1. Protein memiliki berbagai peran penting dalam dunia biologi.

Mereka dapat
mengangkut nutrisi dalam tubuh, membantu percepatan reaksi kimia, dan membangun
struktur makhluk hidup. Semua protein terbuat dari 21 "blok bangunan" yang disebut
asam amino. Asam amino terdiri dari karbon, oksigen, nitrogen, dan hidrogen, dengan
beberapa mengandung atom selenium. Asam amino ini membentuk kelompok amino,
kelompok karboksil, dan rantai samping yang melekat pada atom karbon pusat. Rantai
samping ini yang berbeda-beda dari satu asam amino ke asam amino lainnya dan
menentukan sifatnya. Ada tiga tingkat struktur utama dalam protein: struktur primer
(urutan asam amino), struktur sekunder (alpha helix dan beta sheet), dan struktur
tersier (bentuk tiga dimensi). Banyak protein memiliki bentuk tiga dimensi yang
membentuk struktur berbentuk bola dengan rantai samping hidrofobik yang
terlindungi dari air. Fungsi banyak protein bergantung pada bentuk tiga dimensi
mereka. Ada berbagai visualisasi yang digunakan untuk memahami struktur protein,
termasuk diagram penjajaran ruang, diagram pita, dan representasi permukaan. Protei-
protein ini beragam dalam ukuran, dan pemahaman tentang struktur dan fungsi
mereka penting dalam penelitian biokimia dan pengembangan pengobatan. RCSB
Protein Data Bank adalah sumber daya yang berharga bagi peneliti, menyediakan
informasi tentang struktur tiga dimensi protein dan biomolekul lainnya. Ini membantu
dalam memahami fungsi protein dan interaksi mereka.

Protein memiliki tiga struktur utama yang sangat penting untuk memahami cara
mereka berfungsi:

Struktur Primer (Primary Structure):

o Struktur primer adalah tingkat struktur dasar dari protein.

o Ini mengacu pada urutan linear asam amino yang membentuk rantai
polipeptida protein.
o Urutan asam amino dalam protein ditentukan oleh informasi genetik dalam
DNA.
o Struktur primer memainkan peran kunci dalam menentukan bagaimana protein
akan melipat dan berfungsi.

Struktur Sekunder (Secondary Structure):

o Struktur sekunder merujuk pada pola berulang yang diadopsi oleh rantai
polipeptida dalam protein.
o Dua struktur sekunder yang umum adalah alpha helix dan beta sheet.
o Alpha helix adalah bentuk sekrup kanan yang stabil karena adanya ikatan
hidrogen antara atom-atom dalam rantai amino.
o Beta sheet terbentuk ketika dua atau lebih heliks atau strand berlipat-lipat,
dengan ikatan hidrogen yang mengikatnya.
o Struktur sekunder memberikan stabilitas tambahan pada protein dan
membentuk bagian penting dari lipatan tiga dimensinya.

Struktur Tersier (Tertiary Structure):

o Struktur tersier adalah bentuk tiga dimensi yang diadopsi oleh rantai
polipeptida protein secara keseluruhan.
 o Ini ditentukan oleh interaksi antara berbagai bagian rantai amino, terutama
rantai samping (side chain) mereka.
o Interaksi yang terlibat termasuk ikatan hidrogen, ikatan kovalen seperti ikatan
disulfida, interaksi hidrofobik, dan interaksi elektrostatik.
o Struktur tersier menciptakan bentuk akhir protein, yang sangat penting karena
menentukan bagaimana protein akan berinteraksi dengan molekul lain dan
menjalankan fungsinya dalam sel atau organisme.

Ketiga tingkat struktur ini bekerja bersama-sama untuk membentuk protein

dengan fungsi yang khusus. Perubahan atau kerusakan dalam salah satu tingkat
struktur ini dapat memengaruhi kemampuan protein untuk menjalankan tugasnya
dan dapat memiliki konsekuensi yang signifikan dalam biologi dan kesehatan.

the ability of enzymes to speed up reactions is actually mind boggling the subject of this presentation
is to talk about the ways in which we study the kinetics or the ways that enzymes speed up reactions
in this presentation I will give a little bit of background about the process of catalysis talk about the
flexibility of enzymes and how that enables them to do what they do talk about activation energy
which is another consideration for enzymatic catalysis I'll talk about the mechanism of a specific
reaction for an enzyme called a serine protease and then I'll give the kinetic considerations that we
have during that analytical process and finally talk about the overall overview of using what are
called Michaelis Menten kinetics now when we think about enzymatic reactions there's actually a
series of different ways that that molecules can react in interacting with an enzyme we can have for
example reaction that's a single substrate reaction and a single product a is converted into B we can
have a reaction in which a single substrate is converted into multiple products so for example if I took
a and I split it into two molecules I would make B and C I could take multiple substrates and make
single products which is the opposite which would mean I would be putting two things together to
make a third that third being C is shown here and last I could have multiple substrates and multiple
products in which a and B are converted into two different things C and D now enzymes are as I said
magical in their ability to catalyze reactions and they are so much faster than a chemical catalyst that
it's important to think about the ways in which they're able to accomplish what they accomplish and
so this illustration of an enzymatic reaction goes step by step into some of the considerations for the
ways that enzymes accomplish what they do chemical catalysts I want you to remember are things
that are very fixed a platinum catalyst for example has no breathing it has no movement to it it
simply is a surface on which something can happen and enzymes are fundamentally different from
that in this illustration we see an enzyme shown in green and we see the active site of the enzyme
that is the place where the reaction is catalyzed shown in light green now the enzyme in this reaction
I'm showing you is a reaction of multiple substrates multiple products so we will have a and B as you
can see here that will be converted into two other molecules we start with the enzyme unloaded no
products contain no products and the enzyme and of course no substrates the substrates are the
molecules that bind to the enzyme and they will bind so as to position be positioned at the place
where the reaction occurs the active site we can see here their substrates have started to bind to the
enzyme we see the enzyme again in green we see substrate a that has bound the top portion of the
enzyme and substrate B that has bound the bottom now the interaction of the substrates with the
enzyme will actually cause the enzyme to start to change this is the coastland induced fit model of an
enzyme in the coastland induced fit it says that not only does the enzyme change the substrates into
products but transiently during the catalytic process the substrates change the enzyme and as we
will see that's essential for this reaction to occur so the substrate binding has happened we have
formed at this point what we call the es complex enzyme substrate complex now in the next rate
step you see right here what has happened is we see the reaction going on and the enzyme has
actually changed its shape slightly from the initial binding to bring a and B into closer proximity well
of course for a chemical reaction closeness is an absolutely essential or requirement for the reaction
to occur so the slight change in the shape of the enzyme has converted a and B from being a part to
being slightly closer together these changes of shape can be very large on enzyme terms or it can be
very very subtle but nonetheless the change happens with every reaction now the reaction is
occurring again as we can see because they have been brought into close proximity at this point as
the reaction is going on we have something called the es star complex and we can just simply think
about this as the place where the reaction is now able to occur as we look at this reaction closer we
see during the reaction a part of a has moved from A to B and this has been a transfer of a part of
one substrate to another a is no longer a and B is no longer B at this point we have made what we
call the EP complex we've made the products but we haven't released the products yet so a has
become C and B has become D now the products are still contained within the enzyme but the
products are different than a and B were so just as a and B caused the enzyme to change shape so
too will C and D cause the enzyme to change shape and you can probably see where this is headed
the enzymes going to go back to where it was and that's what happens right here we see in this
reaction now that the enzyme has been changed and it's changed back to its initial state in the initial
state we can think of its fingers being opened like my hand is open and C and D are ready to go flying
away the enzyme now being back in its original state is able to go and bind more substrate it's ready
for the next process now if we think about this our definition of a catalyst that everybody learns in
freshman chemistry is a molecule or an entity that catalyzes a reaction but it's unchanged in the
process that's a principle that is hammered into every freshman chemistry student now we see that
enzymes are actually slightly violating that principle they're being changed transiently during the
process but they end up in this in the end in the same way they started so overall they're not
violating it but they cheat a bit we see in this slide then a summary of all the reactions or the steps in
the process that you've seen before and I don't want to go through those again but I do want to
make the point that you notice that the arrows are going both ways and that means that this
reaction and every step in this reaction is reversible now reversibility of a reaction is a very important
thing to keep in mind when we're talking about metabolic processes or for that matter even non
metabolic processes but especially for metabolic processes because we have to think that is what are
the conditions that would make something go backward we've seen how enzyme flexibility enables
enzymes to accomplish what they accomplish but enzymes do have constraints that they have to
work in I've mentioned in these presentations numerous times now that enzymes and cells all are
governed by the rules rules of the universe that is they can't change the energies of reactions and so
those are true for cells and those are also true for enzymes now enzymes as we will see are tricky
little things I've mentioned how enzymes cheat and enzymes are going to cheat with respect to
energy as well so let's consider a reaction of a going to be in a going to be this is plotted from an
energy perspective on the screen what you see here on the left side of the screen we see a dot
placed on the graph showing free energy that is the energy that's associated with molecule a in the
process of going from A to B we see that there is a change in the energy that the energy is actually
increased and we call this increase in energy activation energy that's necessary to get a reaction
going the reaction proceeds and as the reaction proceeds we can see that the free energy Falls that
we make a product B that down by the end has a lower free energy than a hat that meant that
energy was released in the process of going from A to B and this makes this energy this this reaction
process favorable now it's important to note that this change in free energy that's shown right here
this change in free energy cannot be changed by an enzyme that is there's no change between the
starting and ending points of the enzyme the enzyme does some other things however it's also
important to note here that this height of the peak is really a critical place the height of this peak is
the place where the reaction can reverse and go backwards from where it came that is a can start
and then go back or be if it got enough energy could climb that curve and then go back to a
otherwise a is going to go forward to B and the reaction is going to be is going to be favored it is
going to be occurring now enzymes cheat okay enzymes can change the activation energy there are
no rules about activation energy okay there are rules about beginning and ending energies but what
activation energies changes do is they enable an enzyme to make more molecules more easily go
through that transition that is the magic of enzymes how do they accomplish that well they
accomplish this in a couple of ways one of the ways that they do it is by the fact that they have
binding sites that are very precisely oriented so that the molecules are placed in the close proximity
that they randomly would not be in too close proximity so easily so easily all right and that means
that it takes less energy for them to go through the next step in the process by doing this enzymes
can actually lower the activation energy and make it possible for a reaction to go easier and also to
go faster meaning it's therefore much more likely that the reaction from A to B will be catalyzed you
notice again enzymes have had no change in for overall free energy the energy of a is still a the
energy of B is still be okay only that transitional state has made a difference now I want to go through
and spend some time talking about the mechanism of an enzymatic reaction mechanism is important
to consider because with mechanism we can begin to see how enzymes are facilitating electronic
changes necessary for a chemical reaction to occur the example I will use is an example of a serine
protease a serine proteases are a class of enzymes that cut proteins they break peptide bonds that's
what they do and they break not every peptide bond they see but they break specific peptide bonds
at specific places within the proteins that they bind to all right so that means that they have binding
specificity they don't they don't cut everything that they see serine protease is have flexibility so we
saw in the initial illustration the flexibility of an enzyme and we're going to see it occurring again here
as we talk about the mechanism of the serine protease the electronic environment is very critical for
a reaction in a chemical reaction electrons are being manipulated electrons are being moved around
and to be able to do that one must have the environment for those electrons to readily be able to
move around and we'll see that happening in the active site of the serine protease enzymes also use
coenzymes now in this reaction in this example I'm going to give I won't show a coenzyme but I will
say that coenzymes actually help an enzyme to accomplish what it accomplishes no serine protease
is there they said cleave peptide bonds that's the catalytic action a catalytic thing that they do they
have specificity of cutting again by binding only to certain molecule or certain proteins they only cut
those proteins that they bind they have a common active site all the serine proteases the different
serine proteases have a three-dimensional configuration of the place in them where the reaction
occurs now we'll see that that is important because that configuration is what creates the electronic
environment necessary for the reaction to take place and last of all the serine proteases are very well
studied so we understand the mechanism of their action quite well so let's take a look now at the
mechanism of a serine proteases i've shown on the screen here a substrate for the enzyme this is a
polypeptide chain or protein that these subs that the serine protease will cut the specific cut that's
going to occur here will occur between the carbon and the nitrogen on this molecule and of course
you know from you know the structures we've talked about in other presentations this is the location
of the peptide bond now on the right side of this of this image you can see the central part of a
serine protease now the central part is the place here where the reaction is going to be catalyzed
now it's a little hard to get our head around some of these things so you're gonna see in some cases
I'm gonna stretch bonds and stretch molecules a little bit to actually make things fit so you can
understand this please understand that in an enzyme itself of course they're already better
positioned but it's hard with figures to make things fit as we would like to serine proteases all have a
common feature of their active site and the common feature that they have of their active site is that
they all contain these three amino acid side chains that you can see located in close proximity of
each other now I always like to remind students that when we see something like this it reminds us
that protein folding does occur that is that serine and histidine and aspartic acid which are the three
side chains that we see here are not located close to each other in primary sequence they're brought
into close proximity of each other by the folding of the enzyme to make them physically close to each
other as we see here and the closeness of these is important to start but more importantly the
flexibility of the enzyme with these side chains is absolutely essential to the catalytic function that
will happen okay so we imagine now that we see this folded enzyme and that the rest of the enzyme
is shown in yellow we're looking right now specifically at the active site near the active site we have a
place where the protein is going to bind and the protein that's going to be cut is going to be
interacted with this catalytic triad of serine histidine and aspartic acid The Binding of the substrate to
the enzyme occurs in a specialized site on the enzyme called the s-1 pocket so we've shown here the
s-1 pocket is a sort of a semicircle that's holding on to a part of that protein we can see the protein
that's going to be cut now is at the active site now in the binding of this protein to the active site you
notice that the nitrogen on the histidine has an arrow pointing towards the oxygen towards the
hydroxide we also note that the oxygen that's on the sidechain of aspartic acid is has a little dot next
to the hydrogen on the histidine what's happened here well I'm going from the previous slide to this
slide we can see that what's happened is the enzyme has changed shape very slightly The Binding of
the substrate and remember that binding of substrate changes enzymes has changed the enzyme
very slightly so that the proximity of aspartic acids sidechain 2 histidines has changed that's very
important aspartic acid here the oxygen has a negative charge and the negative charge has moved a
little bit closer to the ring of the histidine is shown here by this small action the electronic
configuration of the ring of histidine is changed and it's that change which is causing now the
nitrogen to be reaching out and what it's going to do is it's going to grab that hydrogen that's on
serine okay so this tiny change in shape that happened on the binding of the enzyme is starting the
process by which the reaction is going to occur so we can see here that the s-1 pocket has facilitated
all this happening I should say in the s-1 pocket that the s-1 pocket gives the specificity of the
enzyme the s-1 pocket will not bind to everything it will bind to specific proteins with specific
sequences within them very very important concept if it doesn't encounter those specific things it
won't bind them and if it won't bind them of course there's nothing to react and the end of this
process will not occur okay so the slight chart structural changes have happened and we now see the
result of this starting to come into play the things that the the entities have moved closer into each
other the electronic environment has definitely changed by this point and what we see is that that
proton that was on the Oh H of serine is now associated with the nitrogen of the histidine ring now
this is the first step in this catalytic process or actually the second step if we count the binding of the
substrate this making of the oxygen with a negative charge on the end of serine is fundamental to
this reaction occurring we call this negatively charged oxygen on serine and alkoxide ion okay that
alkoxide ion that's on serine is extraordinarily reactive it's ready to go do business now we've
stretched that s-1 pocket little bit too that again we're bringing things into closer proximity and that
is important because the alkoxide ion is looking for something to bind to it's looking for a nucleus it's
what we call a nucleophile and the nucleus that it's looking for here is this carbon which is the arrow
that's being pointed from the oxygen - down to the orange carbon so there is actually what's called a
chemical attack a nucleophilic attack that's occurring on that carbon we can see that the electrons
that are double bonded to the oxygen are rearranging as we see the arrow being pointed and the
next step of the process what will happen is that we're going to see a rearrangement in the molecule
okay so we went from this position to this position notice that we had a carbon with a double bond
to an oxygen that now is a carbon with a single bond to an oxygen that molecule is chemically
unstable it's chemically unstable and a chemically unstable molecule has to be dealt with because if
it's not dealt with it's going to cost problems well the enzyme has another pocket in it to deal with
that unstable molecule it's called the oxyanion hole and the oxyanion hole helps that unstable
molecule to fall apart without problem that's pretty cool okay it's going to fall apart without problem
and what's going to happen here as you can see is the nitrogen in blue is going to reach up and grab
that hydrogen that was originally grabbed by the histidine side chain okay so this intermediate that's
in the oxyanion hole is what we call a tetrahedral okay and tetrahedral as we know from organic
chemistry or what happens when carbon has those four bonds that you can see here okay the
peptide bond which is between the carbon and the nitrogen okay is is going to be broken as a result
of nitrogen grabbing that hydrogen here nitrogen has grabbed the hydrogen the grabbing of the
hydrogen from the histidine caused the bond between the carbon and the nitrogen to break so
we've broken the peptide bond and so part of the protein the part of the protein shown in blue is
now free to go and do its business it's released there's nothing attaching it to the enzyme and it goes
and it exits what we have done here is we have actually gone through the first part of the reaction
and in this part of the reaction is what we call the rapid part of the reaction okay the other part of
the protein is attached to serine it's physically attached to syrian it's a covalent bond at this point
now that covalent bond has to be broken in order for the other part of the original protein to be
released and that's what's gonna happen in the slow step of catalysis now the slow step of catalysis
actually has about the same number of steps as the fast step of catalysis but other things have to
happen including the movement of water into the active site in order for the this peptide to be
released well we see that happening here water now has physically moved into the active site there's
a wall a molecule of water and that process that we saw of the nitrogen on histidine taking a proton
is going to repeat itself we see it happening here we see the arrow from the nitrogen on the histidine
pointing to the hydrogen on water so it's going to take that hydrogen instead of taking the hydrogen
that it originally took which is no longer there on Cirie what's going to happen in that process is now
we're gonna have an activated oxygen like we had with the alkoxide ion except for here it's going to
be a hydroxide we're gonna have an activated oxygen that's gonna make a nucleophilic attack on
carbon just like we saw before so there's a nucleophilic attack that's going to happen in the process
of this moving forward here's the attack of the hydroxide and look what happens we see that the
electrons on oxygen are going to rearrange we create at a tetrahedral intermediate as we created
before and now there's the oxyanion hole stabilizing that intermediate we now see that what
happens is that oxygen is going to attack the hydrogen on that group and it's going to pull it away just
like the first peptide did when it does that what happens is the molecules released so we see the
second half of the polypeptide chain released and in addition we have the enzyme returned back to
its original state gone and as it were the cycle is now complete there's about 10 steps going through
what I've described here and the important thing to understand about this is that the enzyme started
in one state it went through a transition and then went back to the original state it was in very much
like the process I've already described but now you've seen it in mechanistic terms when we saw the
image of the reaction occurring we saw these various states that you see on the screen the enzyme
plus the substrate bound together to make the es complex which converted upon the change in the
enzyme to the es star complex which created the EP or the enzyme product complex which
ultimately resulted in the release of the enzyme in the product now I come back to this because we
are going to need to consider some things about the kinetic parameters that is the Meccan of the
speed parameters of the reactions that we're going to study now this rate of formation of product is
really what we're interested in when we talk about how fast an enzyme can make a reaction occur
this is the guts of what we're after we want to know how fast is the enzyme able to do this well if to
do this we need to make some simple assumptions and so we assume in the simple case that the
enzyme substrate complex proceeds directly to enzyme plus product okay so we've simplified this
more complicated equation above to a simpler equation below and this is done to help us better
understand what's going on in the price and the overall mechanism now these constants that are
here won't really enter into our consideration but the cake at that you see in the enzyme going to be
plus P will in fact be an important consideration for us as we talk about the kinetic parameters the k-
kat as we shall see is the rate with which product is forming now let's consider what's happening
inside of a couple of different scenarios of a reaction we can imagine that we have enzymes for
example shown in yellow and we have substrates as little red colored balls that are there we could
have a situation first of all where we have a reaction going on in a condition of low substrate and if
we have a low amount of substrate in a solution we could imagine that there's very few enzymes that
are going to be bound to substrate because the chances of encountering a substrate are reduced in
the middle of course we have an intermediate state where we have a little bit higher concentration
of substrate than we did before and so we can see here that there are more enzyme molecules
bound to and engaged in the process of making the product and the third scenario we could imagine
is high substrate and when we have a situation of high substrate we notice here that every enzyme is
bound to a substrate and that's important because at high substrate concentrations we have
enzymes that are what we call saturated with substrate meaning that once it is bound to substrate
made a product and released it almost instantaneously it grabs another substrate it's not sitting
around and waiting for things now so enzymes interestingly have some kinetic considerations which
is of course what we're interested in studying here but we see now for the first time a projection of
the way that the enzyme is working so I need to explain some things on the graph that you see first
of all we're plotting on this graph a reaction the reaction is plotting the velocity of the reaction on
the y axis versus the substrate concentration that's used in the reaction on the x axis now you notice
the V has a little 0 beneath that and the 0 beneath that I'll explain later but it's called the initial
velocity for our purposes the velocity of a reaction is measured as the concentration of product
made divided by time the concentration of product made for time well we measure concentration in
molar milli molar micro Moeller etc so that would be some molarity per time that is how velocity is
measured the substrate concentration varies because to generate a curve like this I do not one
reaction but I do a series of reactions so let me set that up we could imagine for example that I'm
setting up a series of 20 reactions 20 different test tubes I want to measure the velocity in each one
of those test tubes and what I do is I take into that test tube I place the buffer that holds the
substrate I place the substrate and I place the enzyme now when I'm doing an experiment I want to
have one variable because one variable is the only thing I can really manipulate and measure the
effect of that the variable I have here is substrate concentration I use the same amount of enzyme in
every tube all twenty tubes have the same amount of enzyme they all have the same amount of
buffer and they have varying amounts of substrate starting from very small amounts to very high
amounts I take and I let each one react for an exact same time and then I measure the amount of
product so by doing that I can see the effect of measuring of changing substrate on the velocity and
then I plot it so what you see on the screen is the sum of those plots that is each each point on that
lot on that dot came from a series of reactions that I did and each one of those individual reactions
had a specific substrate concentration and a specific velocity that was reached well not surprising as
we look at this what do we see well on the far left were at low substrate concentration what's the
velocity it's very low and that's what I showed on the original image low substrate concentration
enzyme is sitting there waiting for substrate there's not going to be much velocity when I get to a
high substrate concentration such as they see on the right side of the screen I've got a high velocity
makes sense okay low substrate low velocity high substrate high velocity I want you to remember
that now I'm showing another plot here to illustrate a principle of a reaction on that y-axis I have the
concentration of product we could think of that again it's velocity but on the x-axis now I'm plotting
the time of reaction so I could take one of the tubes that I used in the previous one and for example
look at how fast the product is being accumulated and what happens to that product over time well
we can see on this plot that over the early range of the reaction there's a linear relationship between
the production of product and time okay but after a while what happens is that that curve flattens
off now what that means is that the longer that we let a reaction go it doesn't stay linear forever and
the reason it doesn't stay linear forever because remember enzymes catalyze reversible reactions so
the more we let product accumulate the more likely product will start being converted back into
substrate well that's not what we're interested in studying we want to study how fast the enzyme
makes product so if we're going to study an enzymatic reaction we have to study what's called initial
velocity we don't want to wait too long in order to study the concentration of product because if we
wait too long we're actually starting to study the reverse reaction and that's not what we're after so
that's why we use vo or the initial velocity in our measurements now this is kind of complicated so I
want to step you through it but these are considerations for doing reactions in what are called
Michaelis Menten kinetics we can see here that on the y axis again we have concentration and on the
x axis we have time and before we saw simply the accumulation of product is shown in the orange
icon here at the very beginning of a reaction what is are the circumstances well we have four
different things to think about that can be measured we have the concentration of substrate we have
the concentration of the enzyme we have the concentration of the enzyme substrate complex and
ultimately we're going to have concentration of product which is what we're interested in studying
okay at the beginning the concentration of prod as low as you can see and that's not surprising
because the reaction hasn't had a chance to get started the concentration of es is low because there
hasn't been an opportunity for the substrate to really encounter the enzyme very much the
concentration of the free enzyme that is the enzyme not bound to substrate is relatively high and you
see it's coming down from the y-axis and finally the concentration of substrate is high because none
of the substrate has reacted so at the time zero we have these circumstances going on and these
circumstances turn out not to be ideal for us to measure the enzymatic reaction now as the reaction
proceeds we see changes to these entities we see first of all that the concentration of substrate by
the end of the reaction is low and it's falling during the entire process the concentration of the es
substrate which started out at zero is going higher and we'll see that it will eventually sort of level off
we also see that the concentration of the free enzyme which started out at a relatively high position
is falling and it too will sort of level off in time and finally we see of course that the concentration of
product is going to start at the low and go to high by the very end well I show you this graph not to
complicate the picture too much hopefully but rather to demonstrate what we try to do in studying
enzymatic reactions in the very initial phase I hope I've made the case for you that we're in a set of
conditions called pre steady state now I'll explain what steady state means in a minute but we have a
circumstance where the reaction hasn't had a chance to get started the enzyme isn't doing its thing
and everything in there is changing pretty rapidly the change in substrate the change in enzyme the
change in enzyme substrate complex and the change in product this is going to give us a lot of
variability in a reaction now I said we want to study initial velocity but we want to be careful if we do
it too soon we may not get what we're after here so it's important to think about really studying or
studying reaction at a place where these things have sort of leveled off now under conditions of
steady state what's actually happening is that these other quantities that were varying fairly rapidly
in the very initial phase of the reaction will start to even out and that's very for our consideration so
we can see for example that in that early state the concentration of free enzyme and es complex are
changing the concentration of e is falling very rapidly in the concentration of es is rising very rapidly
however in under steady state conditions as we can see here they have started to flatten out they're
not exactly linear but they're much closer to linear than they were in that pre steady state condition
that turns out to be important for us because what we're interested in studying is the conversion of
enzyme substrate complex into product and so if we have a relatively constant concentration of
enzyme substrate complex then that decay or that falling into product that's actually happened it's
happening at a relatively constant rate that's the place we want to be and that's why it's important
for us to be studying these reactions under steady state conditions steady state conditions of course
again meaning that these quantities are not varying significantly now we can see now the overall plot
of what's happening on here the steady state conditions are where we make our measurements and
we see that this relatively linear portion of the plot for the concentration of free enzyme and
concentration of es complex is happening under the conditions that we measure our enzymatic
reactions okay under the Michaelis Menten kinetics we learned that it's important for us to study
enzymatic reactions that are conditions where our steady states that is we have relatively constant
amounts of es complex under Michaelis Menten kinetics the equation on the on the top applies and
this equation tells us some very important things that we're going to learn in this lecture now vo that
is the velocity of a reaction is equal to v-max and that's something that we'll discuss in a moment
times the concentration of substrate divided by another quantity called km that we'll discuss plus the
concentration of substrate so we've learned two terms here that are going to become important for
us to understand and that's v-max the maximum velocity of a reaction and km which is a quantity
that allows us to measure the affinity that an enzyme has for its substrate well first let's start with v-
max with v-max it's important to and what it is and why it is and how that happens we saw when we
plotted vo versus the concentration of substrate below that we saw that the curve grew and then it
leveled off and the reason it levels off is due to the way that enzymes work and the way that they
interact with substrates instead of an enzymatic reaction going and staying linear with increasing
concentrations of substrate what happens is enzymes get saturated with substrate saturation of
substrate means that the enzyme by is almost constantly bound to substrate meaning that we have
almost everything in the es complex so at very high substrate concentrations the enzyme is
continually releasing product and over time if we add more and more substrate we exceed the
capacity of the enzyme to bind more substrate so under saturating conditions of substrate the
enzyme is no longer able to stay linear and it flattens off so we see this hyperbolic plot now an
example might be a factory that's making products a factory that's making products we'll have a lot
of workers and that those workers are working on something but if they don't have enough materials
to make product then the worker is going to be standing around a fair amount of the time waiting for
material so they can make product on the other hand we could imagine that if we have those same
workers working and they have all the problem is I need to make products they're going to turn out a
certain number of products per day if we increase the amount of materials but we don't increase the
number of workers we're not going to change that maximum amount they're going to get so we see
the same thing happening in the real world that we see happening with enzymatic reactions if we
want to increase the amount of product we have to get more workers perhaps get another factory in
order to make more product now enzymes that don't follow Michaelis Menten kinetics and there are
some include those that bind substrates cooperatively now in another presentation I talked about
how hemoglobin binds to oxygen cooperatively and that means that the binding of one substrate is
affecting the binding of others so when we this happens and of course this only happens for multi
subunit proteins when this happens when the binding of one affects the others then of course we're
going to see a change in the velocity because that's going to change the actual binding conditions of
the enzyme when we have those things happen we can tell them pretty easily because what we will
get is an s-shaped curve for the V versus s plot very much like what we saw with the hemoglobin
binding to oxygen okay well let's now look at these parameters I've introduced the concept of v-max
and we see that eventually the enzyme reaches a place where it's not going to make any more
product over time because it's saturated with substrate v-max turns out to be an interesting quantity
but v-max as we will see has some limitations nonetheless v-max allows us to study some things now
the quantity v-max gives us a maximum amount and we could say well if we want to understand how
much an enzyme interacts with a substrate maybe we should compare v-max 'as well that doesn't
really tell us very much it tells us how fast a reaction goes but it doesn't tell us how well an enzyme
interacts with a substrate because any enzyme will reach v-max as we add an infinite amount of
substrate which is theoretically what v-max is occurring at when it's completely saturated that
doesn't tell us much however the quantity v-max over to where we're getting an enzyme to a certain
point of velocity but not the maximum amount of velocity actually allows us to measure that the
affinity that enzyme has for its substrate if we compare a variety of enzymes and we compare how
much substrate it requires the enzyme to get to v-max over two we get something very interesting
we get a quantity called the km and the km is actually a measure of the enzymes affinity for its
substrate so when I say affinity it's the desire to bind to how well does it bind to its substrate now km
is interesting if we think about two enzymes one enzyme that catalyzes a reaction that has great
affinity for its substrate it really likes that substrate it really grabs that substrate and we have another
enzyme over here that doesn't like its substrate as well okay well which of the two we're going to
bind substrate more readily the first one of course because it's got greater affinity which one is going
to get to v-max over two with a lower substrate concentration well the one that grabs its substrate
more easily so enzymes that have a greater affinity for their substrate are going to have a low km and
those that have less affinity for their substrate are going to have a higher km okay greater affinity low
km lower affinity high camp so km is inversely proportional to the enzymes affinity for its substrate
okay so here we see high km low affinity you see low km high affinity a very important concept to
remember with respect to km and I'd like to think about it about an enzyme that has low affinity we
have to pound it on the head with substrate before it starts to bind it and by pounding on the head
the way that we do that is by adding a lot more substrate now v-max as I said is a very interesting
and important quantity but it actually is not the perfect quantity to measure the speed of a reaction
it's good for the reaction but it's not so good for the enzyme so what does that mean well it means
that v-max when we do a reaction the way I described doing a reaction as we set up 20 tubes and we
have in those 20 tubes buffer we have substrate and we have enzyme and when we're doing a V
versus s plot what we're doing is we're having one variable the one variable that we have is substrate
which means that all 20 tubes have the same amount of enzyme that's great we don't want to have
variable amounts of enzyme but imagine I were to do the same set of reactions and instead of using
the amount I used in the first set let's say that I did the reaction now they used twice the amount of
enzyme for the second set of reactions in each case constant however varying substrate but now
with twice the amount of enzyme what would I see with respect to v-max well if I go back to my
factory analogy and I think about what happened with the factory I said the factory got to a point
where it's saturated it made a maximum amount of product that the workers are gonna put out per
day and it wasn't gonna make anymore what if I had two factories well if I have two factories I would
say well I'd probably expect that I would get twice as much product per day and so if I use a set of
tubes that have twice as much enzyme the parallel follows I would get twice as much product so v-
max is proportional to the amount of enzyme we used it's not a constant for an enzyme but it's a
constant only for a reaction with a set amount of enzyme I'd like to be able to compare enzymes with
a quantity that is independent of the amount of enzyme that I used well fortunately that's fairly easy
to do okay v-max is a velocity and we measure velocity of a reaction as the concentration of the
product produced divided by time if I take the quantity of enzyme that I used in the reaction and I
divide v-max by that quantity and I say quantity in this case meaning concentration the concentration
of enzyme that I used what will happen well the v-max was measured as a concentration of product
and I divided by a concentration of enzyme as long as I use concentration and concentration
consistently the concentrations actually drop out and so what happens is I get a number and the
units on the number are per time so I get something that says a thousand per second what is a
thousand per second mean well I've taken the enzyme out of the equation and now the number that
I get corresponds to the number of molecules of product per enzyme per second so a thousand per
second means every enzyme in that solution is making a thousand molecules of product per second
and that's the fastest that's going to go because remember we started with v-max that quantity is
called Kitcat k-kat is a number that's also called the turnover number but I can come the cake hats of
two enzymes and have a much better understanding about the relative speeds of production of
product that those enzymes have now the idea of cake aunt brings up another thing for us to think
about and enzymes are really remarkable okay we've seen that enzymes can speed up reactions
mind-boggling numbers of times and we've also introduced the concept here of an enzyme having
affinity for its substrate the idea of what a perfect enzyme would mean okay starts to come into
shape we think about what would be a perfect enzyme a perfect enzyme would be an enzyme that
would have as much velocity as possible with as great of affinity for its substrate as possible meaning
that to get to that maximum velocity it wouldn't take very much substrate because the ends that
would be grabbing substrate and converting it into product very readily so a perfect enzyme would
have a high velocity and a low km well we use k-kat as our measure of velocity and km is our
measure of affinity for substrate high cap means high by k-kat means high velocity low km means
high affinity the perfect enzyme will have a large ratio of k-kat to km so if we take those two numbers
and we divide them by each other and we start comparing enzymes we see enzymes have widely
varying ratios of k-kat over km but we also see that there's a sort of a top echelon beyond which
enzymes really don't have a number that increases very much now these numbers vary a little bit
from each other but these are really the top echelon enzymes they don't have a cake at over km
value that's significantly different these are on the order of ten to the seventh two in one case ten to
the ninth but most of them in the range of about 10 to the 8th we don't see enzymes that make it to
10 to the 15th for example why is that well what's happened with these enzymes is they've reached
their maximum efficiency they can't get any more efficient there's two things that limb them one is
they can't with shape and sequence of amino acids make a better active site than what they've made
by evolution in that sense they literally are perfect mutations that change those will always make an
enzyme that's less efficient there's a limit to what that efficiency can be and the second thing is really
interesting it is believed that the reason that we reach a max with this in addition to what I've just
mentioned is that there's something else that's limiting about the enzymatic reaction and the
limiting thing for these enzymes in a solution is one one quantity and that's the rate with which the
substrate can diffuse in water diffusion of course happens with the mixing that we see in its diffusion
that's bringing substrate into the enzymes active site and though that process of diffusion can itself
occur at mind-boggling rates that's what allows enzymes to do what they do it to has a limit and so
these enzymes are so efficient that they're sitting there waiting on water to deliver substrate to them
that's a remarkable thing all right let's take and use now some of these parameters that we've been
talking about with respect to kinetics and understand enzymatic reactions I've shown several times
now the plot of vo versus s and we saw that was a hyperbolic curve and you saw in that curve that at
the very top of that we had something called v-max and if I'm eyeballing that curve I have to ask
myself what I draw in v-max at the right place is it up a little bit is it down a little bit and I have to
make a judgment call with that I'd like to have a more precise way of saying what is the v-max well
one of the tricks or tools that we use to do this is to actually change the analysis of the data a little
bit instead of plotting one instead of plotting vo versus the concentration of substrate that is the
velocity versus the concentration of substrate I take the same data that I had for that vo versus s plot
and I invert it I invert all the data so I do what's called a double reciprocal plot or a lineweaver-burk
plot they were the people who came up with this and when I invert the data like that what I discover
is that that hyperbolic plot becomes linear and that linear plot is much more easy for us to interpret
to determine what these values are when I make such a double reciprocal plot I create a linear plot
of the data and the linear plot of the data I can use a lock and draw a line through the points and
extrapolate through the axes the y axis and the x axis when I do I create an intercept on a y axis and
the y axis has the value of 1 over v-max I can very quickly of course invert that value and I've got B
max on the x-axis the intercept is minus 1 over km so if I take whatever that value the intercept is
and I take minus 1 over that I will get camp very simple plot so lineweaver-burk plot sand there are
other manipulations that people do of grouse lineweaver-burk plot help me to write very readily
determine v-max and km from a set of data I've described so far how enzymes are flexible around
the active site and how that flexibility of the active site facilitates the catalytic process that happens
but enzymes are flexible all over and that flexibility all around the enzyme gives the enzyme some
interesting properties as regards its activity now we can see here on the left an enzyme that is getting
ready to bind a substrate as we've seen before and on the right we see the enzyme after having
bound this substrate has adapted itself to the shape of the enzyme this was the induced fit that I've
been referring to this induced fit makes a lot of sense for the active site as I said but the rest of the
enzyme is also affected by these things by these by these interactions now this is actually manifests
itself in the plot that's shown on this figure right here on this plot we can see the V versus s binding
for an enzyme that's allosteric now remind you that allosteric means that the enzyme is interacting
with a small molecule and having a sec tivity affected in this case the small molecule that it's
interacting with it's affecting it is actually its substrate so this happens with multi subunit enzymes
and then what I'm getting ready to describe very much parallels what I talked about with
hemoglobins finding of oxygen in another of the presentations when he would open binds to oxygen
you may recall that the binding changed as the oxygen concentration increased as the oxygen
concentration increased hemoglobins affinity for oxygen went from low to high and that was
important for the action of hemoglobin the same thing can happen with an enzyme whose affinity
can change depending on its binding of the allosteric in terms of binding of the substrate that affects
it allosteric li now multi subunit enzymes have this happen because a part of one one part of the
enzyme binds the substrate and affects the binding of the substrate on other parts of the enzyme
now there this change that I described to you results in a change in the overall physical shape of the
enzyme not just the catalytic site now this overall change of the enzyme is given a couple of names
first we talked about a state that's relaxed it's called the our state the relaxed state of an enzyme is
the state that really the enzyme is open to binding substrate and is very able to bind substrate the
relaxed state of an enzyme corresponds to a more active state of the enzyme by contrast to the T
state of an enzyme where T stands for tight is the enzyme is tense it is tight it is not flexible and it is
not able to bind substrate as well on this plot for example we could see at the low substrate
concentrations the enzyme is in the T state it's not binding substrate very well but once the substrate
concentration gets high enough the enzyme flips and then it's able to bind substrate better so it's
velocity change actually flips we don't see the hyperbolic plot that we saw before well there's a
couple of ways people have studied and tried to explain this phenomenon going on so I want to
spend a little time going through and explaining ways that we interpret this change they're called the
concerted model and the sequential model so the first of these I'll talk about here is the concerted
model the concerted model is conceptually a little hard to get one's head around we see the enzyme
in this model existing in two states and for the purpose of this illustration we've assumed that this
enzyme has four sub-units enzymes can have many many subunits --is up too easily at least a dozen
subunits in some cases but for this illustration it has four in the t-state we have the enzyme shown in
the squares on the top and the t-state we recall is the least favored of the states this circles below
refer to the enzyme in the our state where the enzyme is relaxed and more able and likely to bind
substrate what the concerted model says is that the flipping between the T state and the our state
happens completely as as shown there as we go from the top to the bottom and there's no
intermediate and what this model says is something that seems counterintuitive because this model
says that the flipping from T to R is not caused by the binding of the substrate but rather the our
state or the T state is favored by whatever the the state happens to be in when it binds substrate so if
I have an enzyme that flips into the our state and it binds substrate the substrate will lock it in the
our state so that it will tend to stay in the our state and consequently be more reactive if the enzyme
binds it in the t-state it's likely to stay in the T state once the enzyme is in the our state it's going to
stay there and keep producing and since the our state is producing more and more of the product
anything that favors our locks in the our state is going to favor the reaction more so the concerted
model is an all or none but the locking into one state or another is central to what it does you can
see here that there's an equilibrium between the two and the equilibrium shifts as we get more of
the substrate binding our locking it into a given state as we go further to the right the our state is
favored because there's more enzymes now in that our state and more enzymes means more
product the our states can flip as I said independently of each other but the bound state is favoring in
this case the our state now the other model that we called sequential model is very much like what
we saw when I described the flipping or the changing of hemoglobin we also refer the states our
state and T State and hemoglobin but we more commonly used this as regards enzymes now in this
model what happens is we have an enzyme that starts out in the T state is shown in the in the four
squares on the left The Binding of the first substrate causes one of the subunits of the enzyme to flip
and that's shown in blue in the second of a model from the left when that flipping occurs the blue
interacts with the other two units of the enzyme and we can see that there's a sort of a purple that
happens and a rounding of those two that's indicating that the blue circle which is in the our state is
affecting the two units around it and causing them to start to flip into the our state well they're
starting to flip favors the binding of more substrate and so we can see sequentially then that the
Blues are becoming more dominant as we get farther to the right the binding of the substrate is a
critical thing for this this enzyme because the binding of the substrate in this model says it's the
cause of the flip now casually when we talk about it we frequently say well this causes the enzyme to
do this or that and when we say that we're sort of loose in the language that we use in this case the
cause is physically causing the flipping to occur and the concerted model the cause is not a direct but
it's an indirect as a result of the locking that I described so the distinguishing difference between the
concerted model and the sequential model is that cause that I mentioned causing the the flipping is
a physical causing the two models that I've just described the concerted model and the sequential
model or just that they're just models in terms of explaining how the T state in the our states come
to be within enzymes it's very likely that no enzyme actually uses exclusively one of the other and
there's a lot of evidence that enzymes may use as sort of a hybrid of these two models enzymes as I
noted at the beginning can bind reactions in different ways and I talked about one substrate going to
one product or to substrate going to one product or in the case I'm going to describe here two
substrates going to two products now when we think of two substrates that can bind to an enzyme
we realize that there's different ways that they could bind for example if we have the reaction a plus
B goes to C plus D we can imagine that maybe an a would have to bind first and then B or maybe be
binds first and then a but what we find is that for some enzymes it really doesn't matter which one
binds first this is called random binding as it's shown in the first example that I have on the screen
random binding means it doesn't matter now some enzymes bind substrates randomly as I'm
showing you here but a lot of enzymes do what's called ordered binding that means that either a
must bind first or B must bind first now that model or that mechanism is significant and the reason
it's significant is it's probably the best illustration that I can give you for the coastland induced fit
model because what order binding tells us that is is that if one of these must bind first before the
other one does that means then that the binding of the first one is actually changing the shape of the
binding site for the second one because if the second one tries to bind first the change hasn't already
happened and that's why the second one can't bind first so ordered binding reinforces the coastland
induced fit model now that might seem to cover all the territory but there's actually a third model
that enzymes use to catalyze reactions and this one's kind of interesting and it has a fun name we call
it the ping pong mechanism and it's also called order a double displacement reaction but the point is
the same the ping pong mechanism is an enzyme that actually exists in two covalently different
states that means that the enzyme is actually physically binding to something and causing a change
most of this happen in the next slide now this illustrates a reaction of a plus C going to B plus D and
we're seeing it's split into two reactions all right in this reaction what's happening is a is starting out
with an oxygen on it and in the reaction of A to B the oxygen is being replaced by an amine so we see
this happening and where is the amine coming from the mean is coming from the enzyme so the
enzyme is carrying the amine and it's carrying it to a so when a interacts was the enzyme the enzyme
swaps the amine that it's carrying for the oxygen that's on a so on the right side of the top equation
we see that a has become B because it now has an amine and the enzyme has grabbed the oxygen it
no longer has an amine so I've colored it with green so that you can see that in the second part of
the reaction C which has an amine is interacting with the enzyme that now has an oxygen and when
that happens they trade places C becomes D where D has a double bond to the oxygen and the
enzyme has become a linked to an amine right so the enzyme has returned to its original state so by
this ping-pong mechanism the enzyme is continually going from a mean two oxygen to a mean to
oxygen and depending upon which state it's in it determines which of the substrate it binds and
swaps with now this type of reaction that I just described to you is a common reaction that's used by
enzymes called transaminases transaminases are enzymes that do just what I've described they swap
oxygens for amines and this is a very important reaction in the metabolism of amino acids because
amino acids get their amines in some cases by the reaction that you see on the top they start out
with a double bonded oxygen and they become an amine a really good example of this is the the
molecule alpha ketoglutarate in the citric acid cycle alpha ketoglutarate can become glutamic acid if
the oxygen on it is swapped for an amine and this way the cell can make an amino acid that it might
need because of this mechanism on the other hand we might have the situation where a glutamic
acid or even another amino acid is needed for energy people that go on low-carb diets for example
don't have a lot of carbohydrates but they're not starving to death because they're eating plenty of
protein proteins providing amino acids and amino acids provide energy as a result of what I'm
showing you here the lower-left reaction has an amino acid that has the amine replaced by a double
bonded oxygen so imagine if you will that the amino acid on the lower left side is actually aspartic
acid aspartic acid can be converted by swapping its amine with an oxygen into exile o acetate and xlo
acetate can be oxidized in the citric acid cycle so this transaminase reaction is important both for
making amino acids and also for metabolizing amino acids for energy the last thing i want to talk
about here are the classifications of enzymes enzymes according to a systematic scheme that has
been developed by the ec commission the enzyme commission have broken all reactions that
enzymes catalyze into six categories and this six category scheme is used to organize and named all
enzymes that are in biology the first scheme a category scheme is that of an oxido reductase that is
an oxidation and a reduction is happening in the reaction that's catalyzed by the first category of
enzymes in this case you can see male 8 which is shown on the left that is being oxidized it's donating
its electrons to nad to form X all acetate and NADH so oxidoreductases will always have transfers of
electrons and will always have an electron carrier involved you can see the nad and the NADH here
the second category of enzymes are those called transfer raises and transfer raises grab a part of one
molecule and move it to another so we can see here for example that we're starting with glucose
we're taking a phosphate off of ATP and we're putting it on to glucose this enzyme hexokinase
catalyzes the first reaction in glycolysis and it's a transfer ace the next reaction involves hydrolysis
and as the name would suggest these enzymes use water to break bonds so we can see in the
schematic reaction here a molecule on the left that has a peptide bond is actually combining with
water to break that peptide bond that's what happens with a serine protease for example water is
being used to split a peptide bond in the fourth category we have enzymes called lyases and Ally A's
as an enzyme that uses a non hydrolytic meaning no water non oxidative way of breaking bonds so
on the Left we see for example isocitrate the enzyme isocitrate lyase which is found in plants breaks
this six carbon molecule into a two-carbon piece called glyoxylate in a four carbon piece called
succinate because water is not involved and because there is no oxidation involved this reaction is a
lie ace the fifth category of enzyme is an isomerase and isomerases our enzymes that catalyze
rearrangements without doing anything else to the structure of the molecule that they're acting on
so in this case we see another reaction from glycolysis the enzyme converts glucose 6-phosphate
which is a sugar with a phosphate on it to fructose 6-phosphate which is the different sugar with a
phosphate on but all that has happened is spend simply a rearrangement of the molecule that's an
isomerase the last category of reaction are those of ligases and ligases are molecules that put things
together so instead of breaking things apart ligase is are making covalent bonds to join things
together so in this case we're seeing ATP energy being used to join urea and bicarbonate to make
your real one carboxylate ligases join things together well we've seen in the reactions here that
enzymes have some pretty amazing abilities in terms of flexibility for catalyzing reactions flexibility as
they affect the mechanism that they use to catalyze things and we've also learned about the
different categories of enzymes that are there in other lectures I will talk about the ways that
enzymes become inhibited