Third lecture on nucleic acids is going to
speak about the stability of DNA and what
are the certain factors that are going to
lead to the denaturation of the DNA or the
renaturation of DNA and how that is extremely
important for designing of drugs for the cleaving
of the DNA, as I mentioned in the last class
where we want to consider that the protein
synthesis is inhibited or stopped. If we go
back to look at the double helical structure
of DNA, we have our sugar phosphate backbone
and the specific bases linked together.
Apart from the hydrogen bonding present here
we are going to have certain other interactions
that are going to be responsible for the double
helix to maintain its structure in solution.
The forces that actually result in the DNA
structure are electrostatic forces that are
mainly repulsion forces. Can you guess why
we would call them repulsive forces? Why would
the electrostatic force in the case of DNA
or the structure of DNA that I showed on the
previous slide we call repulsive? What do
we have in the back bone? We have sugar phosphate.
What is the charge on these phosphates? They
are all negative so the strands were tend
to be as far as possible which is going to
result in an electrostatic force, that is
going to be basically repulsion.
We have hydrophobic forces and hydrogen bonds,
obviously between the bases and also possible
between the other hetero atoms present in
the bases with the water that is around and
we have stacking interactions between the
bases at different levels. Considering the
properties of the Watson Crick base pairing,
the base pairings are planar. In the resulting
planarity that we have between the G C and
the A T pairs, we are going to see a stacking
interaction that again can be distracted with
any agent that penetrates this region between
the bases. We have these four types of forces
that actually determine the DNA structure.
The base pairs are usually found in the interior
of the helix which is going to result in the
stacking interactions between the bases.
The charged and the hydrophilic sugar phosphate
backbone are on the exterior which results
in the electrostatic repulsion of the strands.
The phosphates from the two strands would
obviously be as far away as possible why because
they are negatively charged and they would
reduce the electrostatic repulsion if they
would be as far apart as possible. Some of
this is neutralized by poly amines. Why would
they be neutralized by polyamines or magnesium?
Because they would be positively charged they
would counter act the negative charge of the
phosphate and result in stabilization. Usually
we have magnesium and polyamines that would
result in the stability of DNA. So there are
certain forces that keep the DNA structure
together, keep it stable and we are going
to learn now of what agents can actually disrupt
the structural DNA.
The stacking interactions stabilize the double
helix as much as probably close to the base
pair hydrogen bonds. Because what do we have
in those cases? We have an interactions. We
have a series of such interactions. The stacking
interactions actually stabilize the double
helix as much as the base pair hydrogen bonds.
The stacking interactions are not sufficient
to over come the electrostatic repulsion of
the phosphates. The phosphates will remain
as far apart as possible that would be stabilized
by some positively charged divalent ions or
poly amines and the hydrogen bond between
the purines and pyrimidines guarantees that
there is going to be complementarity of the
strands.
This is extremely important in DNA because
you understand when we go from a double helical
DNA to forming the dotted DNA. Then what happens?
You have to have perfect complementarity.
It is not like RNA where you can have a single
strand and you can have a complementarity
for some region of the RNA but not for the
whole region. So we have these stacking interactions,
we have the electrostatic reaction interactions
and we have the hydrogen bonding interactions
and this is what holds the DNA together.
And we also have, this is one picture which
we saw previously where we also have an additional
hydrogen bonding between water that goes through
the minor groove of DNA. We have the major
groove that is this large gap here and the
minor groove. The minor groove also twists
around with the double helical structure.
We have these hydrogen bonds also formed in
addition to the other types of bonds or the
other types of interactions that are observed.
DNA usually is quite stable; it actually resists
attack in acid and alkali solutions. Can you
tell me why DNA is more stable compared to
RNA?
What are we deferring in this? We have our
base and phosphate. We have continuation here.
What do we have? In RNA we have this; in DNA
you are missing that. What is linked here?
You have the other strand here, you have the
phosphate here then you have the phosphate
again another sugar another base and so on
and so forth. What happens here is this is
susceptible to hydrolysis but DNA can resist
hydrolysis which is why ribonuclease does
not work on DNA; it cleaves only RNA, in the
mechanism that we learnt in or enzyme mechanism
classes. DNA actually is quite stable and
in mild acid solutions at pH 4. In this case
we have a different kind of hydrolysis where
by the purine bases themselves are hydrolyzed
since the OH is absent at the positions unlike
RNA.
What happens in this case the glycosidic bond
to the purine bases are hydrolyzed. We also
have the protonation of the purines bases.
What is going to happen if you are going to
have the protonation of the purine base bases
at the acidic solution? You are going to disrupt
the hydrogen bonding. As a result of which
you are going to have this hydrolysis which
leaves your purines protonated that is going
to act as a good leaving group. You are going
to have an isomerization of the depurinated
sugar. This is the hydrolyzed sugar where
you are going to have what is this and what
does this signify? What is this and signify?
It signifies that this part of a DNA strand.
You do not have the OH here so this is part
of the DNA strand and this occurs under mild
acidic conditions where there is a protonation
of the purine. The purine is lost and what
happens is this sugar now that is on the left
hand side which is depurinated isomerizes
to an open chain fall. What have you done
in mild acid conditions? You have not only
depurinated your DNA; you have also opened
up the sugar so that could basically be a
problem. What happens in RNA?
This is RNA now. In RNA what we have here
is we have the case where we have the OH at
the position. The OH at the position is going
to give you what? This is your OH at the position.
What is going to happen in that case? You
are going to have easy hydrolysis possible
with an OH--. You can have hydrolysis at
the OH position. What happened when we considered
the ribonuclease mechanism? Histidines 12
and histidine 19 were important in donating
a proton. One acted as an acid and one as
a base. The roles were reversed in what was
called the hydrolysis step. What do we have
in a sense? We have a trans phosporlization.
This is what is happening. What is this we
have a cyclic phosphate intermediate which
results in the phosphate being transferred?
You see how the phosphate has been transferred.
Where was it originally? You have either it
go back to its original form or it is cleaved
and so we have this part, which is the rest
of the chain and this part is a previous part
of the chain. We either have this phosphate
remain here or go over to the position. This
is not possible with DNA, you understand that.
Why is it not possible? Simply because you
don't have the OH there, so it is not possible
but this is easier for the case of ribonuclease.
When we look at our RNA, we have it as very
unstable and in alkali solution basically
because of that OH that is present. It results
in hydrolysis a possibility of hydrolysis
always of the phosphoester diester backbone
and this renders your RNA susceptible to strand
cleavage.
What is going to happen is your RNA strand
that was a single strand is going to easily
cleave, because of the OH being present there
under mild even on the alkali conditions it
is going to break up. And we already consider
the enzymatic hydrolysis of RNA where there
are ribonucleases that are going to cleave
RNA in a similar fashion. In this case it
is going to be the histidines that are going
to be important in the cleavage mechanism
whereas in just considering an alkali solution,
it is going to be your OH-- that is going
to attack the OH which you cannot do in the
case of DNA because it is absent.
We looked at this representation and as I
mentioned before when we are looking at enzymatic
cleavage we are talking about nucleases. We
are talking about ribonuclease which is going
to cleave ribo nucleic acid. We are talking
about deoxy ribo nucleic nucleases which are
going to cleave deoxy ribo nucleic acid. There
are two types of nucleases. The two types
are exonucleases and endonucleases. Exonucleases
chop off nucleotides from the ends, so when
you have a nucleotide and you are chopping
it off from the end then you have an exonucleases.
You have endonucleases when you remove the
internal phosphodiester bonds that is something
is within the chain. Again you have two types
because you have two bonds.
You can remove at the positions or you can
cleave at the position. If you act on the
hydroxyl group of a nucleotide it is type
a, if you if it acts on the hydroxyl group
it is Type b. So the 2 types of nucleases
are exonucleases and endonucleases. Exonucleases
are like, what is an analogous case for proteins?
An exonucleases, an analogous enzyme for proteins.
Remember when we did the c terminal we had
a carboxy peptidase. What did that do? Chop
off from the carboxy terminal of the poly
peptide chain but if you have trypsin or chymotrypsin
what did that do? Chop off in the middle it
cleaved in the middle. So an analogy for exonucleases
in the case of a poly peptide chain would
be that for carboxy peptidase.
The endonuclease is for two types. We have
the end and the end. The we have it acting
a type a acts on the hydroxyl group so we
have our A G. What is this path? This is the
of this. This is the of C. We have A G C T
in this sequence we have a end for each we
have a end for each. This is the end of the
gene but it is the .
What do we have here? The end and the end.
This is 3 so this phosphate, what is this?
This is the 3 of C. This is the 3 of G. If
you cleave here first of all you are in an
endonucleases and type. When you are cleaving
this is the end you have to remember this
is the end. This is the end of G. what is
this? C, so if we now look at the cleavages
what do we have here? If the cleavages here
it is cleaving at the end so it is Type b.
If it is cleaving here where, the red dotted
lines are it is cleaving at the end. It is
type a for example the phosphodiester present
in the snake venom is a type a endonucleases,
it just chops off the RNA.
These enzymes are actually used for cutting
DNA and RNA into manageable sizes. They are
used a lot in microbiology molecular biology,
where you have these specific genes that are
tailored to what you want to make or which
protein you want to make. You want to make
a mutation in a polypeptide chain. You know
the genetic code, you know what amino acid
you want and you know what bases you want
for that amino acid to be made. What you have
to do is in your DNA sequence you have to
change that set of bases. This is routinely
done and it is called side directed mutagenesis.
You call it recombinant DNA technology where
you have the set of bases that you can change
the protein that you are going to synthesized.
If you want to change a specific amino acid,
the rest of the chain is all the same.
You cleave at a specific position of your
original DNA you change it to what ever you
want it to be and then you have the protein
expressed in bacteria and once it is expressed
in bacteria then you have the mutated protein,
because the DNA what you do is you use the
machinery of the bacteria to make the protein
for you, that is what you are doing. You have
the DNA; you have changed the particular sequence
of the DNA. Once you change that what is going
to happen a different protein is going to
form, why because it is going go from DNA
to RNA to protein. So the message that the
messenger RNA is going to get is going to
be different than the original case because
you have already changed a base or a set of
bases.
When you use the bacteria to make the protein
for you, the protein is going to be the changed
protein. A lot of routines studies are done
in protein chemistry to understand the effect
of certain amino acids. For example in ribonuclease
you know that histidine 12 is important for
your activity. You change histidine 12 to
alamine and then you check for protein activity,
you won't get any activity why? Because
histidine 12 is crucial for the ribonucleictic
activity of the protein. In this way you figure
out which amino acid residues are important
in determining the mechanism of the reaction
is basically. These are different types of
nucleases basically.
We have rattle snake venom here or snake venom.
These are mostly nucleases. This cleaves DNA
and RNA that is why it is snake venom in the
first place. It cleaves exo (a). What is that
mean? The end chopping off one nucleotide
at a time and there is no base specificity.
So it will just chop off your DNA or your
RNA. Rendering any protein synthesis impossible,
you have pancreatic ribonuclease A that is
on the end. It has a preference for pyrimidine,
it is the type of the endonuclease because
it cleaves in the middle. It is a Type b so
this is as much as we are going to do about
the endonucleases or the nucleases in general
because we study the mechanism of ribonucleases
in detail. We are going to study what interactions
of DNA can be disrupted by disrupting the
chains or by separating out the chains. What
we have is there are certain terminologies
that they have used here. We have dsDNA. What
is this mean? ssDNA, dsDNA Double stranded
DNA going to single stranded DNA and because
this is what going to happen, suppose we add
such an agent that is going to disrupt all
hydrogen bonds. What is going to happen? The
chains are going to separate so we are going
from dsDNA to ssDNA. We have the strands,
hydrogen bonded separated. The process is
that we are rather the terminologies that
we are going to see are melting, denaturation,
strand separation.
Then the terms that describe the changing
from ssDNA to dsDNA. What are we doing then?
We are reforming. It is called annealing,
renaturation and sometimes hybridization.
We even have a process that is called zippering
we just have one you start linking one set
of bases and the rest zips up by itself. Now
how can DNA be denatured? DNA can be denatured
under extreme conditions of temperature or
pH. What do we do? We want to disrupt any
of the interactions that are responsible for
stabilizing DNA. We want to disrupt either
the hydrogen bonding or the hydrophobic interactions
or the stacking interactions whatever intercalation
just to separate the strands.
Denatured DNA is less viscous than native
double helical DNA and the bases exhibit greater
UV absorption. What we have here is denatured
DNA is less viscous. Why would that be? When
we have normal DNA, we have a double stranded
DNA that in solution would render the solution
more viscous. Why, because you have two strands
that have to be kept together always because
of the stacking interactions hydrogen bonding
or whatever forces are holding it together.
So the solution is going to be more viscous.
Once you separate the strands, what can happen
is within the strands you can get some coiling.
What is going to happen? Is your solution
of the DNA is going to be easier for it to
flow, so it makes it less viscous.
The bases exhibit greater UV absorption. Let
us go to the analogy of the protein. You have
a protein a tripped fan. Where do you monitor
the UV of proteins? At 280 Nano meters. You
have a tripped fan that is embedded in the
center of the protein. You unfold the protein;
the tripped fan can be seen. Your absorption
is going to increase the same thing here.
The absorption the UV absorption that you
see for the nucleotides is due to the bases.
If the bases are always involved in an interaction
within themselves you cannot see as much but
as soon as you open up the strands. What is
going to happen? You will have greater UV
absorption. We have the bases exhibit greater
UV absorption and the DNA is less viscous.
This transition from double stranded DNA to
single stranded DNA is very commonly called
the helix coil transition because you are
going from the helix to the coil. This is
exactly what is happening. You have a double
helix. You are disrupting it, DNA denaturation
and you have a coil. So this is helix to coil
transitions, a dsDNA going to an ssDNA. This
is an actual picture. You see how this strand,
this is actually the double helix and how
the double helix has opened up here, where
the arrow is can you see that the double helix?
There is a strand going down here then it
is single helix again and then slightly double
helix not well all of it is double helix.
It looks like a single thread here but it
is opened up here. So what has happened here?
It has denatured. It finds, usually DNA repairs
itself. If there is some problem it will form
the double helix back again itself.
We can follow the denaturation of dsDNA by
spectroscopy. The bases have a maximal absorption
at 260 nanometers not 280 like proteins. In
double stranded DNA the absorption is decreased
due to the base stacking interactions because
you cannot see as much as you see when they
are single stranded. When DNA is denatured
these interaction are disrupted and you see
an increase. This is called as a hyper chromic
effect why because it is more. And the extent
of the effect can also be monitored by a function
of temperature.
Let us see what we get. So this is native
DNA the blue line at 25 degree centigrade.
Where is the maximum? 260, if I did the same
for proteins what would it look like? I would
have something that comes down here and it
goes up here. I would get the maximum at 280
nm for a protein. When the DNA is denatured
you increase the temperature so you disrupted
the DNA interactions. They are now single
stranded. You have rendered a helix coil transition.
What has happened to the absorption? It has
increased and you now know why it increases.
You have a relative absorbents of DNA that
increases on DNA denaturation because there
are stacking interactions, there are hydrogen
bonding interactions in the double stranded
DNA that are not going to allow a an absorbents
as high as it could be in the case of a single
stranded.
We have denaturation. What happens in some
cases is you cannot reverse the situation.
It is irreversible denaturation. What happens
is if the temperature is rapidly decreased,
then the change in the viscosity of the absorption
that is absorbed cannot be fully reversed
and the change occurs over a broader range
of temperatures. We will see what that means
and sometimes what happens is because now
if you just look at say the strand like this.
What you are intending? If you separate these
strands out all together then what is going
to happen and if you result in a cleavage
of the strands 2 it is unlikely they will
be coming together. What happens is with increase
in temperature or with certain agents also
it is not possible for the renaturation of
DNA. The overall renaturation actually depends
upon the average length of the DNA segments.
If the segments are small, the possibility
that they are going to find a complementary
base strands and join up to form the double
stranded helix. Then it is also depends upon
the concentration of the DNA. If the strands
are too far apart then it is unlikely that
they are going to find the partner DNA and
also the complexity of the DNA. What do we
mean by the complexity of the DNA?
We have to remember for complementarity in
the bases, so for looking for complementarity
in the bases, it might not always be possible
to find the same stretch of DNA that is going
to act or form the double helix factor together
again. We have the over all rate of renaturation
determined by these specific factors. The
concentration of DNA, the average length of
the DNA segments and also the complexity of
the DNA. Under certain conditions DNA can
be renatured. When we say renatured in this
case what do we mean, how is it different
from the renaturation of proteins? When we
denature the protein, we are unfolding the
poly peptide chain but when we are denaturing
DNA, we are separating the strands but for
the protein the amino acids are still linked
to one another.
When we remove the denaturing agent for example
urea what ever has been usually temperated
denaturation is always not renaturable but
suppose we have urea in this solution and
we have denatured the protein. We have just
prepared poly peptide chain back again. We
removed the denaturing substance. What is
going to happen? The protein will fold back
in this case the DNA has to find its complementary
strand. So under certain conditions can be
renatured where the complementary strand can
be brought back together. Why, because only
then are you going to get the proper double
stranded linear double helix rather the double
helix structure because you have to have the
ladder formation first, where you are going
to have complementary base pairs and you have
to have the correct hydrogen bonding pattern
reproduced again. Only then can you renature
the DNA back to where you started from. This
occurs at a temperature called the annealing
temperature very efficiently that is Tm -25
0C. What the Tm? It is called the melting
temperature of DNA.
The melting temperature of DNA is referred
to as the Tm. What are you monitoring here?
We are monitoring the melting of the DNA,
melting of DNA is basically the separation
of the strands helix coil transition that
is going to render the DNA structure disrupted.
So we have to have a specific measure of how
we can do that? What can we measure here?
What did we use when we measure the denaturation
at for the temperature cases? We measured
the relative absorbance. So I can measure
the absorbance.
What nanometer wavelength am I going to use?
260 nm. This is going to be my temperature
scale. I am going to monitor the 
absorbance with temperature as I increase
the temperature. What is going to happen to
my double stranded DNA? It is going to come
apart as it comes apart, what is going to
happen to the absorbance? It will increase
that is exactly what happens. It will increase
but will it keep on increasing? It will come
to a point where all the bases are exposed.
What will happen to the absorbance then? It
will not increase any more basically. We will
get some thing like this.
What sort of a DNA do I have here? A helix,
a dsDNA. What do I have up here? A single
stranded DNA, a coil. I have a transition,
the midpoint of this curve is what is your
Tm. Basically somewhere here would be the
Tm of this. What did we do in the previous
one that I showed you? We had the DNA curve,
we were monitoring at different wavelengths
and we found out that the maximum absorbance
was at 260 nm. This maximum absorbent at 260
nm increases when we increase the temperature.
What is now done is we are increasing the
temperature and I am monitoring the absorbance
at 260 nm. We know that the strands are being
separated so because the hydrogen bonds are
being disrupted between the bases. You are
disrupting the structure. You have this increase.
The mid point of this is the Tm. What we are
going to look at is factors that are going
to affect the Tm.
Before we get into that we have the renaturation
that we were talking about. We have a Tm -25
0C that is going to be an efficient annealing
temperature where the strands are brought
back together. Whatever the Tm is -25 0C,
it is kind of a rule of thumb. There are two
steps in the renaturation first there is a
nucleation, this nucleation is where the two
strands find a region of complementarity and
then they form a short double helix.
Then there is zippering in either direction
from the paired region of complementarity
the double helix is elongated and you understand
that only if it finds a correct set, otherwise
what will happen? It will form a bulge suppose
what are we looking at here. We have two strands
A T C G. We have other strands floating around
but it is found this part that is complemented.
We have T, what do we have here? A G C so
this part it forms a double helix within this
part and then it zips up in both directions.
It is going to form something like that once
it zips up but if it so happens that this
part is complementary but this part is not
then what are you going to have? You are going
to have some part that looks like a proper
double helix but some part that is sticking
out like that.
Then you would have a bulge, you would not
have a proper renaturation. It has to find,
the nucleation step is important that is rate
limiting in finding the two strands where
you have the complementary region and it zips
up in both directions and if it finds the
right kind of base pairs obviously it is going
to be very fast and it is the first order
reaction and it is pretty rapid. We have different
melting temperatures. This we would call then
a denaturation curve like the one I showed
you here. What do we have here? A denaturation
curve. Why is it called a denaturation curve?
What do we have here, helix. We have coil
here. We have double strand here and single
strand. We have denatured it, so this is a
denaturation curve and the midpoint of that
will give me the Tm.
What I have 3 denaturation curves. I have
3 corresponding melting temperatures of 3
different DNA samples that have differences
in their G + C content. What is the G + C
content? G C base pair content, the G C percentage
has increased from left to right and this
has resulted in an increase in the Tm. Why?
What happens in the G C pairing? In the GC
pairing I have triple bonds. I have double
bonds in my A T pairing. The higher amount
of G C that I have, it is going to be more
difficult for me to disparate the strands
so the melting temperature is going to be
higher. The GC content is important in determining
what the melting temperature is. So the higher
that you are going to see other factors also
of DNA but the higher the GC content, the
higher the Tm. Because it has to disrupt in
that case, free hydrogen bonds to separate
the strands.
We have to look at the influences on the melting
temperature, the GC content. We know why?
The higher the GC percentage the higher the
Tm because the GC base pairs have 3 hydrogen
bonds and are thus stronger than AT base pairs
that have 2 hydrogen bonds. What are the factors
do you think might be important? You know
what interactions stabilize the DNA. The basic
idea in this case is going to be disrupting
those interactions. What else is stabilizing
the DNA? We have electrostatic repulsion.
What is salt going to do? The salt is going
to shield the charges, lessen the repulsion
between the phosphates and the higher the
salt concentration lesser the repulsion then
higher the Tm. What is going to happen? The
higher the salt concentration, suppose you
have a large amount of magnesium what is going
to happen? The ions are going to shield the
phosphate charges on the backbone and this
backbone is going to remain as far apart as
possible, because you have the phosphate.
If you have salt ions that are going to stabilize
this, what is going to happen?
It will not be very easy for you to separate
the chains because these chains if say you
have further negative charge here. What is
going to happen? There is going to be repulsion
between the chains. Repulsion between the
strands is what is going to take it apart,
because essentially what you are doing is
you are going and you are resulting in a helix
coil transition that is what you are looking
at. If you are looking at a helix coil transition
is what you want is you want destabilization
of this. You want destabilization of your
strands. Now you want this repulsion to be
minimum. What is going to happen to the repulsion
here if you add salt? If you add Mg+2, the
higher the salt concentration the higher the
Tm because you are stabilizing the strands.
When you have pH, what is going to happen
when you have different pH? At low pH what
are you doing? You protonate the bases, as
you protonate the bases what is going to happen?
You are disrupting the hydrogen bonding. Again
the same thing when you increase the pH you
are disrupting the hydrogen bonding. What
is that? How is that going to affect the Tm?
It will decrease the Tm because you are resulting
in a larger disruption. All you have to think
about is what effect each of these are going
to have on the forces that are stabilizing
the DNA.
We have organic compounds. Organic compounds
can act in different ways. One thing is they
can intercalate between the bases. What are
they going to do? They are going to disrupt
them in that case, the stacking interactions.
They can also form more favorable hydrogen
bonds with the bases and in that case. What
are they going to do? Again disrupt the stability
of the DNA. Disrupt the hydrogen bonding either
the electrostatic repulsion or the hydrogen
bonding or the stacking interactions which
ever type or which ever type of interaction
is disrupted will result in the separation
of the chains. What we learnt was, we learnt
how the overall structure of DNA and RNA is
formed by the different, the nucleotides basically
coming together.
We have the sugar phosphate backbone in both
cases just the sugars being different in the
deoxy type, the deoxy ribose type and the
ribose type. The bases are the purines and
the pyramidines for both cases. We just have
a change thymine to urosil, when we go from
DNA to RNA. Then we looked at the hydrogen
bonding patterns in each cases, where we have
2 hydrogen bonds between A and T and 3 hydrogen
bonds between G and C. We saw how the stability
of DNA can be disrupted by certain factors
and what the melting temperature? What affect
the G C content and the A T content had on
the overall DNA stability. Other factors like
we have temperature also that we have to look
at, we have the G C A T content. We have the
pH. We have the addition of ions and the addition
of organic solvents. This completes our discussion
on nucleic acids, we will begin our bio energetics
in our next class.
We begin our lecture on bioenergetics which
is the final part of this course. We will
be speaking on certain aspects of the energy
of systems and mostly later on the metabolism
of carbohydrates. If we consider the bioenergetics
of life, we consider the thermodynamics of
energy conversions in living systems.
We have seen before how we look at ATP as
the source of energy and it will be more apparent
now when we see how this energy of the breakage
of the high energy phosphate bond is actually
going to give us a lot of the energy that
is required to drive these processes. We have
the free energy from ATP and other high energy
compounds, then the free energy from electron
transfer from one molecule to another in ordinary
oxidation reduction reactions where again
we will be using some compounds that we have
considered that have been derived from the
vitamins.
Then we will be looking at some metabolic
pathways and involved in that we will be looking
mostly at the breakdown of glucose, how glucose
is broken down in the body and in that there
will be some organic reaction mechanisms and
there will be some studies based on how the
energy is utilized in the processes in glycolysis
or the tricarboxylic acid cycle where we have
the final breakdown of glucose.
When we consider bio chemical energetics usually
the energy is actually harnessed by a process
that is called oxidative phosphorylation.
This is an aerobic process. Aerobic process
means it is a process that requires oxygen.
This oxidative phosphorylation is the process
by which ATP is formed. And you realize that
since ATP is the currency of energy we require
its formation at a very high level as well
because its breakdown is going to finally
drive a lot of other processes that are going
on.
Therefore the formation of ATP is extremely
important and oxidative phosphorylation is
the process in which it is formed as electrons
are transferred from NADH or FADH2 to oxygen
by a series of electron carriers.
So what happens is this pyrophosphate is often
the product of reaction that needs a driving
force. Now if you look at both the examples
that I showed you both of them are breaking
down at ATP which means that ATP has to be
produced somewhere. If you do not have enough
production of ATP obviously none of these
reactions are going to be possible.
So we are going to look at generally how ATP
is formed in a very simplistic manner. Now,
we have looked at these molecules before.
We will go into some of the details of what
we looked at before. We considered these when
we considered vitamins and coenzymes. NAD
plus is a coenzyme, that reversibly binds
to enzymes, that is what is a coenzyme. FAD
is a prosthetic group that remains tightly
bound to the active site of an enzyme. We
will see how these are utilized basically
in the process of oxidative phosphorylation
to actually give you the production of ATP.
And these flavins, now we will look at what
these molecules are. The flavin nucleotides
are tightly bound to what are called flavoproteins.
So the proteins that have FAD or the flavin
nucleotides are called flavoproteins and these
are required in the reactions of oxidative
phosphorylation to give you enough energetics
or whatever is required for the production
of ATP.
Now, this is something we studied before where
we are looking at vitamin B3 and Niacin here
where we have two cofactor forms that were
NAD and NADP, they are not tightly held, they
are the cofactors and they are reused for
reaction after reaction. We do not use them
in the raw form here, what we have to transform
them to is to NAD plus or NADP. Now what happens
in these reactions is we have an oxidized
form and a reduced form.
What are these forms? When we are looking
at Nicotinamide Adenine Dinucleotide, we know
that we have the Nicotinamide Adenine and
Dinucleotide here (Refer Slide Time: 56:00).
The nucleotide has a single phosphate, a Dinucleotide
has these two phosphates and we have a Nicotinamide
and Adenine....What is this membrane potential?
We have a membrane potential developed so
when an ion is transported from negative to
positive delta psi which is the membrane potential
is positive because you are going from a negative
value to a positive value. So what is the
delta psi? It is a positive value. Now, when
you have the matrix side, the matrix side
is the N side that has a low proton or low
hydrogen ion concentration, a low proton concentration,
a negative electrical potential. The intermembrane
side which is the C2 concentration in this
case has a high proton concentration and has
a positive electrical potential.
So when you are looking at the delta G values
you have an RT ln, what is your product in
this case? It is going to the intermembrane
side so it is C2 by C1. You have a Z F delta
psi which is nothing but your NFE, it is a
potential. Now, when you consider the low
H plus concentration and the high H plus concentration
you can link the logarithm of the hydrogen
ion concentration with the PH. So if we just
work this out I have my delta G. My delta
G is RT ln C2 by C1. Where is my C2? My C1
is low H plus. My C2 is high H plus. And you
have to remember that my high H plus is in
the intermembrane space and you are still
pumping in H plus to that space. Why, because
you have to make ATP. This (Refer Slide Time:
58:38) is plus your Z F delta psi.Now if we
want to convert, what do we know? We know
that the pH is equal to minus log of H plus
concentration, we know this. So all we have
to do is relate this with the pH.
