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Hello welcome back to our lecture series in rate processes. So today, we will take up
this topic on kinetics of some specific reactions; and under the heading under this heading,
we will talk about enzyme inhibition. Till now we have talked about reaction rates, rate
laws, effect of temperature on reaction rate then complex reactions we also talked about
theories of reaction rate then we talked about kinetics of some specific reactions; and under
this heading we will deal with this enzyme inhibition. We already have talked about enzyme
kinetics that is the bio catalysis, where enzyme is the bio catalyst like chemical reactions
normal chemical reactions in biochemical system. This enzymes are acting as catalyst.
So under this heading enzyme inhibition, we will talk about what is meant by enzyme inhibition
and various aspects of this. Now before going into the details of it, let us have some recapitulation
now we have dealt with this Michaelis Menten kinetics, where we talked about this K m that
is Michalis constant. Now if this Michaelis Menten equation is written in this form there
is in this form means 1 by V is equal to K m by V max into 1 by S, where S is substitute
concentration plus 1 by V max. So, this is m, this is x and this is c; and this is y.
Now K m is a constant. So this is called the Michaelis constant, now small K m means tight
binding; there is binding interaction is tighter, so like suppose you have got this is your
enzyme; and say this is your substrate, so they are bound tight together. So, it is difficult
to separate them up, may be. So when K m is small, then this is more this
interaction is more that is binding is tighter; and high K m means weak binding. So when K
m there is Michaelis constant is high, then binding interaction is big. Now this is useful
I mean this K m is very useful you know to compare you know this various reactants; and
they are you know how efficient, you know some enzyme is with respect to some reactant
or with respect to around different reactant. Now so for, for example, if we think of this
Hexokinase this enzyme now when the substrate is D fructose it is 1.5 mill molar k m is
1.5 mill molar whereas, for d glucose it is .15 mill molar, so the value of this, I mean
this for D glucose is K m value of K m for D glucose is less that means you know here
it is tight binding. It is also useful to compare K m for a common substrate used by
several enzymes like Hexokinase and Glucokinase for Hexokinase D glucose case; it is .15 milli
molar whereas, for Glucokinase d glucose it is 20 milli molar.
So value this value is higher compared to this value. So, we can we can compare you
know compare whether the binding is stronger or the binding is weaker. Now enzyme inhibition,
now enzyme inhibitors are important for a variety of reasons. Now it can be used to
get information about the shape of the enzyme active site you know you have got suppose
you have got enzyme
like this, it is basically a protein; and say this is your active site .So this active
site in this active site suppose you are your reactant fits, and then chemical reaction
takes place and after that your product is formed plus your enzyme is returned back.
So inhibitors are used you know to find information about the shape of the enzyme active site.
This site may be, and the residues in the active size. So there are residues in these
active sites. So may be in this region in this in this region
this active site, so residues are there, so which residues are there and which residues
are important compared to the other that we can that we can get information about, then
then it can be used to get information about the mechanism, so to get the information of
the mechanism or how this reaction is taking place.
So mechanistic part is also you know we can we can find out the information of that. It
can be used to get information about the regulation or control of metabolic path ways. So using
in in using various inhibitors, we can find out this things; and also for drug design.
These studies are very important that is enzyme inhibition studies are very important.
Next types of inhibition, one is called you know you know when when it is the question
of inhibition that means we need an inhibitor. So reversible inhibitor is basically a substance
that binds to an enzyme to inhibit the reaction but, can be released in a reversible fashion
see usually involves formation of non covalent bonds may be some vander waals interaction
may be hydrophobic interaction or may be some electrostatic interaction.
So these are the three non covalent mode of modes of interaction by which this inhibitor
can bind with with enzyme. Next is irreversible inhibitor what is an irreversible inhibitor?
It is a substance that causes inhibition that cannot be reverse. So it is a it is a reversible
formation of bond that is usually it involves the formation or breaking of covalent bonds
that is breaking up covalent bonds may be within enzyme or may be formation of new bonds
that is a it is basically a chemical transformation of your enzyme.
So therefore, the enzyme loses its activity, and in a it is an in I mean irreversible fashion
but, for reversible case since the binding is not a covalent interaction I mean bond
is not a covalent bond so may be may be it can be reverse back. So because, because like
a hydrophobic hydrogen bonding may be electrostatic or van der waals interaction. These are the
modes of interaction for reversible inhibition.
Now so when we talk about irreversible or reversible you know inhibition you know there
there are other things that we should take into account, one is you know there are there
are three types of broad classification with respect to inhibition, one is competitive
inhibition, next is mixed or non competitive inhibition, third is uncompetitive inhibition.
So competitive inhibition mixed or non competitive inhibition and the third one is uncompetitive
inhibition, so competitive, non competitive, uncompetitive.
So these are the three types of you know inhibitors. So maybe you can you can call like competitive
inhibitor may be in another case you can call non competitive inhibitor or the way uncompetitive
inhibitor.
So let us look into the the chemical scheme for enzyme inhibition. See, in this particular
stage scheme enzyme plus substrate producing enzyme substrate complex, then it produces
the outcome outcome that is your product with a say rate constant of k b, this is associate
I mean k is forward rate constant for ES formation and this is the backward rate constant, I
mean the disassociation of ES complex. The movement you add some inhibitor what is going
to happen? that may be this inhibitor directly minds to the enzyme producing enzyme inhibitor
complex like this EI giving rise to E plus I back to EI, so this is one one reaction.
So this is K i and this is for your I mean inhibitor disassociation process or may be
or or may be the reverse processes enzyme inhibitor complex formation, and the followed
process is enzyme inhibitor disassociation. You can think in in other way also I mean
you can write this reaction in a in a reverse fashion. Another situation is enzyme substrate,
then it minds to inhibitor producing enzyme substitute inhibitor then it it gives you
know in a reversible fashion enzyme substitute plus inhibitor. So the corresponding equilibrium
constant can be written as this. We are defining one quantity alpha which is 1 plus i by K
i or alpha prime is 1 plus i by K i prime, and in this case v can be written you know
in this fashion and if we plot plot in this way 1 by v verses 1 by S, then we see that
there are three situations when alpha is greater than 1 and alpha prime is equal to 1 this
1 we are we are getting alpha is equal to alpha prime is equal to 1 this is another
situation. The second situation is alpha is equal to
1 alpha prime is greater than 1 I mean this this this the second means this one in in
second you know for the card b and here you see that alpha it is equal to alpha prime
is equal to 1. So this may be another situation, third situation is that like this and you
see that difference is that you see that for this 1 the intercept same for both cards you
see that this two intercepts are different and also it is cutting the 1 by by S axis
at different points here also they are cutting at different points here we see that this
intercepts are different but, they are cutting at the same 1 by S axis, so how this is coming?
we we will be discussing later on.
So these three situations may be may be may be may be considered. Irreversible inhibition
the inhibitor binds with the essential part of the enzyme active center by covalent bond
resulting in loss of activity for example, say this schematically written in this way
that say this is 1 reactant this is your enzyme E. So it is producing something like this
and HX something else also another another you know resulting substances produce but,
you see this this is a covalent bond formation, so that means enzyme activity since this is
a permanent bond formation, so enzyme is no longer enzyme is in no longer free, enzyme
is no longer free to react with another substrate. So the reaction after after this reaction
happens no further reaction is taking place, so that means the enzyme losses its its activity.
So this is called a irreversible inhibition it is not a not in a reversible fashion so
backward reaction is impossible or even if it happens it happens to an infinitesimally
small extent, so that that is practically not you know of any significance.
So this is basically a schematic representation of irreversible inhibition. Now inhibition
pattern an inhibitor may bind at the same site as substrates that means suppose you
have got this enzyme say this is your your substrate and say this is another similar
looking inhibitor. They have got resemblance in their structure so may be what is happening
that it will come over here that is it will compete with this, so this one also this one
these two are having the probability of binding to this or occupying this this space.
So so they are structurally similar so they are having structural resemblance and as a
result of which they will bind to the same active site. Next situation is an inhibitor
may bind at different site, and there by altering the catalytic activity although substrate
binding remains identical. So suppose your enzyme is this and your inhibitor binds at
some other place in such a way that suppose here it binds your inhibitor, and it modifies
your active site or active site activity in an indirect fashion, so that reaction gets
slowed down. Although apparently this particular site there is active site is not distant but,
because of this binding some internal structural change may happen as a result of which this
enzyme activity may get reduced. These are third situation is that many inhibitors may
do both of these that at the same time it may bind here also bind at a different place.
So they are doing in a in a in a in a parallel fashion may be. As I told you that the type
of inhibitors I mean inhibition is competitive, non competitive or mixed and the third one
is uncompetitive.
So competitive inhibitor it competes with the substrate suppose suppose you have got
similar kind of you know substrate inhibitor then what is happening that enzyme does not
know which one which one whether this 1 is is is its right substrate or this one is its
right substrate but, two as this is is our right substrate.
So what is happening that it becomes difficult since they are having structural resemblance
it is difficult for the enzyme to discriminate between these two, and as a result of which
it is said that these two compete for the for the same active site. Malonate is a competitive
inhibitor of succinate for succinate dehydrogenase like the scheme shown over here. Although
you know succinate for succinate after adding succinate or in presence of succinate dehydrogenase
it produces Fumarate but, here no reaction occurs but, there are having some structural
resemblance because, it is a you know diacid this is also diacid although it is only one
CH 2 spacer there are two CH 2 spacers. So for the formation of a double bonding meet
another C. Since it is not available over here so no reaction occurs but, although they
are having structural resemblance, so they are competing each I mean competing for the
same enzyme there is succinate dehydrogenase.
Next competitive inhibition the scheme is like the classical scheme there is the this
is the normal scheme enzyme, substrate, enzyme substrate complex, then enzyme back then product.
Now since the inhibitor is present there along with your substrate what is happening? Now
this inhibitor again binds with your enzyme, so what will happen? with with a with binding
constant K i producing EI. So a competitive inhibitor lowers the amount
of unbound enzyme available to substrate binding and as a result of which K m for substrate
increases, K m per substrate increases means you’re in you know binding is less as I
told at the very beginning, now what will happen? the other situation is that if you
add your substrate to a huge extent huge extent means that the substrate concentration is
very high, then what will happen? that concentration if we if we take the concentration ratio of
substrate to inhibitor then the ratio for substrate to you know substrate is more I
mean more amount of substrate is is there. So what is happening that may be almost all
the enzyme molecules are you know are surrounded by many substrate molecules compared to I
inhibitor. So what is happening, so probability of binding
of the substrate molecule increases since you increase the concentration of your substrate,
so that this path is you know more accessible than this path so this is happening parallel
but, since the concentration of your substrate is very high is very high therefore, this
is the majored channel so competitive inhibition can be can be minimized with high substrate
concentration. It cannot be eliminated but, it can be minimized that is the effect can
be minimized with high substrate concentration. So what is happening for your competitive
inhibition a competitive inhibitor lowers the amount of unbound enzyme available to
substrate binding and they are by K m for substrate increases, and if we increase the
concentration of your substrate then competitive inhibition remains but, the effect is minimized
the effect is minimized because, more of substrate are there so probability that the substrate
will bind enzyme is more as a result of which your normal reaction path is more promoted
compared to this inhibition path.
Next so the corresponding a plot 1 by V verses verses 1 by S you see that this is for your
no inhibitor present no inhibitor present and this is for your competitive inhibitor
present. Let us go back to the earlier slide this one you see here you see that this is
1 by V axis intercept for both red and blue curve same but, corresponding you know meeting
point at on the 1 by S axis you see the intercept on x axis it is 1 by K m minus one by K m
you see this is nearer to this point and this is farther.
So an intercept is 1 by V max so maximal velocity remains the same, and the slope you see K
m by V max so V max 1 by V max same for both, so K m is different so Michelis constant is
different so for your competitive inhibition if it is a case of competitive inhibition
you will be getting a curve I mean plot like this in absence of inhibitor and in presence
of an inhibitor. So this type of plot you may expect for a competitive inhibition as
I told you in the in the here also you see so this is a case of this is a case of competitive
inhibition so three possibilities are there, so first possibility we are just a we have
just explored.
So pictorially competitive inhibition for a unimolecular unimolecular reaction enzyme
substrate enzyme substrate complex then product then product will be reduced and enzyme will
be freed to come back to here and do the do this in a cyclic fashion, so in presence of
a competitive inhibitor what is happening that your enzyme is here they look very similar
you see this part and this part they will do very similar although this side is different
it is pictorially you know demonstrating this way you see that this one is having the option
to bind here you know take this place or may be this one can attach over here. So what
is happening that if it attaches then substrate cannot access this this pocket for the reaction
to take place. So basically enzyme competitive inhibitor
complex now what what you can do is you can increase the concentration of this substrate,
so probability wise if if it is very high, then competitive inhibitor cannot compete
concentration wise and as a result of which more of substrate will bind to enzyme to give
give rise to products so there by you can minimize, so it is a unimolecular case that
one pocket one substance, now if it is a case of two pocket and two substance then you see
this is your substrate and this is another substrate, so you see that substrate one substrate
two it is producing enzyme substrate complex then giving rise to products.
Now if you have if you have you’re a competitive inhibitor over here so which will compete
with this S, then in place of S this competing inhibitor will take this this place, so that
to have this further reaction you know since inhibitor is here so for that reaction basically
cannot take place. These these are basically pictorial representation so the pictorial
representations of the enzyme pocket.
Next is uncompetitive inhibition what is the uncompetitive inhibition? that your uncompetitive
inhibitor binds to the enzyme substrate complex. It it does not bind to your enzyme, so what
happens is that once enzyme substrate complex is formed, then this has got two options either
it will go it it will go this way to produce a product or it will move this way to give
rise to enzyme inhibitor substrate complex or sometimes it is called as ESI or EIS, so
what happens the decrease in V max and K m, now you see if you increase the substrate
concentration suppose there was one S initially now increase it to 6 1 2 3 4 5 6 so it will
happen that if you increase this substrate concentration more of ES will be formed and
the more this is formed that is enzyme substrate complex is formed it has got more option to
more option to move to this pathway. So that means you you cannot minimize this
state because, you know you can you can stop this process because, this process is very
much dependent on the availability of ES so therefore, this effect of this uncompetitive
inhibition cannot be cannot be minimized cannot be minimized with cannot be minimized with
high substrate concentration. So if you go back to competitive inhibition you see that
here the inhibitor binds directly with enzyme directly with enzyme but, in case of uncompetitive
it does in a different fashion that it binds with enzyme substrate to produce enzyme substrate
inhibitor a different complex and once it is formed it cannot give rise to this this
that is your product and there is a change in V max as well as K m,
so uncompetitive inhibition you see substrate this is another substrate this is the you
know third substrate, so what is happening these three are you know binding with enzyme
to produce to give rise to product P this is a product then P 2 and and also, something
over here. So in presence of uncompetitive inhibitor say substrate binds giving us the
enzyme substrate and then uncompetitive inhibitor binds over here once it binds then this S
2 cannot access this site. So reaction stops over here you see that this
uncompetitive inhibitor inhibitor binds with enzyme substrate complex not with the free
enzyme its action is important when substrate is bound already bounded the enzyme with the
enzyme, so when substrate is not present its action is you know it is not designable you
cannot cannot see its action but, the movement substrate is present in binds it stops the
enzyme activity it stops the enzyme activity
This is uncompetitive you see for uncompetitive this inhibitor increases, and the corresponding
plots you see that this this numbers this this intercept on a y axis it is you know
changing and also this intercept is changing, so both V and also V max and also Michaelis
constant these are changing for an uncompetitive situation, so for your competitive you see
it is one point on your y axis and two different points on your x axis you see here three different
points on y three different points on x axis of course, in negative fashion so this is
uncompetitive situation.
Next next or non competitive inhibition now the situation is the inhibitor may bind to
both free enzyme as also the enzyme substrate complex. The affinity of the inhibitor to
the two complexes could be different if binding of inhibitor results in a change of affinity
for the substrate K m will be changed and as a result I know and the the name is mixed
inhibition, if K m is changed but, if only V max affected then it is called non competitive
inhibition. So two situations mixed inhibition K m will
be changed non competitive inhibition V max will be affected, the scheme is kinetic scheme
is this enzyme substrate producing enzyme substrate complex giving rise to your enzyme
back and product now this enzyme can bind to both I mean this ES as also E with K i
for E and K i prime for ES so giving rise to EI for here, and enzyme inhibitor substrate
complex for here but, remember for your uncompetitive situation is like EIS,
so only the enzyme binds over here with enzyme substrate but, not with your enzyme but, here
non competitive or mixed fashion you see that enzyme binds with ES and I mean your inhibitor
binds with ES and E with different you know rate constants I mean equilibrium constants
EIS and EI so if K m is changed is called mixed if only V max is affected it is called
non competitive inhibitor.
You see mixed inhibition substrate enzyme this is another substrate, so it is basically a bimolecular scheme, so substrate
binds with enzyme producing enzyme substrate another S 2 also binds over here giving rise
to your product you see this is a case of mixed inhibition I mean non competitive or
mixed inhibition you see non competitive inhibitor substrate substrate 2 first substrate binds
then in presence of non competitive inhibitor this is produced or in another situation your
enzyme first noncompetitive inhibitor binds then in presence of your substrate it gives
rise to this, so basically this enzyme free enzyme and enzyme substrate inhibitor they
are in equilibrium higher these two may be intermediates.
So these four are in equilibrium so that is why basically you can you can put you know
basically enzyme inhibitor then enzyme inhibitor substrate just put one substrate over here
so that these four are in equilibrium, and only enzyme substrates channel can give give
rise to this enzyme plus product so probability wise enzymes are free enzymes substitutes
is reduced and reaction rate is lowered.
So it is called the mixed inhibition. So so this is the scheme enzyme substrate enzyme
substrate then giving rise to I means enzyme plus substrate giving rise to enzyme substrate
then product plus enzyme in presence of inhibitor what is happening? enzyme inhibitor substrate
then this is an equilibrium with enzyme inhibitor then enzyme inhibitor is equilibrium with
this one. So this is the this is this is the equilibrium
scheme you see as I have shown over here this is the equilibrium they are these four species
are in equilibrium now what is happening that if you put so you see here there is one channel
through which you can get your product. So if you increase your substrate concentration
to huge value huge number then what is happening? That possibility of this step I mean this
channel is a little increased although these steps are there.
So you can partially overcome the effect of this inhibition mixed inhibition by increasing
your substrate concentration so what is happening as a result of which what is happening you
see that V max is decreased and K m may get increased or may get decreased you see these
are three situations you see this is increased inhibitor is increased you see and this cutting
point is here in between not on your on y axis.
So three distinct situations are there this is your mixed or you know non competitive
come back to earlier slide that this is one case, this is the third case, this is the
second case uncompetitive this is the non competitive or mixed inhibition, so for your
mixed inhibition like your competitive inhibition is a kind of competitive kind of but, not
exactly a competitive it is a although it is a non competitive but, it has resemblance
to your to to competitive that you can partially overcome although competitive inhibition can
be greatly overcome with high substrate concentration here also you can you can cannot fully overcome
but, you can partly overcome with high substrate concentration.
So this is a typical plot for your mixed inhibition. So general a non competitive inhibition you
see no inhibitor present this one non competitive inhibitor you see it is here K m is remaining
same k m is remaining same so K m is remaining same means you see this is a non competitive
inhibition, if K m is affected it is called mixed inhibition you see here K m is affected
k m is affected. So here K m is not affected so it is called
your non competitive inhibition, so if this cutting point is somewhere over here then
K m will be affected but, if if this cutting point is here then you know K m is not affected
you see only V max is affected you see 1 by V max over here there is another one I mean
the the different 1 by V max, so V max is affected.
So that is why it is a non competitive inhibitor again go back to competitive inhibition you
see the typical plot for competitive inhibition this one you see K m is changing but, V max
is remaining same and a non competitive K m is fixed V max is changed and the this is
because, your scheme has changed scheme means the reaction scheme has changed and as a result
of which your you know the plot is different. So let us go back to this slide, this is your
non competitive inhibition this is your competitive inhibition this is uncompetitive inhibition
so basically we can we can explain these three you know crabs arising out of a you know you
know using using enzyme kinetics when when we talk about enzyme inhibitors and three
types of three broad classifications are there competitive, uncompetitive and non competitive
that is competitive, uncompetitive and non competitive.
So for your competitive inhibition V max is not changed only K m as I told you K m is
you know K m is increased and K m is increased means you’re you know binding is less binding
affinity is less less as a result of the presence of your inhibitor, for your uncompetitive
it is a different situation that you get two parallel plots as if these two are you know
occurring in an unconnected fashion as the third one is has got connection over here
you see that K m is not changed but, V max V max is changing.
So now why have we a have we learnt this enzyme inhibitors what is the necessity of a exploring
this one? now many drugs are enzyme inhibitors enzyme inhibitors means sometimes you need
to inhibit the action of some of the enzymes within our body for some you know for some
reason or in some cases suppose in presence of some inhibitor some physiological you know
process are impaired. So in that case you need to remove your inhibitor.
Now many drugs are enzyme inhibitors, so their discovery and improvement is an active area
of research in biochemistry and also in pharmacology. A medicinal enzyme inhibitor is often judged
by its specificity and its potency. Now by it’s a specificity and potency means specificity
means how specific its action is and a high specificity and potency ensured that a drug
will have few side effects and thus it will have low toxicity. Toxicity means side effects
suppose we are putting a drug we are giving a drug which has got side effects means it
is parallely affecting other a biochemical channels. It is affecting our our interest
I mean our our I mean the channel which we are interested in along with other channels
which which is not a desirable. So we need to you know we need to minimize
the side effect and you need to minimize the toxicity, so that’s why high specificity
and a potency is very important. Now enzyme inhibitors also occurred naturally and are
involved in regulation of metabolism and natural enzyme inhibitors can also be poisons and
are used as used to defend a predators or or as ways of killing prey therefore, natural
enzymes are also you know very important and that is natural enzyme inhibitors are also
very important . So summing up what we have learnt let us again
come back from come back to starting point that we started with with this Michaelis Menten
kinetics. We talked about the significance of K m already we have a discussion in in
in a in an earlier classes about this Michaelis Menten kinetics. Now the significance of smallness
or largeness of K m is discussed with the help of you know line we were bar plot we
tried to explain various types of you know inhibition effects.
Now you know enzyme inhibitors they are why why they are important because, which in a
you know information’s are necessary I mean that I mean basically you want to get information
about there is I mean active sites and also the residues that are important in the active
sites we want to get information or we want to get idea about the mechanism that is why
studies on on inhibition is very important how metabolic pathways can be affected? With
the help of inhibitors. we can study that and also most importantly
drawn design because certain drugs are enzyme inhibitors that means if we put that drug
or we if we administer the drug then it will bind to the active site of the enzyme or it
will it will trying to change you know the the local site of your active region of the
enzyme by some secondary effect may be it is binding to a distance point from the active
site and it changes the activity. So the study of this enzyme inhibition is
very important. Types of inhibition whether it is reversible or irreversible talked about
it reversible inhibition means it is a non covalent bond formation and irreversible inhibition
means it is basically a formation of a formation of a covalent bonds or rearrangement of number
of covalent bonds within the enzyme or may be in in the enzyme substrate or enzyme inhibitor
complex, now a now a competitive inhibition, non competitive inhibition and uncompetitive
inhibition. we have talked about then the basic kinetic
scheme we have given and three different situations can be can be obtained out of it one is for
competitive another is for uncompetitive and third one is for non competitive inhibition
we have given the idea of irreversible inhibition that is irreversible irreversibly this enzyme
is transformed to something else. Now different inhibition patterns we talked
about we talked about your in details taking each situations like for your competitive
inhibition how it is operative? How this can be minimized? And the linear if I part plot
for your competitive inhibition and its characteristics then with the help of pictorial diagram.
we try to explain the competitive inhibition then we went to competitive I mean uncompetitive
inhibition and the corresponding diagram liner we have bar plot then we came to non competitive
inhibition and with the help of this diagram I tried to explain the corresponding liner
we have bar plot it is the non competitive case how does it look whether we means whether
we are getting a different K m or not. So it looks like that we are getting same
K m but, different V max for your non competitive case although for next case its different
you see here this different K m and different V max, and the importance of enzyme inhibitors
we try to give you some idea why we need to study and actually this inhibitors studies
are very important for rational drug design. So this is very important this is very important
means the study of enzyme inhibitors is a very important.
So with this words we would like to I would like to conclude this session, so in the next
session we will talk about this kinetics of autocatalytic reaction and oscillatory reactions
so till then good bye.