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Kevin Ahern: Exam prep's coming along?
I've got two announcements, well, actually maybe three.
So we'll get everything in order here.
First, I do have a review session scheduled.
It will be in ALS 4001 on Saturday at 3:00 p.m.
I will videotape that.
We're set there.
That was number one.
I said "three," didn't I?
Number two, I've had several people comment
about the extra credit question that I have thrown out at you
and, yes, it will be on the exam.
There is a sort of a solution to it in the book,
but I'm looking for more than what that book says.
The book talks about how the change in structure that can occur
is similar for allosterism as well as
what's happening in cooperativity.
But there's actually a little bit more to it than that,
and it's that little bit more that I'm looking for.
So there's something else that's similar that is important
for that phenomenon that I described to you.
So think about that.
The last thing is the logistics of getting it set up
are such that there are 250 of you.
We aim, as best we can, to get the exam in your hands
as quickly as possible.
To do that, we need your cooperation.
I'm going to tell you how I want you to sit
when you come into the room, okay?
Starting with this aisle, right here,
you are Number 1, on this far edge.
I want everybody sitting at least two away from everybody else.
In fact, I want everybody sitting in the odd-numbered ones,
so 1, 3, 1, 3, okay?
For this one over here, it starts 1, 3, 5, 7, 9, et cetera.
Student: This is 11.
Kevin Ahern: I'm sorry?
Student: This is 11.
Kevin Ahern: Well, I'm just saying,
count from the end is all you have to do.
Just count from the end,
and count in an odd number, okay?
And that is the end I want you to count in from.
Same thing here.
So start 1, 3, 5, and 7.
Everybody got that?
So if you come in and you do that,
and I won't have to get you up
and moving you around and so forth,
we can get the exam out quicker.
The same things hold up there.
So 1 over there, 1 there,
and then 1 over here.
Everybody clear on that?
So it's important to do that.
I will also tell you that you'll find that
I'm very picky about time on the exam.
I do that to give everybody an equal chance.
I can't have some people taking two minutes
to fill out their exam while they're waiting
in line to come up
and turn their exam in after I've called time off.
So when I call time off,
I will expect everybody will immediately stop writing.
If I see anybody writing after I say "stop,"
then I will take points off.
I'll warn you about that during the exam,
but it's important that you stop when I say "stop."
I don't want anybody having a time advantage over others.
The first exam there are sometimes time issues.
Don't spend too much time on any one question.
I've tried to make it shorter
so that you won't have the issues with time.
But, nonetheless, problem solving sometimes
takes some people longer than others.
So don't spend too much time on any one question.
Again, I want time to be equalized for everybody
as much as I can,
and that's why I do it.
I don't do it to be mean, much as you might think I do.
What else can I say?
Be sure, obviously, one of the biggest recommendations
I have is put your name on your exam as soon as you get it.
This happens every year.
I get somebody
and they've got to write their name on their exam
after I've said time to stop.
"It's just my name!"
Well, I can't tell if you're writing your name
or you're writing answers.
So if I see you writing, even if it's your name,
after time has expired, you're going to lose points.
So get your name on there.
Don't wait to do that.
Yes?
Question?
Student: Well, I was going to ask if we're required to use pen
and then I realized it might be on the syllabus.
Kevin Ahern: Can you use pen?
You can use pen.
You can use pencil.
You can use crayon, as far as I'm concerned, okay?
As long as...
[laughter]
Student: Yes, crayon!
Kevin Ahern: Yes, you're welcome to use crayon.
We may get a good laugh out of it as we're grading it,
but as long as we can read it.
The biggest issue that we have is reading what you've written.
If we can't read your name
and we have to figure out what your name is,
you'll lose some points.
I tell everybody every time,
"Print your name clearly,"
and I still see these scrawls people put on there for their name.
I'm thinking, "You want a zero?"
You don't want a zero, right?
Make sure we can read your name.
Put 'em in big block letters.
I mean, that's just a no-brainer.
So that's pretty much the stuff for the exam.
As I say, come in,
get seated where I told you to get seated appropriately.
If you get down here
and there's no seats down here,
then the logical thing to do is [whispering] go upstairs.
[loudly] "Where should I sit?"
Well, [unintelligible] it out up there, okay?
It's pretty straightforward.
So that's the logistics for the exam.
Hopefully everybody aces it.
I'll be delighted if that happened, absolutely.
I want to finish up the stuff on enzymes.
I went through that Lineweaver-Burk business
with the inhibition and so forth last time,
so I've only got a couple of things to talk about
and they actually relate to our very next topic, anyways.
So this breakpoint for the exam
was a very good breakpoint that I made there.
First of these is a chemical modification
that can be done to proteins.
You've seen a couple of chemical modifications already.
You saw cyanogen bromide, for example,
could cut a polypeptide at methionine residues.
That was a covalent bond that was broken.
You saw that mercaptoethanol could
reduce disulfide bonds between cysteines,
and that was a covalent bond that was changed.
Well, the next one I want to describe to you
is a covalent bond in which something is added to a protein.
This addition of things to proteins
can serve some useful purposes.
This compound here is called DIPF,
and it's got a longer name that I'm going to require you to know,
since I can't even recall it off the top of my head, as it is.
Diisopropyl fluorophosphate?
Phosphofluoro...
I don't know.
Who knows.
DIPF, right?
This is the compound, right here.
You don't need to know the structure,
but the important thing about this compound is
it reacts with the side chains of serines in proteins.
It reacts with the side chains of serines in proteins.
Since the side chains of serines are OH's,
this is what we end up making, over here.
Well, why do we care about this?
As we will see, sometimes serine plays a very important role
in catalysis of an enzyme.
There's a whole class of enzymes known as serine proteases.
If that serine plays an essential role, that is,
the OH plays an essential role,
and I basically destroy the OH,
you could imagine I would have a pretty serious effect
on an enzyme that used or that had that serine,
if I destroyed it by adding DIPF.
One of the ways that I can tell,
or one of the easy tests I can do about
does this enzyme have any serines that are important for
catalysis is I can take an enzyme and I can treat it with DIPF.
Then I can use that enzyme in a reaction
and say does it still work.
Is its activity affected?
If its activity is affected,
then I've got some kind of evidence that serine may,
in fact, have some important role in the catalysis,
catalytic action of the enzyme.
Yes, sir?
Student: Would you be able to tell if it's a competitive
inhibitor or if it's a non-competitive inhibitor with just this?
Kevin Ahern: So your question is, I'm not sure.
An inhibitor is something different, now.
I'm actually modifying the enzyme here.
So I think you're asking me if DIPF is an inhibitor of some sort.
Is that what you're asking?
Kevin Ahern: Okay.
DIPF, per se, is not an inhibitor.
Anything that would covalently bind to an enzyme
would not fit into either of those categories
because, remember, that competitive and
non-competitve inhibition are both reversible things.
They're not covalent bonds.
When we have covalent bonds, we have other things going on,
and that actually leads me to my next topic, in just a second.
But everything I've talked about in terms of inhibition,
so far, are reversible reactions.
And as I say, that's important to have reversible
because remember the example I gave
where I treat a cancer patient with methotrexate.
That goes onto the enzyme.
It inhibits the enzyme, but if I don't flush that out,
I'm going to kill the patient.
The fact that it's reversible
I can flush it outóallows the patient to live.
If I can't flush that out,
as I would have with a covalent reaction,
then the patient's going to die with the treatment.
So I don't want to have that.
So these are reversible inhibitions that we do,
at least what I've talked about so far.
There's one that's not.
But, anyway, to answer your question,
this is not a specific inhibitor.
It might end up inhibiting the enzyme,
but not by a competitive or non-competitive mechanism.
I'm just using this now as a tool.
This will work on many enzymes
because if the enzyme has a serine that's essential for activity
and I cover that hydroxyl group up,
the enzyme isn't going to work.
Oh, yes. Question?
Student: Just a question... even if we know that it reacts
at that particular side chain,
how do we know that it's the inactivation of the side chain
and not the creation of stereo hindrance at the active site
that deactivates the enzyme?
Kevin Ahern: His question is a little bit more detailed,
which is, how do I know that it's the inhibition
of the serine side chain and not some other secondary effect,
like maybe it's blocking access to the active site
or the binding site of the substrate and so forth?
We don't.
So it's only evidence, is all it is.
It's not proof that we have that.
So when we go study an enzyme, we get many, many pieces
of things together before we make a decision
in terms of what actually has happened with that.
Good question.
Yes?
Student: With the process you just talked about,
with the serine modification, is that reversible?
Kevin Ahern: No.
Remember, this is a covalent bond.
So when we've got covalent bonds,
we're essentially talking about irreversible processes.
Speaking of covalent bonds,
that brings us to our last type of inhibition.
The last type of inhibition is known as "suicide inhibition."
It's kind of a very visual image
that you get with suicide inhibition.
Suicide inhibition occurs, first of all,
when the substrate makes a covalent bond to the enzyme.
In this case, the substrate is this.
The difference between suicide inhibition
and the DIPF inhibition is that
a suicide inhibitor resembles the substrate.
it resembles the substrate.
The enzyme binds it as if it's the substrate,
and only after it has bound this suicide inhibitor
does the covalent bond occur,
and the covalent bond links the molecule to the enzyme.
Now, suicide inhibitors will be specific for specific enzymes.
DIPF will work with any enzyme that's got serines.
Alright?
Suicide inhibitors will be specific for specific enzymes.
This inhibitor right here, bromoacetol phosphate,
will only inhibit this enzyme.
It's not going to affect other enzymes.
And the reason?
It has a specific shape,
and that specific shape fits in the active site of the enzyme,
and only that will fit.
It's called "suicide inhibition" because it's not reversible.
Once we've made that link, here,
this enzyme is dead in the water.
So people say, "Well, suicide inhibition,
is it non-competitive or is it competitive?"
And the answer is, it's neither, again,
because those are reversible processes.
We can wash it away, we can get it away,
and we can see the phenomenon that we see
because they're reversible.
We can't see it when we've got a covalent suicide inhibitor
a suicide inhibitor, in general, period.
Everybody with me?
Yeah?
Student: So what happens to the enzyme
after it's no longer useful?
Kevin Ahern: What happens to the enzyme
after it's no longer useful?
Cells have a garbage cleaning mechanism, as it were,
that will take non-functional proteins and break them down.
There's a structure called a proteasome.
We don't talk about it much in this class,
but a proteasome is designed to basically recycle your proteins.
It will take those proteins,
digest them with proteases to recover the amino acids
back out of them.
So cells are pretty efficient.
Yes, sir? Back in the back.
Student: What happened to the inhibitor?
Is it destroyed when it destroys the enzyme?
Kevin Ahern: What happens to the inhibitor?
It's all going to depend on the chemical stability
of the inhibitor itself, and I don't have an answer for that.
It's going to depend from one to another.
Yes, sir?
Student: So will the net effect just be moles of inhibitor
versus moles of enzyme?
Kevin Ahern: The net effect?
Oh, yeah, actually, that's a good question, also.
So the net effect of this, will it be,
if I have excess inhibitor compared to enzyme,
that's going to be the maximum effect I'm going to have,
and the answer is, yes.
So it's just a concentration phenomenon, that's all it is.
A really good example of a suicide inhibitor is penicillin.
Penicillin works by inhibiting an enzyme
that bacteria need to make their cell wall.
It's a suicide inhibitor.
It binds to that enzyme
and bacteria got no chance.
Bacteria make more enzyme,
as long as you have excess penicillin
which is what he's talking about here
as long as we have excess penicillin, it binds to that, too.
Bacteria can't make their cell wall, they can't divide,
bacteria will die.
So penicillin is the prime example
of a suicide inhibitor that I think about
when I use it to describe the material here.
I thinkóthere it is.
Well, unfortunately, in the old book,
they had the structure of the penicillin where
you could see the bonding form
and they don't have it here,
so you can't see it.
Penicillin has an odd four-member ring that is very reactive,
and it binds very much like the natural substrate to the enzyme,
but it makes a covalent bond to the enzyme once it is bound.
That's pretty much what I want to say about suicide inhibitors
and the very last about what I want to say
specifically about enzymes, in general.
I will turn to mechanisms of catalysis
if there are no other questions.
Okay, let's do that.
You saw up close and personal how hemoglobin worked,
and we've talked, in general, about how enzymes work.
Now we're going to spend a couple of lectures looking up close
and personal at how a couple of enzymes work.
This means that we're going to get a little mechanistic,
and I'm usually the first to say
I'm not overly fond of being mechanistic,
but there are some mechanisms that we need to go through
in order to understand at least general principles
for how enzymes work.
So that's what I'm going to do here.
I'm going to spend most of the mechanistic considerations
in today's lecture.
I'm going to give you most of those today, so bear with me.
I will try to give you a very clear overview of what,
out of the mechanisms, I think are clearly important.
I start by talking about this class
of enzymes we've mentioned a few times already,
and these are the proteases.
Proteases are enzymes that break peptide bonds
in other proteins.
There's a class of proteases that has a very,
very similar mechanism from one protease to the other,
and this class of protease is called "serine protease."
As its name would suggest, serine proteases
have a very important serine residue,
and, as we shall see, the serine proteases have
this serine residue play a role in the catalytic process.
It's actually in the active site and doing its thing.
What we're talking about in any kind of
proteolytic degradationóthat is, breaking peptide bonds
is we're breaking bonds between amine groups
and carboxyl groups making the peptide bond.
These are hydrolysis reactions.
When I say a hydrolysis reaction,
I'm talking about a reaction in which
water is added across the bond to break it.
So water is being added across the bond to break it.
You will notice that adding water across the bond
recreates the carboxyl
and it recreates the amine.
Those were joined together
and we don't have a free carboxyl when we made the peptide bond,
but when we break it with a protease we go back to the carboxyl
and the amine.
That's an alpha carboxyl and an alpha amine.
The enzyme that we're going to focus mostly on,
at least upfront, is called "chymotrypsin."
It's one of the enzymes I mentioned earlier, in a table.
I said you didn't need to know the specificity of it
because it actually can work on a variety of different enzymes.
So, for example, chymotrypsin
will cut this polypeptide right here.
It will also cut it over here.
So it will cut, in this case, adjacent to phenylalanine
and adjacent to methionine.
For our purposes, if we think about these
as being mostly hydrophobic side chains, we'll be in good shape,
and I'll show you a little bit more detail about that later.
So this guy will cut in both of those places.
Did you have a question?
Student: Oh, I was thinking, it can cut on either side, right?
Kevin Ahern: It cuts on the carboxyl side whenever it cuts.
So you see the carboxyl side.
There's the carboxyl group.
It's not cutting here, but it's cutting on this side.
We know that chymotrypsin has a serine
that plays a very important role in the catalytic process,
partly because if we treat chymotrypsin with DIPF
its activity essentially goes away.
So this is one piece of evidence that
this serine residue is very, very important
in the catalytic process of chymotrypsin.
There's other things that we know,
but that was one piece of evidence that that was the case.
We can see that same reaction going on
that we talked about before.
There's that covalent intermediate,
and chymotrypsin is knocked out of action as a result of this.
I always like to stop at this point
and say something about my profession.
I'm a biochemist
and I can say, with all honesty, that biochemists are lazy people.
We're really lazy people
and most scientists are lazy people.
We like to do things the easy wayóI think it's human natureóif
we can, compared to the hard way.
If I try to study the breaking of a peptide bond by an enzyme,
it's very difficult to do.
There it is, but I don't have any easy way of determining that
peptide bond got broken.
Maybe I have to run a gel
and see that I create fragments or something when I treat it
with the enzyme, but that takes hours
and so forth to do.
I want a very simple assay to tell me in seconds, literally,
is the enzyme working, is the enzyme doing its thing, because
if I can do it in seconds, then I can do a lot more analyses very quickly.
So what biochemists have come up with in the case of this
enzyme is they've created an artificial substrate that
chymotrypsin recognizes.
Chymotrypsin will actually act, right here, on this bond.
Even though that isn't exactly a peptide bond, it fits in the
enzyme's active site well enough that the enzyme will actually
break that bond.
When it does, it creates these two molecules right here:
this guy and this guy.
You notice this guy is written in yellow,
and the reason is because this guy makes a yellow color
when it's freed from the other guy.
So by measuring how much yellow color I get
and how fast I get it, I can study the kinetics
of chymotrypsin's action.
Otherwise, I've got to spend days doing a single data point
and it's really a pain to do,
and I don't want to have to do that if I don't have to.
Yes, Shannon?
Student: So if you did a spectrophotometric assay of that,
then it could tell you how much it was working?
Kevin Ahern: Yes.
So her question is, if I did a spectrophotometric assay
which is basically just measuring the amount
of yellow color producedóI can measure the reaction,
and that's exactly what I do.
So it's very simple for me in the lab to measure
how much color I produce.
I do that.
So that's what happens
and that's what people did when they started studying
the mechanism of chymotrypsin's action.
When they did that
and they looked at a very short time scaleólook at this,
milliseconds, thousandths of a second
they discovered something very odd.
The odd thing that they discovered was that
this is product, its absorbance, so in this case
it's basically the concentration of product that's being made,
so we're thinking about velocity going up on the y-axis
and we're thinking time going on the x-axis
they see this two-phase curve.
There's two things that happen here.
They see, first of all, that the reaction occurs
in what they call a "burst phase,"
meaning that a lot of product is produced very quickly.
You see the steepness of this line.
And then it sort of bends over
and goes at more of what we call a "steady state,"
meaning, well, it's going up
but we're just not seeing that rate
kind of like we saw at the start.
When scientists saw this for the first time,
they recognized that there'd have to be two things happening
in the catalytic action of this enzyme:
a slow phase and a very fast phase
(the fast phase, of course, coming first).
So there's a fast phase to the catalysis
and a slow phase.
This tells us that the catalytic process has more than one step.
To have a fast phase and a slow phase,
I have to have at least two steps.
We'll see there's actually seven or eight steps.
But the fast phase and the slow phase...
so that was a very important step in beginning to understand
how it is that chymotrypsin does what it does.
Now, we know today that chymotrypsin does something
and many enzymes do this,
as we shall seeóchymotrypsin does something very interesting.
I've talked already about how enzymes are flexible
and that flexibility gives rise to extreme increase
in catalytic action.
That's one way that we can explain that enzymes accomplish
the magic that they accomplish.
I'm getting ready to show you another.
Enzymes, remember I said,
when we talked about the Koshland induced fit model,
I said that the substrate transiently changes the enzyme?
And then it goes back?
Well, this is a very important transient change.
We see in the catalytic action of chymotrypsin
that it becomes covalently attached
to part of the substrate during its catalysis.
It's transient.
It gets released later, but that is an important step.
If we look at what's happening here,
here's that artificial substrate that we had.
I'm sorry, it's actually over here,
the artificial substrateóback up.
The artificial substrate is right here.
The enzyme acts on it
and it spits out this yellow thing very quickly.
The enzyme gets trapped in a covalent bond
with the rest of the molecule,
and only more slowly gets released.
So now we see fast step, slow step.
While the enzyme is waiting to get rid of this,
it's not catalyzing anything, it has to come back over here.
So this accounts for the slow part,
the regeneration of the enzyme.
Yes?
Student: So this is a covalent bond...
Kevin Ahern: Yes.
Student: ...forming?
So how is that different than a suicidal?
Kevin Ahern: How is it different than a suicidal?
It's transient.
So the enzyme, as part of its action, gets released from that.
I guess it was a little confusing
when I said a covalent bond you don't get released from, right?
But those are chemical changes.
That is that there's not a catalytic action
that's involved here.
As we'll see, this is a part of a big catalytic action,
so it's only a very transient thing, but it's a good question.
Student: Okay.
Kevin Ahern: Okay?
So I guess this is the exception.
This and many enzymes that use this
as their catalytic action have this exception.
That is, they will become transiently covalently linked,
but they get released.
DIPF, it's not part of a catalytic action,
so it can't get released.
Yes, sir?
Student: The first portion looks like an SN1 nucleophilic attack,
but what about the deescalation
of the second part that's slower?
Kevin Ahern: Let me go through the mechanism.
I'll just talk about the mechanism.
So this is just a general scheme of the overall reaction.
Before I show you the mechanism,
I've got to tell you a little bit
about the active site of chymotrypsin.
Chymotrypsin has three amino acids
that play very important roles in the catalytic process...
very important roles in the catalytic process.
They're called the "catalytic triad."
They are aspartic acid, histidine and serine.
The numbers that you see by these
are their position in the primary structure.
This is amino acid number 102, this is amino acid number 57,
and this is amino acid number 195.
The fact that they're all three brought into close proximity
to each other, that happens because of folding, right?
Folding brings together things that aren't close
in primary sequence,
and they're brought into very close proximity to each other.
We'll see that all of the serine proteases have,
in fact, this catalytic triad.
All the serine proteases have the same catalytic triad.
They have the same basic geometry.
In the catalytic action of this enzyme,
there are several things to consider.
Notice what's happening here.
We have the enzyme in its starting state.
Here's the OH of serine.
Here's histidine.
Here's aspartic acid.
Over here, this histidine has pulled the proton
off of this serine
and created a nucleophile.
This guy over here, this O that has lost its proton,
still has electrons.
It's negatively charged.
It is a nucleophile.
It seeks a nucleus.
This is extraordinarily reactive
and it's called the "alkoxide ion."
Well, how did we get from here over to here?
What's the difference?
The answer is the binding of the substrate...
the binding of the substrate.
When the proper substrate binds in the enzyme,
what's going to happen to the shape of the enzyme?
It's going to change very, very slightly, right?
These slight changes make big differences,
because that slight change in position
now repositions these guys so that this guy,
the aspartic acid gets closer over here.
This pushes electrons over to this half of the ring, as it were.
This is slightly more negative,
which is the mechanism for pulling this proton off.
So this slight rearrangement has changed the geometry of these
very slightly so that this proton on the hydroxyl can get pulled.
Everybody with me?
The reason that that happens
and I get asked this question all the time,
"Why does it happen?"
it happens because the proper substrate
has bound in the active site.
Once that happens, that slight change happens, and ***!
Yes, Connie?
Student: What was the nucleophile's name?
Kevin Ahern: The nucleophile is called the alkoxide ion.
Let's now look at the overall mechanism
and I'm going to step you through it.
There.
Bear with me.
I'm going to go through it.
Then I'll come back
and I'm going to go through it again, okay?
So let me go through it
and tell you where we are with this stuff.
In this case, the proper substrate has bound
into the active site.
This change has started to happen
and we see that this guy here is going to grab this proton
and make the alkoxide ion.
We're looking right in the middle of that process
as we look right here.
Now, the proton here has been removed,
and they haven't even shown you the intermediate
of the alkoxide ion, which I think is unfortunate.
But there's the alkoxide ion, I told you, is a nucleophile.
That nucleophile seeks a nucleus
and the nucleus it seeks is the carbon of the carbonyl group.
It literally attacks that carbon.
That creates this unstable intermediate, right here.
It actually has a tetrahedral structure,
which isn't really important for our purposes,
but this unstable intermediate falls apart in the next step.
It falls apart.
Do I want to have an unstable intermediate in my enzyme?
Well, I may want to be careful about that,
because if I'm not careful, that unstable intermediate
may react with my enzyme.
So the enzyme protects itself with something called
an oxyanion hole.
It's basically a stabilizing structure
that keeps the intermediate from reacting with itself
and allows the intermediate to fall apart on its own.
So the oxyanion hole is doing that.
One of the questions I get is,
"Where's all this happening?"
This is happening in the active site.
The active site is a chamber of the enzyme.
Active site.
Oxyanion hole is a little room off the active site.
It's right there at the active site.
At this point, we have very quickly broken this bond.
So we've just broken the peptide bond.
You see what's left right now, which is one half of that peptide
is linked covalently to that hydroxyl.
The other one is only hydrogen bonded and leaves.
The hydrogen bond is not strong enough to hold it,
so one half of it is gone.
The other half is stuck to the enzyme.
And the question about why is this the slow step,
it's the slow step because we've got to go back
and we've got to regenerate another nucleophile
and we've got to wait for water to get in there.
And that's what's happening here.
So now we've got to get this guy released.
Water has to diffuse into the active site.
When it comes in, here's our nitrogen
that likes pulling protons.
Guess what it does?
Grabs a proton off of water, makes a reactive hydroxide,
which is also a nucleophile, attacks this guy,
and look what happens.
It breaks the bond.
We go back to our starting material.
This step takes a while because we have to get the water in there
and everything positioned appropriately.
Those are the general steps that are happening in that process.
I'm going to go through them again, but before I do that,
I'll take questions.
Yes, sir?
Student: The oxyanion hole, is it just a portion,
like a clamshell top that comes down
and it's dealt a positive to stabilize
the negative charge on the carbonyl carbon?
Kevin Ahern: It is there, yeah, basically to stabilize it.
That's correct.
You're right.
I'll show you an example of an oxyanion hole
in just a little bit.
So let me step through it one more time.
"What do I need to know about this?"
I'm not going to ask you to draw this whole structure.
There's a relief.
There's a big sigh of relief that goes through the room.
Put this on the exam for Monday, right?
Extra credit, draw the...
Student: Oh, god.
Kevin Ahern: No, no.
I would get killed if I did that.
So let's think about this.
There are several key steps that I think are important.
First of all, we have the catalytic triad.
The catalytic triad consists of aspartic acid,
histidine and serine.
They work together to create a nucleophile.
The way they do that is by the proper binding of the substrate
causes the histidine, ultimately,
to pull the proton off of the serine.
That creates this O-minus.
The O-minus is very reactive.
It attacks the carbonyl carbon.
We can think of that as sort of Step 1 or 1.5.
That attack on the carbonyl carbon creates an unstable
intermediate that is stabilized by the oxyanion hole.
Because it is not now going to react with the enzyme,
the instability it takes out on itself
and it cuts its own leg off.
Right?
The leg goes flying away
and we're left with the other half stuck there.
We can think of that as Step 2 or 2.5.
What's that?
Student: The intermediate is stabilized by the hole?
Kevin Ahern: The intermediate is stabilized in the hole, that's correct.
So we've just finished the fast step of the process.
Now we're in the slow step.
The slow step, we've got to get water in here,
we've got to get it oriented,
and we've got to get it activated.
That happens here, as you can see.
Water comes in.
Here's our proton puller.
There's the nucleophile we create.
And, again, the carbonyl carbonóyou think you get picked on...
think about the carbonyl carbon.
It's getting picked on twice here.
It's getting picked on here.
That creates an unstable intermediate that, in this case,
falls off of the enzyme.
When it falls off, it's released
and we're back where we started.
Mechanisms are things that you can spend thousands of words on,
but the reality is, if you sit down
and analyze what's going on with them they will, hopefully,
make much more sense than words can tell you.
So look at the major features that I've talked about here
the catalytic triad, the binding, the alkoxide ion creation.
What did the oxyanion hole do?
What was the fast step?
What was the slow step?
What role does water play in that process?óand basically,
you've got the mechanism.
That's basically what happens here.
Yes, sir?
Student: I hesitate to ask you another question, but...
Student: ...at the same point, it looks like with the dependence
on [unintelligible] like a nucleophilic attack
with the different steps, this could really be screwed up
by pH going plus or minus in either direction.
So what is an approximate given range?
Kevin Ahern: That's a very good question.
For chymotrypsin, its range, as I recall,
is fairly physiological,
and I don't know how wide that range is,
but that's a very good question.
There are serine proteases, for example, trypsin,
that can work in a fairly acidic environment,
and chymotrypsin in a not-so-acidic environment.
So there are ranges that this mechanism will work in, but
I can't tell you the full range.
I don't know that.
So I want you to sit down
and look at that.
Write things out.
I find it's really helpful to write things out.
These are all just individual steps, up close
and personal, so I'm not going to go through each one of those.
I think that's a bit of overkill.
Here's the oxyanion hole that's there,
and you can see this is sort of a representative structure.
There's some stabilization of this negative ion right here
by these protons, but it's not an overly charged structure, no.
There's something else that's important
for us to understand about this enzyme.
It's kind of cool and it's not difficult to understand.
I said that these changes happen
when the proper substrate binds to the enzyme.
How does the enzyme know the proper substrate?
Well, you have this idea in your head,
and it's correct, that the enzymes have a specific shape
and they will only accommodate certain shapes
properly into there.
In the case of this enzyme,
it has something called the "S1 pocket."
Like the oxyanion hole,
the S1 pocket is right there at the active site.
The S1 pocket is the one place
that different serine proteases differ from each other.
They have different shapes
and will bind different molecules, as a result.
You're looking at one of those pockets, right here,
and this pocket is kind of nice and deep.
What you see is the side chain of a phenylalanine,
I think, that's in here.
This will accommodate phenylalanine very nicely.
It may not accommodate something in here that's very charged.
This is a relatively non-polar environment.
That is, there's no plus or minus charge in there.
You saw some serines,
but no plus or minus charges in there.
Trypsin has a very different S1 pocket,
as I will show you in a bit,
but it uses the same catalytic mechanism.
It's a serine protease, just like chymotrypsin is,
but instead of cutting next to hydrophobic amino acids,
trypsin will cut next to lysine and arginine,
as I hope you got from my lecture before.
How is that difference accomplished?
Well, when we look at that S1 pocket
that trypsin has and we compare it
to the S1 pocket that chymotrypsin has,
trypsin has a carboxyl group at the base of it.
The negative charge attracts the positive charge
and they make a nice little bond,
and that's what determines that it's got the right thing bound.
Now, I show you this structure to show
you the important structural similarities
of these two enzymes.
I mean, that might look like there's some difference.
There's a difference out here.
There's a little bit of a difference there.
But, overall, those two structures
are not very different from each other.
That's not totally surprising
because they're going to have
similar mechanisms of action.
Structure makes function.
So if they have similar mechanisms,
it's not surprising they would have very similar structures.
The place where we would expect
that they would differ would be in the S1 pocket,
and that's indeed exactly what would happen.
Somebody's going to ask me where is the S1 pocket on here
and I don't know off the top of my head.
I think it's down here,
but I'm not sure of that.
Student: This is chymotrypsin
and trypsin overlapping?
Kevin Ahern: That's right, chymotrypsin
and trypsin overlapped.
This shows the S1 pockets,
chymotrypsin, trypsin, elastase,
showing them very schematically,
very schematically.
Chymotrypsin, fairly non-polar,
no pluses or minuses down here,
so it accommodates phenylalanine nicely.
It'll accommodate methionine nicely.
It doesn't like pluses
and minuses down there.
Here's trypsin.
It's got a carboxyl group at the bottom.
It really likes if it's got a positively-charged
side chain like lysine or arginine has.
Elastase is sort of like chymotrypsin,
except it's got some things
jutting into it that keep big side chains from fitting in.
So this guy here can't take a phenylalanine.
It won't cut next to a phenylalanine
because these things are blocking the access of the ring.
This guy likes to cut next to alanines,
a very tiny hydrophobic group that fits in there.
So the S1 pockets really help
us to understand how the specificity
of an enzyme is set up.
Questions about this?
Okay.
So that's cool.
Kevin Ahern: Put it back up there? Yeah, sure.
I'm going to tell you one more thing
of medical implication,
then I've got a song that I think you'll enjoy.
Actually, no, I don't have that.
Maybe we'll just do the song.
We'll finish early one day, how about that?
So I have a song.
This song, I have never sung to a class before.
So it's a brand new song about serine proteases
and I hope that you will sing loud because,
I will tell you what.
I'll make a deal.
If I hear you sing loudly today,
we will have a second,
not just one, but a second
extra credit on the exam.
Student: Whoo!
Kevin Ahern: Are we set?
Okay, let's go then.
It's to the tune of "Rudolph the Red-Nosed Reindeer."
[singing "The New Serine Protease Song"]
Lyrics: All serine proteases
work almost identically,
using amino acid
triads catalytically.
First they bind peptide substrates,
holding onto them so tight,
changing their structure
when they get them in the S1 site.
Then there are electron shifts
at the active site.
Serine gives up its proton
as the RE-ac-tion goes on.
Next the alkoxide ion,
being so electron rich,
grabs peptide's carbonyl group,
breaks its bond without a hitch.
So one piece is bound to it.
The other gets set free.
Water has to act next to
let the final fragment loose.
Then it's back where it started,
waiting for a peptide chain
that it can bind itself to
go and start all o'er again.
Kevin Ahearn: Okay.
Have fun.
[applause]
Captioning provided by Disability Access Services
at Oregon State University
[END]