphoglycerate

14. Next the phosphate on 3 phosphoglycerate is moved from carbon 3 to

carbon 2 by the enzyme phosphoglycerate mutas, thus forming 2phosphoglycerate.
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.