Serine & Cysteine Proteases

Uploaded by lamechivanes on 05.04.2010


Suppose you just identified that there's a serine protease
as an important biological target
for therapeutic applications that needs to be inhibited.
The first thing you'd want to do,
and what we're gonna do in this webcast,
is understand the molecular mechanism
of the normal mode of action of that enzyme.
And for serine proteases, we can look at a prototype,
a prototype enzyme that behaves as typical,
as a typical serine protease,
and the prototype that we're gonna look at
is α chymotrypsin.
Α chymotrypsin has 241 amino acids in its polypeptide chain
and it cleaves peptide substrates
at a specific location.
The substrate selectivity is in an internal position
that makes it an endoprotease or an endopeptidase
as opposed to an exopro,
protease which cleaves at the end of the chain.
And beyond that it has even more specificity.
It will cleave after an aromatic residue.
And what does it mean to cleave after
an aromatic residue?
Well as we're traveling along from the N to C direction,
well, once we hit the α carbon that has an aromatic
phenylalanine, tyrosine or tryptophan residue,
then the peptide bond immediately following that
is where the cleavage will take place.
That's known as the sessile bond.
In α chymotrypsin, it's the serine 195 group
that plays the role of a nucleophilic catalyst.
It'll form an intermediate ester group,
and there's a couple of other residues that are key.
Histidine 57 functions in a, as a base
and that pKa, the pKa of that histidine is modulated
by a closely related art, aspartate group at 102.
That's, those three groups, serine, histidine and aspartate
come together to what's known as the catalytic triad.
Let's take a look at the molecular mechanism.

There's the catalytic triad, aspartate 102,
histidine 57 and the serine 195,
which is gonna function as the nucleophile.
The bond that we're gonna cleave,
go ahead and highlight it in your notes,
it's that bond right there.
And the first step is going to be
general based catalyzed nucleophile addition
to the carbonyl group of that peptide bond.
Those arrows look something like that.
Again, notice that we're using the sp2 nitrogen
of the imidazole ring of histidine
to do the nucleophile addition to the polarized π bond.
That's going to end up making for us the imidazolium cation,
and again, it's the role of the aspartate group
with its negative charge to help stabilize
that positive charge, making it a stronger base.

The... the serine group has added in
to make a tetrahedral intermediate
at what was the carbonyl carbon and the oxyanion, the O-
is stabilized by the amide residues, ah, from serine 195
over to residue 193, the glycine NH bond.
Those two NH bonds are hydrogen bond donors
and they're pointing right in at that oxyanion
to help stabilize that intermediate.
The next step is a reverse of the electron flow,
so just turn that electron flow right around
and we're gonna do a β elimination.
Go ahead and highlight that bond.
That's the bond that leaves,
and now we're using that imidazolium group
that now has a positive charge as the general acid.
So it's a general acid catalyzed β elimination,
and in the, as a consequence, what we end up making
is, ah, the released peptide amino side of the, ah,
the peptide fragment and an ester group.
So we've got the imidazole returned to its neutral state,
and we have the, ah, ester formation
between the carbonyl, ah, and that serine oxygen atom.
So we've got a protein bound intermediate
and that's typical of nucleophilic,
or covalent catalysis.
Here's where water enters in.
Water in going to hydrolyze that ester group.
We're basically going to repeat that mechanism all over again,
but rather than adding in the serine group,
we're gonna add in a molecule of water.
So imidazole is gonna again function as the general base.
We'll make an oxyanion tetrahedral intermediate
and an imidazolium cation,
which picks up one of the water, ah, protons.
The tetrahedral intermediate is formed
and we again have an oxyanion
that's stabilized by what known as,
what's know again as that oxyanion hole.
Finally, what we're gonna do
is turn the electron flow back around, do a β elimination.
And we now have released the carboxylic acid portion
of the substrate.
And so we've broken that peptide bond into two,
added the elements of water across that peptide bond,
and we've returned our residues,
our catalytic triad, back to their original form.
So if we just look at, ah, summarize
some of the key things.
There's the oxyanion hole that stabilizes the intermediate.
Chymotrypsin's very effective
at stabilizing the transition state.
Even though it's the deprotonation
of the serine hydroxyl group it,
that forms or that functions as the nucleophile
with, with it's pKa of that oxy, that ah,
hydrogen on oxygen of 16
and it's being deprotonated by histidine,
that is quite a weak base to deprotonate such a weak acid,
it's able to be stabilized by that aspartate 102 group
and help modulate the pKa in making that
imidazole ring of histidine a stronger base.
To get an idea of the role of that aspartate group,
there can be, ah, we can examine a mutant enzyme
where we've replaced that aspartate group at 102
with an asparagine group.
And if we just look at the relative rates,
the wild type, the naturally occurring
with the aspartate 102 in place has a relative rate of one,
10,000 times slower is the rate
where we've made that one atom, oxygen for nitrogen,
actually, it's three atoms if you count the two hydrogens,
but oxygen for nitrogen substitution
changes the relative rate by a factor of 10,000.

Let's take a look at the structure of this enzyme.
So, peeking in from the outside,
you can see that the active site is pretty much located
on the surface of this enzyme.
There's the, ah, histidine residue.
The green is going to be the catalytic triad.
When we look at this a little bit closer
you can see the, ah, different, there you three,
see all three of the residues that form the catalytic triad.
Over here we can examine, ah, a ribbon diagram
of that catalytic triad.
There's the aspartate group.
Right above that is the histidine,
and all the way at the top we see the serine.
So those three groups are lined up, ah, in a row.
There's another view.
You can look at a couple of different orientations.
There you can clearly see all three groups lined up in a row,
serine, histadine and the, ah, aspartate group.

Here we can see, ah, the oxyanion hole
and its relationship to those three groups.
At the bottom we have the aspartate,
the imidazole ring of histidine and the serine group,
and colored in yellow is the catalytic, ah, triad,
ah, positioned into the oxyanion hole.
So in yellow are the two nitrogens that form
the oxyanion hole and that will help to stabilize that,
ah, ah, tetrahedral intermediate.

And finally, we can spin that around and zoom in on it,
looking at a couple of different orientations.
What you can see is that scaffold,
that protein scaffold of chymotrypsin
is able to position very accurately the, those groups
so that they're just in the right location
to bind the substrate and to, ah stabilize that intermediate.

Finally, I'll just leave you very quickly with
the closely related family of enzymes
knows as the cysteine proteases.
Papain is the prototype that we would study there.
And the mechanism is very similar except that
rather than using the serine hydroxyl group,
it's a cysteine thiol group.
And if you remember the pKa
is, for the thiol group, is much, ah, lower.
It's a much stronger acid than is the serine hydroxyl group.
And so because of that, there's no longer the need
for that aspartate group and, ah,
cysteine proteases function without a catalytic triad,
they just have a histidine and cysteine as opposed to,
ah, an aspartate, histidine and serine.