Okay. Now, serine proteases, as I said, cleave peptide
bonds, that's the catalytic thing that they do.
They have specificity of cutting,
again, by binding only to 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 will 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 the serine proteases.
I have shown on the screen here a substrate
for the enzyme. This is a
polypeptide chain or protein
that the serine protease will cut.
The specific cut is going to occur here
will occur between the carbon and
the nitrogen on this molecule.
And of course, you know from the structures we
have talked about in the other presentations
this is the location of the peptide bond.
Now on the right side 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 at some of these things.
So you are gonna see in some
cases, I am gonna stretch
bonds and stretch molecules a little bit
to actually make things fits
so you can understand this.
Please understand that in an enzyme itself,
of course, they are 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
acids 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 the serine and histidine
and the aspartic acid
which are the three side
chains that we see here
are not located close to each
other in primary sequence.
They are 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 then the rest of the enzyme is shown
in yellow. We are 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 gonna 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 call the S1 pocket.
So we have shown here the S1 pocket that is a sort
of a semi circle that's holding on to a part of that protein.
We can see the protein that is 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 hydroxide.
We also note that the oxygen, that is on
the side chain of aspartic acid has a little dot
next to the hydrogen on the histidine.
What's happened here? Well, then
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 acid's
side chain to 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 as 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
happen 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 S1 pocket
has facilitated all this happening.
I should say in the S1 pocket, that the S1
pocket gives the specificity of the enzyme.
The S1 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
won't bind them, of course, there is nothing to react.
At the end 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, 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 of serine is now associated with
the nitrogen of the histidine ring.
Now this is the first step in this catalytic
process. 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, an alkoxide ion.
Okay? That alkoxide ion that's on serine is extraordinary reactive.
It's ready to go do business.
Now we have stretched that S1 pocket
little bit to remind us that again
we are bringing things into closer proximity
and that is important because the
alkoxide ion is looking for something
to bind to. It is looking for
a nucleus. It's what we call a nucleophile.
And the nucleus that it is looking for here
is this carbon, which is the arrow
that's being pointed from the oxygen
minus 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 in the next step of the process
what will happen is that we are 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 cause problems.
Well, the enzyme has another
pocket in it to deal
with that unstable molecules
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 gonna 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 tetrahedrals we know from organic chemistry
are 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,
is going to be the broken as a
result of nitrogen grabbing that hydrogen.
Here, nitrogen has grabbed the hydrogen.
The grabbing of the hydrogen from the histidine
cause the bond between the carbon
and the nitrogen to break.
So we have 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 it's
business. It's released.
There is nothing attaching it to the
enzymes 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 serine.
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 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 this peptide to be released.
Well, we see that happening here. Water
now has physically moved into the
active site. There is 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 gonna take that hydrogen instead of taking the hydrogen
that is originally took, which is no longer there, on serine.
What's gonna happen in that process is now
we are gonna have an activated oxygen
like we had with the alkoxide ion except
for here it's gonna be a hydroxide.
We are gonna have an activated oxygen that
is gonna make a nucleophilic attack
on carbon just like we saw before.
So there is a nucleophilic attack that's going to
happen in the process of this moving forward.
Here is the attack of the hydroxide
and look what happens. We see that the electrons
on oxygen are going to rearrange.
We create a tetrahedral immediate as we created before.
And now there is the oxyanion hole stabilize in that intermediate.
We now see that what happens is that oxygen
is going to attack the hydrogen on that group and it's
gonna pour away just like the first peptide did.
When it does that, what happens is the molecule released.
So we see the second half of the polypeptide chain released
and in addition the enzyme returned back to it's original state.
Gone and as it were.
The cycle is now complete. So it is about 10
steps going through what I described here
and the important thing to understand about
this is that the enzyme started at one state.
It went through a transition
and then went back to the original state it was in.
Very much like the process I have already described
but now you have 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.