The final chapter of this course deals with
metabolism.
In metabolism we need certain nutrients.
We are going to learn about especially in
the metabolism of carbohydrates how the food
that we are taking in is broken down.
Metabolism actually as you have learnt from
your school days is comprised of catabolism
which is the degradation of nutrients to generate
energy and starting materials which is what
we are going to do basically and anabolism
is the biosynthesis of biomolecules from starting
materials which also actually happens in our
body because we create proteins and proteins
are synthesized in the body which is a process
that would require anabolism.
Catabolism is the breaking down of the nutrients
i.e. ultimately going to provide the energy
for the actions or whatever work that we do.
The metabolites that take part in these processes
actually are substrates and intermediates
and of course there will be a large number
of enzymes that are going to be actually part
of the whole system.
If we just look at this whole picture, I will
show you another one also where we will see
how the whole process actually takes place.
We have the energy containing nutrients that
we taken in our diet; carbohydrates, fats
and proteins.
In the catabolism that is in the breakdown
of these processes or the breakdown of these
units we actually have energy depleted end
products that are finally carbon dioxide,
water and ammonia.
And we have in this process then the use or
the utilization of certain aspects or certain
cofactors that we have already looked.
We have ADP and ATP in a large extent.
We have NAD+, FAD and NADP+ and so on and
so forth.
All of these actually will then either breakdown
or form in different ways giving us finally
the chemical energy.
Then we have certain precursor molecules that
are also found in our body where we have the
amino acids, the sugars, the fatty acids and
the nitrogenous bases that in a process of
anabolism that is in the formation of the
macro molecules will ultimately lead to the
cell macro molecules such as proteins, polysacoharides,
lipids and nucleic acids.
Eventually it is just like a whole cyclic
process that it is the food that we take that
is ultimately broken down and then after the
breaking down the bits and pieces some of
them get lost in energy depleted products,
some of them form the precursor molecules
further formation of the generation of other
proteins, polysacoharides and lipids and nucleic
acids.
Now if we look at actually the whole process
it is extremely interesting.
This is like a gear system.
We have the process of Glycolysis which we
are going to study in our breakdown of glucose
in the metabolism that we study.
The Glycolysis leads to something called the
TCA cycle which is the Tri Carboxylic Acid
cycle that has electron transport.
As we just looked at the electron transport
has a proton pump to it.
The proton pump then uses ATP synthase to
produce cellular work where we get work.
This oxygen again comes in from the blood
utilized in the ATP synthase in the electron
transport system where we have the reduction
of the oxygen to water.
This is the way all of these are interconnected
actually.
And if you just look at a whole picture of
all the metabolic pathways there is actually
a beautiful picture on the net that has all
the metabolic pathways that actually take
place in our body.
What I am going to give you is just probably
a small like a drop in the huge ocean of all
those metabolic pathways that actually take
place.
What we actually intake?
We are looking at stages of catabolism.
Our intake is proteins, polysacoharides, lipids
that is fats, proteins and carbohydrates basically
that is what we intake.
The smaller blocks of proteins, what is the
breakdown of proteins going to give you?
It is going to give you amino acids.
The breakdown of polysacoharides is going
to give you monosacoharides, the breakdown
of lipids is going to give you fatty acids
or glycerol.
Now, each of these again are further going
to be broken down and converge to acetyl coenzyme
A which is a extremely important part in the
catabolic pathway that is going to take you
again to the citric acid cycle that requires
the oxidation of acetyl coenzyme A. So basically
what we are getting at is we are going to
look at just this part here; monosachorides
the breakdown of glucose that is what we are
going to look at.
But if you consider the ampting polysacoharides
that are present in the body that are possible
for breaking down you realize that each of
them is an enzymatic reaction that is required
for the breaking down from its polymeric form
to the monomeric form which is finally going
to be broken down into other forms.
So what we have is we have our lipids, polysacoharides
and proteins, lipids breaking down into fatty
acids that form the acetyl CoA, polysacoharides
breaking down into glucose the monomaric unit
in the process of Glycolysis forming pyruvate
which is what we will see in the process of
Glycolysis that we will study, proteins again
breaking down into amino acids and again are
utilized in this acetyl CoA which finally
is used in the Tri Carboxylic Acid cycle which
is going to be the breakdown of the Tri Carboxylic
Acids the oxaloacetate and so on and so forth.
Then we have this process where we have FADH2+
NADH giving you with oxygen oxidative phosphorylation
in the production of ATP.
This is a part that we have studied in a bit
detail.
Now what we are look at is we are going to
look at the glucose going to pyruvate in the
process called Glycolysis and in the event
producing ATP also but requiring ATP also
in a number of steps and finally acetyl CoA
that is going to be utilized in the Tri Carboxylic
Acid cycle that is also known as the Krebs
cycle.
Now, before I get into the process of Glycolysis
this is something that we looked at before
when we considered the vitamins.
I mentioned that each of these vitamins you
now realize is the precursor for a large number
of cofactors and prosthetic groups.
Coenzyme A is another such compound that is
extremely important in the formation or the
transfer rather of the acetyl group that is
the transfer of the two carbon system for
any of the processes that occur.
What we have in coenzyme A is actually an
ADP part an Adenine Dinucleotide part, a phosphorylate
1 because there is a phosphor at this position
if you can see and we have pantothenic acid
that is one vitamin and to it we have mercapto
ethanylamine attached.
Now this SH if acetylated is called acetyl
CoA.
So whenever we look at any metabolic pathway
you will see acetyl CoA come into the picture
a large number of times.
But you have to remember it is derived from
that vitamin and it is nothing but a phosphorylated
ADP linked with the oxygen to pantothenic
acid that is linked with marcaptoethylamine
and this unit is coenzyme A. If this thiol
group is acetylated it becomes acetyl CoA
and that is exactly as how it is referred
to in all of the metabolic pathways acetyl
CoA.
So it is this acetyl part that is important
and the rest is you realize is derived from
phosphorylated ADP, vitamin pantothenic acid
and marcaptoethylamine.
Now if you look at all the catabolic processes
that occur the Glycolysis path way will lead
us to pyruvate, pyruvate all of the breakdown
getting to acetyl CoA, acetyl CoA being the
main part of the TCA cycle that ultimately
breaks it down to carbon dioxide and water.
Now this is obviously going to involve a large
number of enzymes and we will look at each
of these enzymes and how they act.
Glycolysis: the process of Glycolysis takes
place in the cytosol of cells and glucose
enters the Glycolysis pathway by the formation
of glucose-6-phosphate.
Glucose-6-phosphate means that we are going
to have a large number of steps that are actually
going to get you to pyruvate.
There are ten steps involved which mean there
are ten enzymes involved and we have this
form pyruvate and we have three steps that
are regulated in the formation of pyruvate
from glucose in the ten steps that perform
the process of Glycolysis.
So we have glucose go to pyruvate in ten steps
and we have three of those steps that are
regulated and it is occurring in the cytosol
of the cells.
If we do not have oxygen then this pyruvate
forms lactate.
This is the overall reaction.
We have glucose + 2 ADP + 2 Pi that is phosphate
+ 2 NAD + go to 2 pyruvate + 2 ATP so you
are generating ATP here also + 2 NADH + H2O.
So we are going to look at each of these steps
and see how you have ATP consumption, ATP
production and finally we are going to take
an account of where we are using ATP where
we are producing ATP.
Now before we get into that we have to see
why enzymes are involved in the metabolic
pathway.
We have kinases, isomerases, aldolases, dehydrogenases.
Kinases are a specific class of transferases
and kinases are those that transfer a phosphate
group, this we have looked at before or I
have mentioned before.
We will also see how Hexokinase will be a
part of our Glycolysis mechanism.
So we have kinases that transfer a phosphate
group from ATP to a specific substrate.
We have isomerases, what are isomerases going
to do?
They are going to convert one isomer to another
and that is what the function of isomerase
is.
If we look at aldolases they are going to
catalyze aldol condensations.
All of you know what aldol condensations are.
So we have kinases, isomerases, aldolases,
we have dehydrogenases.
dehydrogenases we have looked at also where
we have the removal of hydrogens by oxidation.
Then we have the other enzymes, we have a
few more we have mutases that are actually
group transfer enzymes.
They transfer phosphate from one position
of the substrate to another position.
Say you have a glucose-1-P to form a glucose-6-phosphate
you will need a mutase.
So the mutase is going to be a group transfer
enzyme that is going to transfer, the common
use would be the transfer of phosphate to
a different position an example being glucose-1-P
mutase which would actually transfer the phosphate
from carbon 1 to carbon 6.
Forming from glucose-1-P it would form glucose-6-phosphate
in that case you would need mutase.
If you want an enolase you would convert a
C double bond C group to an alcohol because
you have to remember that when we are looking
at each of these steps we are finally going
to breakdown glucose into carbon dioxide and
water, that is our final aim.
So we have to look at how this can be accomplished
in the biological way with the use of these
certain enzymes.
We have synthase that is also known as synthetase,
what does that do?
It just combines two molecules together, synthesis.
We have ATPases that are going to hydrolyses
ATP to ADP and Pi and this is in reverse to
ATPsynthase which was, what was ATP synthase
doing?
It was taking ADP and Pi and producing ATP,
this hydrolyses ATP to ADP and Pi.
So these are the pathway enzymes that are
going to be used in every step of the way
as we go along.
We have step number 1 which is the use of
Hexokinase.
Hexokinase is the first step in Glycolysis
after you have broken down polysacoharide
to glucose.
We now have glucose, we are not going in to
how the polysacoharide is broken down or how
even the other dietary nutrients are broken
down like fats or amino acids, proteins or
whatever.
We are just going to look at the metabolism
of carbohydrates just looking at the breakdown
of glucose.
The glucose breakdown will ultimately lead
us to pyruvate and it will involve ten steps,
this is step number 1 where from glucose we
form glucose-6-phosphate.
That means the 6 carbon atoms is no longer
OH it is now phosphorylated.
It is phosphorylated by taking a phosphate
from ATP, in the event ATP becomes ADP.
This is a coupled reaction.
We will look at the energetics of these steps.
The glucose going to glucose-6-phosphate has
a delta G that is positive.
The ATP to ADP has a delta G that is negative,
and the coupled reaction will give a favorable
forward reaction to this and the reaction
actually involves a nucleophilic attack of
the hydroxyl OH that is attached to carbon
6 to the gamma phosphate of ATP resulting
in the formation of ADP and in this case the
enzyme that is involved is Hexokinase and
Hexo means it is going to act on a 6 membered
sugar ring.
It is a Kinase and it helps in the transfer
of the phosphate and the ATP binds to the
enzyme as a complex with magnesium.
Now, why would we have magnesium there, what
is ATP?
ATP has a large number of negative groups.
There are three phosphates one after the other,
what magnesium does is it interacts with the
negatively charged phosphate oxygen atoms
and provides charge compensation and also
promotes a favorable conformation of ATP at
the active site of the Hexokinase enzyme because
you have to realize that when we are looking
we are not going to look at the details of
how the enzyme is working.
But what we have here is we have magnesium
in the set so that the ATP can be favorably
interacted with the magnesium because we are
finally going to break the final phosphate
bond here.
And what is going to happen, where is the
phosphate going to go?
It is going to be attached to the sixth carbon
atom of glucose in the formation of glucose-6-phosphatefrom
glucose, so that is our step number 1.
So step number 1 is glucose to glucose-6-phosphate
the enzyme is Hexokinase and you have ATP
going to ADP and Pi is attached to sixth carbon
so ADP.
Now this reaction is highly spontaneous because
the phosphoanhydride bond of ATP is cleaved.
That is a high energy bond as we call it and
the phosphate ester has a lower delta G of
hydrolysis.
Now what happens in this case is you have
what is called an induced fit.
Remember, we studied induced fit for the enzyme
mechanisms the way they work.
There is a lock and key mechanism and an induced
fit mechanism.
Usually the enzymes of the glycolytic pathway
have induced fit.
They are not ones that would just sit with
a lock and key where an exact substrate would
come and sit there.
It happens such that the binding of the glucose
to this Hexokinase promotes a conformational
change in the protein in the enzyme rather
and it stabilizes a different conformation
where by the ATP is positioned in such a way
that the terminal phosphate can be transferred
to the substrate that is glucose.
So what happens is you normally would have
Hexokinase and as soon as the substrate glucose
comes into the picture there is the ATP that
is positioned favorably so that the phosphate
can be transferred from ATP to glucose.
And we also have this important point where
water is excluded from the active site.
What would happen if water would be there?
The ATP would be hydrolyzed.
We don’t want the hydrolysis of ATP but
we want the phosphate to be transferred only
to glucose.
So once the substrate comes to Hexokinase
it changes its conformation so that it can
accommodate glucose at the same time positioning
ATP in such a manner so that it is favorably
interacting and remember what happened, how
did the reaction take place?
It was the sixth OH the OH attached to the
sixth carbon atom that actually went and attacked
the phosphate along here.
This is step number 2.
In step number 2 you have glucose-6-phosphate
go to fructose-6-phsphate.
So what happens is you have the formation
of an isomer so the enzyme involved is an
isomerase.
What is this isomerase?
It is a Phosphoglucose Isomerase and what
is happening here is the glucose-6-phosphate
forms a fructose-6-phosphate where instead
of the aldehyde now the aldose you have a
ketose.
You have the CH2 OH up right here now so you
have glucose-6-phosphate go to fructose-6-phosphate.
The enzyme involved is Phosphoglucose Isomerase
and the mechanism actually involves acid base
catalysis with the ring opening via an ene
diolate intermediate and finally we have ring
closure.
So we have an isomerase so what was the first
step?
The first step was glucose to glucose-6-phosphate
and the next step is glucose-6-phosphate to
fructose-6-phosphate.
What do the enzymes require?
In the first step we need the kinase and in
the second step you need an isomerase.
Third step; look at what is happening.
We had fructose-6-phosphate go to fructose-1.6-bis
phosphate.
So what is happening?
I have the addition of another phosphate at
the one position so what is the enzyme that
I need?
I need a kinase and that is what you have
to identify.
You have a process you know the substrate,
you know the product what is the enzyme involved.
You know in this case that you have prepared
fructose-6-phosphate from glucose-6-phosphate
and it was just an isomerazation so you needed
an isomerase.
You are going now from fructose-6-phosphate
to fructose-1.6-bis phosphate which means
you have added another phosphate which again
means the breakdown of another ATP and that
you need a kinase.
And what kind of a kinase do you need?
A fructokinase.
Why a fructokinase is because you have a fructose
and you are adding a phosphate to a fructose
that is it.
So you have fructose-6-phosphate that is going
to form fructose-1.6-bis phosphate and this
process is actually highly spontaneous and
this phosphofructokinase reaction is the rate
limiting step of Glycolysis.
We will see how that is later on when we study
the whole process and this enzyme is extremely
tightly regulated.
And when we go through the whole system you
will see that there are some processes in
the Glycolysis steps of reactions that are
reversible and there some that are irreversible.
Once glucose has got in to glucose-6-phosphate
there is no way it is going to get back to
glucose.
The next step what we have here now is we
have fructose-1.6-bis-phosphate.
Now what happens to fructose-1.6-bis-phosphate
is we have it in the ring formation here.
In the next step there is going to be the
breakdown of this, how carbons do we have
here?
We have 6, glucose has 6 carbons, and we have
6 carbons here.
We have 1, 2, 3, 4, 5, 6 and what is going
to happen now is in the next step first there
is ring opening then the six membered ring
is going to break down into two three member
rings.
Because you have to remember that finally
we have to get to carbon dioxide and water.
So unless we start chopping up it is not going
to be possible.
Hence we have our glucose and now we are going
to go to a step where we are going to break
the fructose-1.6-bis-phosphate into two three
carbon units.
The process here is a reverse of aldol condensation.
It is aldol cleavage.
We have here the fructose-1.6-bis-phosphate
which is now not in its ring form but in its
open form where you can see the ketone.
It is a ketose so it has to have this C double
bond O. there are two phosphates attached
to it now, one at the one position and one
at the six position.
We have now an aldolase which a reaction that
is going to involve aldol cleavage and there
is going to be a reverse of aldol condensation
which means you are going to have a break
at this position here where we are going to
have the formation of two three carbon units
one of them is Dihydroxyacetone phosphate.
If you look at this, this is acetone CH3 CO
CH3 is acetone, this is acetone CH3 this is
acetone Dihydroxyacetone is that.
Now if we have Dihydroxyacetone phosphate
we have phosphorylated one of these.
So that is exactly what we have.
Here we have Dihydroxyacetone phosphate and
we have Glyceraldehyde-3-phosphate.
This is Glyceraldehyde you recognize the CHO
of the aldehyde the CH OH and CH2 OH which
would have been here but we now have it phosphorylated.
So the phosphor now is distributed in the
two three carbon units.
So this is the ketone form, this is the aldehyde
form.
What are these?
These are isomers.
So you can go from one to the other by an
isomerase enzyme.
What is this isomerase going to be named?
it works on a three carbon unit so a triosephosphate
isomerase.
If you look at the nomenclature it is actually
very simple.
All you have to know is what the substrates
are and what the products are.
In this case we have an aldolase with the
reaction being an aldol cleavage.
We have the formation of Dihydroxyacetone
phosphate and Glyceraldehyde-3-phosphate.
So this step is where you have the breakdown
of the six membered or the six carbon unit.
Now, if you look at how the six carbon unit
or the aldolase actually works there is a
lysine residue in the enzyme aldolase.
Obviously it has to be there because the aldolase
is what is acting on the substrate.
What is the substrate?
The substrate is fructose-1.6-bis-phosphate
that is your substrate.
What are your products?
Your products are Dihydroxyacetone phosphate
and Glyceraldehyde-3-phosphate.
So you have Glyceraldehyde-3-phosphate and
Dihydroxyacetone phosphate which later on
you will see that it is written as dhap and
g3p.
So we have a lysine residue in aldolase that
is present at the active site.
What this lysine does is a keto group of fructose-1.6-bis-phosphate
reacts with the amino group of the lysine
and it forms a protonated Schiff base then
there is a cleavage between carbon atoms 3
and 4.
We are not going to go into details of all
this but what you have to know is you have
the breakdown, the breakdown from the six
carbon to the three carbon by the enzyme aldolase.
The enzyme has a lysine residue that interacts
with the amino group of the lysine and where
is this lysine it is in the active site of
aldolase.
It interacts with the keto group of fructose-1.6-bis-phosphate
and it breaks it up, that is cleavage between
carbon atoms 3 and 4.
Therefore this is just the triose phosphate
isomerase that is going to be inter converting
dihydroxyacetone phosphate and Glyceraldehyde-3-phosphate.
This protein actually is pretty interesting
in its structure also but we not going into
the details of that, it is called TIM and
it has a TIM barrel actually associated with
it.
We have the Glycolysis that is going tocontinue
from Glyceraldehyde-3-phosphate.
Now what is that triose phosphate isomerase
doing?
It is converting Dihydroxyacetone phosphate
to Glyceraldehyde-3-phosphate.
This is an isomeration reaction.
The equilibrium constant is such that it favors
Dihydroxyacetone phosphate.
So this is favored but the Glycolysis steps
the further steps actually continue from Glyceraldehyde-3-phosphate.
Therefore if the equilibrium shifts to Dihydroxyacetone
phosphate what does it mean?
It means I may not have sufficient Glyceraldehyde-3-phosphate
to continue with the Glycolysis.
I will repeat that once more.
We have the enzyme triose phosphate isomerase.
The equilibrium constant of triose phosphate
isomerase is such that it favors Dihydroxyacetone
phosphate.
When you have equilibrium your equilibrium
either shifts your left or right to the reactants
or products.
In this case Dihydroxyacetone phosphate is
preferred based on its equilibrium constant.
But the process of Glycolysis only will continue
with Glyceraldehyde-3-phosphate.
So what has to be done is your equilibrium
has to shift to your right.
So what is usually done is this Glyceraldehyde-3-phosphate
as soon as it is formed it is then utilized
in the next step.
thus if it is utilized in the next step what
happens is your equilibrium is shifted to
right which means some more of the Dihydroxyacetone
phosphate has to be isomerized to Glyceraldehyde-3-phosphate.
It is regulated.
You see how it is regulated.
The equilibrium is such that it shifted to
the left.
But if you require the breakdown of the Glyceraldehyde-3-phosphate
you break it down.
What happens then is your equilibrium is moved
in such a way that the Dihydroxyacetone phosphate
forms in the Glyceraldehyde-3-phosphate.
Then what happens is there is removal of the
Glyceraldehyde-3-phosphate and subsequent
spontaneous reaction.
But it doesn’t happen as it is.
The equilibrium shifted to the left but with
the removal of the product that is Glyceraldehyde-3-phosphate
it obviously moves to isomerize more of the
dha.
This is the intermediate you have which is
the enediol intermediate where you have the
ketose/aldose conversion which actually involves
acid base catalysis and the isomerase that
actually takes place is a Phosphoglucose Isomerase
or rather what happens here is when we have
Dihydroxyacetone phosphate what are we talking
of is we are talking of ketose.
When we have the Glyceraldehyde-3-phosphate
we are talking of an aldehyde so there has
to be an enediol intermediate that is going
to take you from the ketose to the aldehyde.
It is similar from fructose to glucose.
What is glucose?
Glucose is an aldose and fructose is a ketose.
You have an isomerization there are also that
takes you from glucose to fructose.
It is exactly the same thing but here you
are working on a three carbon atom instead
of a six carbon, so that is the difference.
We have the Dihydroxyacetone phosphate, we
have the Glyceraldehyde-3-phosphate and these
are formed from an enediol intermediate.
So when we are talking of the triose phosphate
isomerase we are talking of this equilibrium.
It is this equilibrium that we are talking
about.
So the equilibrium will actually shift to
the left side but with the removal of the
Glyceraldehyde-3-phosphate what will happen
is there will be more formation of the Glyceraldehyde-3-phosphate
that will eventually be used in the other
steps of Glycolysis.
This is what we are going to do for Glycolysis
today because there are some other discussions
I want to make regarding Hexokinase.
These are the steps.
We have just broken down the six membered
ring, let us put it that way.
We will see now what is going to happen to
the three carbon ring.
Now the three carbon system will eventually
get into the Tri Carboxylic Acid cycle and
that Tri Carboxylic Acid cycle will ultimately
produce carbon dioxide and water so you haven’
broken down.
But what we started off with is we started
off with glucose.
In the first step we had glucose go to glucose-6-phosphate.
Now since it took up a phosphate it required
the breakdown of an ATP, it is just summarizing
the steps that we have done so far.
So we have glucose to glucose-6-phosphate
that required the breakdown of an ATP and
the enzyme was Hexokinase, step number 1 in
our Glycolysis.
Step number 2 was the formation of fructose-6-phosphate
from glucose-6-phosphate, it is nothing but
an isomeration where we have an aldose go
to a ketose.
The enzyme is a Phosphoglucose Isomerase.
Step number 3 is where we have our fructose-6-phosphate
form fructose-1.6-bis-phosphate.
What is happening there is I am adding another
phosphate.
In the addition of a phosphate I have to breakdown
another ATP and I need another kinase.
So I have fructose-6-phosphate that will take
up phosphate from the ATP into forming fructose-1.6-bis-phosphate
with the help of phosphofructokinase.
The next step now is the breakdown of the
six carbon to 2 3 carbons and we have aldolase,
aldolase is going to breakdown fructose-1.6-bis-phosphate
into Glyceraldehyde-3-phosphate and Dihydroxyacetone
phosphate.
And if you notice here it is Glyceraldehyde-3-phosphate
that will allow the Glycolysis to continue.
So we need an isomerase that is going to transform
a Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate.
Now in the steps that we wrote here if you
notice the steps that involve the addition
of the phosphate are irreversible.
So, glucose forms glucose-6-phosphate with
the breakdown of ATP to ADP that step is one
way.
That means once glucose enters the cell and
forms glucose-6-phosphate it is struck into
being broken down.
If it is not required to be broken down then
glucose will actually go for storage as glycogen.
But once it forms glucose-6-phosphate it has
to be broken down.
So, if we look at the features actually of
Hexokinase this enzyme is inhibited by it
is product.
Now biochemically that makes absolute sense
because if Hexokinase is inhibited by glucose-6-phosphate
then what is it going to do?
It is going to prevent the formation of glucose-6-phosphate
from glucose so it is going to be regulated
in nature.
Therefore as soon as there is sufficient glucose-6-phosphate
are sufficient glucose to be broken down it
will stop itself from acting because you don’t
want all the glucose to form the glucose-6-phosphate
because once the glucose-6-phosphate is formed
it has to be broken down it is entered the
glycolitic cycle there is no way it can go
back.
But if Hexokinase is inhibited then what happens?
Glucose can still go elsewhere and be stored
as glycogen in the liver sink.
But once Hexokinase has acted on glucose there
is no way it can go back.
So glucose-6-phosphate acts as a inhibited
to the Hexokinase so that further glucose
breakdown is not possible.
Hexokinase is inhibited by its product glucose-6-phosphate
and the way it inhibits Hexokinase is by competition
at the active site as well as by allosteric
interactions at a separate site on the enzyme.
What does that mean?
It means that it will change the active site
conformation to such an extent that it will
not be able to bind the substrate glucose.
And remember I showed you a rough cartoon
of Hexokinase where it has an induced fit
as soon as glucose comes into the picture
then the ATP is positioned in such a manner
that it forms glucose-6-phosphate.
But if glucose-6-phosphate actually inhibits
Hexokinase then it will either inhibit by
sitting at the active site itself or it could
inhibit at another position where it could
affect the active site so that the substrate
cannot bind.
So once the substrate cannot bind it means
that the enzyme is inhibited and if the enzyme
is inhibited then the product will not be
formed.
If the product is not formed in this case
it means that the glucose breakdown does not
occur, as simple as that.
So it is extremely tightly regulated.
Thus the cells trap the glucose by phosphorylating
it.
Once glucose is phosphorylated it is trapped,
it has to be broken down.
This can be prevented if this glucose-6-phosphate
inhibits the enzyme.
If Hexokinase is inhibited then glucose cannot
form glucose-6-phosphate.
So the product inhibition of Hexokinase ensures
that cells will not continue to accumulate
glucose from the blood if glucose-6-phosphate
within the cell is insufficient quantity.
Because once your glucose-6-phosphate is of
insufficient quantity it means that this can
continue in the glycolytic steps.
But if you need it then only glucose is broken
down.
Unnecessarily glucose is not broken down in
the cell.
It is only when the glucocarbohydrate metabolism
is required is glucose broken down in the
cell because once this first step of the glycolytic
cycle takes place it is irreversible.
Now Glucokinase which is a variant of Hexokinase
is found in the liver.
This has a high Km value, all of you know
what the Km value is now Michaelis Menten
constant for glucose and it is acted only
at high glucose concentration.
So the Glucokinase acts at high glucose concentrations.
Hence when there is a high level of glucose
concentration the Glucokinase enzyme comes
into the picture.
The Glucokinase enzyme is not subject to product
inhibition by glucose-6-phosphate.
What does that mean?
It means that Glucokinase will act on glucose
because it is not inhibited by glucose-6-phosphate.
So any glucose that comes into contact with
the Glucokinase will have a phosphate transferred
to it, as simple as that.
Why is that?
It is because the Glucokinase is not subject
to product inhibition by glucose-6-phosphate.
So Glucokinase actually works at high glucose
concentration.
So, when there is high glucose Glucokinase
will transfer a phosphate to the glucose at
high glucose concentrations.
That means where is this Glucokinase found?
It is found in the liver.
And what it does is it takes up and phosphorylates
glucose even when the glucose-6-phosphate
is high.
Why is that?
It is because it is not inhibited by glucose-6-phosphate.
Glucose-6-phosphate has got nothing to do
with Glucokinase. if it was Hexokinase then
glucose-6-phosphate would have inhibited the
enzyme.
But Glucokinase is not inhibited by glucose-6-phosphate
so it is immaterial whether the glucose-6-phosphate
is high or low it does not matter.
Liver Glucokinase is actually inhibited by
a Glucokinase regulatory protein.
What does that do?
You have to remember that when you are considering
an inhibition it is to prevent the enzyme
from forming its product.
Now naturally there are certain steps that
you would not want the product to be formed
for example in the Hexokinase step.
In the Hexokinase step you realize that breaking
down of all the glucose present is extremely
unnatural and you wouldn’t obviously want
that to happen because you want some stored
glucose.
So the product inhibition in the case of Hexokinase
is an extremely clever way of preventing the
glucose from being broken down.
But in the case in the liver the Glucokinase
is present not Hexokinase.
It is a variant of Hexokinase but it acts
only when the glucose level is very high.
Now the liver Glucokinase is subject to inhibition
by another protein and the protein will regulate
when Glucokinase is going to act.
So it is not inhibited by a product it is
inhibited by another protein that is going
to regulate the action of Glucokinase as to
when it should be acting on a glucose and
when it should not be acting on a glucose.
This Glucokinase regulatory protein actually
would work then when we have low glucose because
high glucose we would actually have Glucokinase
work on it.
So what happens in this case Glucokinase with
its high Km value for glucose allows the liver
to store glucose as glycogen when the blood
glucose level is high.
So, when the blood glucose level concentration
is high it will allow the storage of glucose
as glycogen.
Now what happens is therefore this is what
we would have in the liver where we would
have the glucose acted upon by the Glucokinase
to form glucose-6-phosphate it would not be
broken down or rather Hexokinase would be
inhibited by glucose-6-phosphate but not Glucokinase
and then we would have from the glucose-6-phosphate
once this is formed in the cell it is trapped.
So we have a regulatory mechanism that actually
works here 
and then glucose-6-phosphatase catalyses hydrolytic
release of Pi from glucose.
So what happens if you look at the step here
you had glucose-6-phosphate formed but there
is another protein that is glucose-6-phosphatase
that can release the phosphate but this happens
only in the liver where the glucose can be
stored.
In the other cells what happens is, the enzymes
Glucokinase and glucose-6-phosphatase are
both found in liver but not in most other
body cells.
Why would it not be in most other body cells
because there you wouldn’t get the energy,
glucose has to be broken down?
If all the steps prevented the breakdown of
glucose then obviously you would not get energy.
But in the liver the excess glucose is stored
as glycogen because they are the specific
enzymes that are present in the liver that
can form glycogen from glucose.
So what we actually looked at is we looked
at the first few steps of Glycolysis where
what we have ultimately come to today is we
have broken down the six membered ring of
glucose to 2 3 membered rings.
Now we are going to see how those three membered
rings or rather Glyceraldehyde-3-phosphate
actually will form pyruvate and finally how
that will in anaerobic conditions goes to
lactate or goes through acetyl CoA to the
Tri Carboxylic Acid cycle where it is finally
broken down.
We will see that in our next class, thank
you.We continue our lecture on glycolisis.
We started of yesterday where we considered
the different metabolic processes that actually
go on in the body.
And if we look at the first slide here the
overall catabolic processes look at the break
down of lipids polysaccharides and proteins.
Now the break down of lipids gets into components
of fatty acids and glycerol.
The polysaccharides break down to monosaccharide
and the proteins break down to amino acids.
Now in the anabolic processes we have these
broken down amino acids and other factors
that actually get on into building up the
other macro molecules that are required for
our bodily functions.
Now what we are interested in is the breakdown
of the monosaccharide particularly the process
of glycolysis that takes glucose and breaks
it down into pyruvate.
Later on we will see how this pyruvate then
gets on into the tricarboxylic acid cycle
or the Krebs cycle to finally get to water
and carbon dioxide.
And an off shoot of that is the production
of ATP where we studied oxidative phophorylation
in the different complex processes that require
a number of electron transfer cofactors as
well as certain enzymes.
The overall equation for glycolysis is the
breakdown of glucose into 2 pyruvate.
Now what we have here is we see how ATP is
produced.
As we continue with all the steps we will
see how ATP is produced in some of the steps
but in the first set that we did, we found
ATP consumption.
This is the number of steps that we considered
in our last class.
We had glucose going to glucose-6-phosphate,
the enzyme being hexokinase, we have to remember
that kinase is a transferase that transfers
a phosphate group, so in this process we had
the breakdown of ATP to ADP and the phosphate
was transferred to glucose.
This then went on to form the ketose from
aldose so we had fructose-6-phosphate that
had the enzyme phosphoglucose isomerase acting
on it because this is an isomer, the aldose
and the ketose that we have here.
The fructose-6-phosphate then went on to form
fructose1, 6-bisphosphate where we required
another ATP to be broken down and the enzyme
used there was another kinase but in this
time it was phosphofructokinase.
After this particular step we have aldolase
come into the picture.
aldolase is what actually breaks up the 6
carbon membered ring into 2, 3 carbon membered
rings.
How is it getting oxidized in this case?
Glyceraldehyde is getting oxidized to form
glycerate.
What is getting reduced?
NAD plus is getting reduced to NADH and the
enzyme is the dehydrogenase.So we have Glyceraldehyde-3-phosphate
to Glyceraldehyde-3-phosphate dehydrogenase
and in the event it forms 1,3-bisphospoglycerate.
Now that you have formed the glycerate you
have to lose the phosphate to form the pyruvate.
So the first step in the loss of one of the
phosphates is the phosphoglycerate kinase
going to lose the phosphate that is attached
to the carboxylic first carbon atom of the
glycerate and you have three phosphoglycerate
formed.
After you form three phosphoglycerate there
is a mutase reaction which shifts the phosphate
moiety from the third carbon to the second
carbon.
So you have three phosphoglycerate formed
two phosphoglycerate then another enzyme that
helps in the dehydration is enolase that results
in phosphoenolpyruvate so after phosphoenolpyruvate
you have the enolic form of pyruvic acid which
then forms pyruvate after the loss of the
phosphate and who takes up this phosphate,
ATP.
So that comprises the whole series of steps
where glucose is broken down into pyruvate.
Now what we have to see is we have to do a
balance of energy.
We have to see how many ATPs are taken out,
how many ATPs are produced, and we have to
see whether the actual breakdown of glucose
is giving us any energy at all.
There is one thing that we have to remember
that per glucose there are two Glyceraldehyde-3-phosphates.
What is the previous step?
We have two of these and in the triosephosphate
isomerase we know that the equilibrium is
shifted to this side because this is being
consumed in the further steps.
