#13 Biochemistry Catalytic Mechanisms II Lecture for Kevin Ahern's BB 450/550

Uploaded by oharow on 26.10.2011

Ahern: So the exams are not graded.
The TAs have exams of their own
and I have told the TAs that, fingers crossed,
I would like to have exams back sometime on Friday.
That may not be until afternoon, I don't know at this point.
And I'm not even sure if that's possible.
But the aim is to get things back by Friday.
When exams are available, what I will do is I will send
an email out to the class announcing that they're available
and announcing where to pick them up, okay?
So they won't be available here, for example.
They won't be available in my office.
But I will announce where to pick them up.
You will need your ID to pick up the exam.
So keep that in mind
and the minute, I can assure you
the minute they are ready to go,
I will put an email out to the class
and get them to you.
I don't like to have exams out too long
before you get a chance to look at them.
So I always like to ask,
just sort of informally with the class,
what did you think of the exam?
Student: Good exam.
Ahern: Good exam?
I'll comment one thing, I have never had an exam
where I had fewer questions.
There were maybe 10 questions I got on the exam
and that was for a class of this side, really unusual.
So I hope that's a good sign.
But you don't have to like my exam,
I don't hate you if you don't like my exam.
Student: Why do you videotape your exams?
Ahern: Why do I videotape my exams?
I find it decreases the looking around factor
by a factor of about 100.
[student laughs]
It does.
Student: I figured, but.
Professor Ahern: Yup.
I don't mean to be mean, but there's just too many eyes here
and not enough of our eyes.
And what I have found is that really works very well.
That's why I videotape those.
I find most students are honest.
I've only had a handful of situations where in this class,
where I've had dishonesty as an issue.
And the only students I've ever had
with serious dishonesty issues actually
were in smaller classes interestingly enough.
But it has happened here
and I do have taped evidence when it does happen.
So that's why I do it.
Student: Was the zwitterion question a trick question?
Ahern: Was the zwitterion question a trick question?
How many zwitterions,
how many amino acids can form zwitterion?
None. They all can.
That's not trick.
You guys should know that, right?
Is the answer "None" a trick?
I'm sorry, I don't think so, folks.
There are no amino acids, everything, so think about it.
What is a zwitterion?
A molecule that has a net charge of zero?
Doesn't every amino acid have a PI?
and what's PI?
It's the pH at which the charge is zero.
I know I will get some answers on that that don't say none
and so I hope that's very few,
but that's something that you should know.
Yes, Shannon?
Student: What did you have in mind for the last question?
The one about the Kcat.
Ahern: What did I have in mind for the one about the Kcat?
Oh, yeah, yeah, yeah, okay.
So the question, and by the way,
I will post the key outside my door
after we give the exams back.
I don't post those now
because students get all anxious until they see their exam.
So I don't post the key until the exams are back.
When the exams go back, there will be a key outside my door.
But answer your question about Kcat.
How can you have two apparent Kcats?
and the answer is it depends on how you calculate
the concentration of the enzyme.
If you take Vmax and divide it
by the total concentration of enzyme,
you will see a reduced Kcat
compared to the uninhibited enzyme.
Because much of that concentration of enzyme is not active,
it's inhibited.
However, if you take the inhibited out of it
and you take Vmax and divide it by that
modified concentration of enzyme,
you will see exactly the same Kcat
as if you have an uninhibited enzyme
because you're only measuring the velocity
of the uninhibited enzyme when you do that.
So that's the answer to that.
That was the thinker question
and I like you to think,
I like you to think about the principles
that you've hopefully learned in the class.
How about length?
Was length a factor or not?
Student: Yeah.
Ahern: How many thought it was too long?
Oh, that's relatively small, okay.
I really did try, this is the one exam
where time is sometimes a factor,
and so I was going to have 3 of the longer answer questions
and I decided not to do that
and I made them shorter.
So I had 2.
There's trade offs in all these as I've told other classes.
So one of the things that happens
is the fewer the questions I have,
the more points each question is worth.
And so people are always worried about that.
So I have to try to strike a balance.
And this balance, I think works fairly well.
I'm glad to hear there weren't too many at least
who indicated time problems.
Yes, sir?
Student: Have you ever offered alternate formats
like putting out a 5 part exam
and saying choose the 4 you want to attack?
Ahern: Have I ever done alternate formats where I say okay,
pick the ones you want to do?
I have done that.
They cause logistical problems for grading
and, to be honest, I'm not sure that's the best way to test.
I don't rule them out, but in my opinion,
the, when I'm giving an exam, I'm sampling.
And so I'm sampling your knowledge
and while that sampling can be large
and I can do what you recommend there.
My concern is, well, what I always see is there's a bias.
Everybody decides to do this question over here.
And it seems to me that when I have that bias, it suggests
that all of the questions aren't equal in difficulty
and so that kind of makes me think
well maybe that's not the way to go.
But I have considered it
and I have done it on some occasions.
And I won't rule it out.
I always reserve that as an option.
But I've shied away from it for that reason.
Anybody hate the exam?
You can say it.
As I said, I don't...okay,
there you go.
Do you want to say anything?
Student: 20 point questions suck.
Professor: 20 point questions suck, okay.
Student: If you don't know it,
that's like 2 letter grades gone.
Professor: Yeah, that's why I like having
more questions rather than fewer.
Next term, the format of the exam changes in 451.
Most people like 451 better because
the maximum number of points on a question I think is 3.
Student: There we go.
Professor: We have a lot of shorter answer questions.
But we're working problems,
we have to work through problems and that's why.
If you're going to spend a fair amount of time on something,
it should be worth more points.
I mean I can make a 3 point Henderson Hasselbalch question,
but if you spend 20 minutes of your time working on it,
that wasn't a good investment of your time, right?
Other comments or questions?
Student: You said this is the exam
was the one where time is an issue.
Ahern: This is the one exam where time is sometimes an issue.
Student: Okay, so the second midterm
we'll have a little extra time?
Ahern: So the second midterm will be exactly the same format.
The final will be exactly the same format.
So all the exams are the same format,
but what I think happens on the second and third exam
is that you've gotten used to Henderson HasselBalch format.
The working of the problems,
which usually is the time limitation,
isn't as much of a factor because you've seen
how to do these sorts of problems before.
And so I rarely, I won't say never because you,
in a class of this size, I could give an exam
where I ask you to sign their name
and people would say they didn't have enough time.
But literally, I rarely have much of an issue
with the other two exams.
And usually I'll tell you.
When I ask that question to this class,
I would say I've seen as many as 2/3 of the hands
go, 'I didn't have enough time.'
So I was very careful to try to make sure
I didn't ask you too many things.
But it meant I had to have 20 point problems
because we have to have a 100 point exam.
Yeah, Elizabeth?
Student: About the extra credit question...[inaudible]
Ahern: The extra credit question, okay.
I'll answer that
and then I'll go onto other things here.
So the extra credit question for hemoglobin, alright.
So most people got, the book sort of eluded to the fact
that in both cases, you had shape changes.
That you're changing from R to T or T to R
and so fourth, and that's true.
But the root of that change is the binding of a molecule.
And that's the answer.
Because in the case of hemoglobin,
you're binding a molecule exactly in the same manner
that the enzyme is binding a substrate.
And so the parallels of those
with respect to concentration and so forth,
that's really the reason you see those two curves
being essentially the same.
Everybody's all [Ahern makes groaning noises].
Alright, I hope everybody got how I start my lecture.
Student: No.
Ahern: No!?
Student: [Inaudible]
Ahern: I said if you said something about starting the exam,
we would give credit.
Or starting the class, we would give credit, yeah?
Student: I said, "How's everybody doing Today?"
That's always the next thing you say.
Ahern: That's the second thing I say.
So the first thing I say is "Okay folks, let's get started."
I don't live consciously, I just blurt it out.
[class laughing]
Alright, well let's turn our attention.
Thank you for your feedback.
That's not a lot of feedback,
but I do appreciate feedback and I'm always happy to listen
to what you have to say about exam formats.
And I do take suggestions.
The suggestion about having other possible choices is one,
as I said, I've done and I won't rule out.
But other thoughts or feedback,
I'm open to 'em, very much appreciate that.
Okay, last time I talked about, in some detail,
mechanisms by which this, these S1 proteases worked.
And I hope you've had a chance to look through those mechanisms
and sort of lay things out.
And I know it probably wasn't the first thing you did
when you went home after the lecture on Friday,
but I think it will pay you to go through and analyze that.
Because you're going to see when you do that
is by understanding that mechanism by which S1 proteases work,
you will see similarities in mechanisms
that other enzymes use to catalyze reactions.
And I'm going to talk about some of those today.
But before I do that,
I want to talk a little bit more about S1 proteases
because so far all I've told you about them
is that they are a class of enzymes.
And they're a class of enzymes that are called S1,
are called serine proteases, I'm saying S1 proteases,
they're serine proteases.
They're called serine proteases
because they all use serine in the active site.
So they all have the catalytic triad.
But there are some related
proteases that have things like that
that I want to spend a few minutes talking about today.
One of the things that we think about with the proteases,
the serine proteases, is the fact that as I said,
they all have serine, they all have histidine,
and they all have aspartic acid
as a catalytic triad at the active site.
So one of the questions people have asked is,
"Well, what is the relative importance of each of these
"in the catalysis of these enzymes?"
And so using genetic techniques today,
it's very easy to alter the genetic code
for any of these proteases
and change which amino acid
is presence at any given place.
Doing that, researchers have changed,
for example, a serine residue of 221,
which is the serine, gives it its name, to an alanine.
Or changing the histidine position 64 to an alanine.
Or changing aspartic acid at position 32 to an alanine.
Or changing all 3 to an alanine.
And when they do that,
and they compare the activity, so this is the log of Kcat.
So Kcat of course is a good measure of velocity
and the wild type enzyme has an activity up here.
I know this is a log scale.
So this isn't like these are half,
this is 1, 2, 3, 4, 5, 6, 7 orders of magnitude,
meaning that the wild type is 10 million times
more active than the enzyme
that has its serine changed to an alanine, okay?
So obviously that serine is a very important residue.
You might wonder how in the world
it even happens if it doesn't have a serine,
and I won't go into that, but this tells us
that serine is very, very critical for the catalytic acid.
One 10 millionth as active when that serine
is changed to an alanine.
What this graph also tells us
is that histidine is also very critical.
And that's not surprising because,
as you saw in that catalytic,
excuse me, in that catalytic mechanism,
histidine had to pull that protein,
protein, proton off of the serine
to make the alkoxide ion.
If you don't have something that can pull that proton off,
then it's a much tougher go.
Student: Is the 221, is that what amino acid number is?
Ahern: That's the number of the amino acid in each case.
So without something that can pull that proton off,
the enzyme is just as dead in the water
as if it didn't have a proton
that could be pulled off in the first place.
So these are important.
When we look at removing the aspartic acid,
we still see about 5 or 4 or 5 orders of magnitude lower.
But this tells us that that aspartic acid residue
is not as important as the other two.
The other 2 are much more important.
And that's not totally surprising
because the aspartic acid
was mainly crowding that histidine.
That tells us it's not absolutely essential
for its catalysis although it does play
some importance in that.
Yes, Neil?
Student: [inaudible]
Ahern: You're saying would this be equivalent
to what would happen if I put DIPF there,
is that your question?
Student: No, [inaudible]
Ahern: Would this one respond to DIPF?
Student: [inaudible]
Ahern: This one?
Would it respond to DIPF?
You mean if I made the mutation?
Student: Yeah.
Ahern: Well if I made the mutation,
I wouldn't have serine in there anymore, right?
So it would not be possible to treat with DIPF.
I can only treat something that has serine
and expect DIPF to have an effect.
Yes, sir?
Student: Are there studies done on [inaudible]
such as this where you can replace the serine,
histidine, or aspartic acid
with something other than alanine
and perhaps increase the function?
Ahern: Okay, so it's actually a very good question.
His question is what
if I mean if I go from serine to alanine,
at least they look similar,
but they're not chemically similar.
What if I change the serine to a threonine for example?
Threonine also has an hydroxyl group.
The question is A, would I see activity?
and B, might it even be better?
and the answer is it's all possible,
of course, but my production would be,
in this case, that it would not be,
because we're thinking about
very precise orientations inside of that active site
and threonine is going to have
a slightly different configuration.
It's possible it will be better
but my suspicion is it will probably
be somewhere in here.
And his question is also good because when we think
about the process that gave rise to this proteases
in the first place, they were mutation and selection.
Mutation and selection.
We think of mutation as mostly being detrimental
because most mutations give rise to things
that aren't functional,
kind of like what these individual ones are here.
But some mutations actually give rise
to more functional enzyme.
That's how enzymes evolved evolutionarily
in the first place and so that tells us
something that it's possible, it could happen.
But histidine to alanine here,
that's a pretty big change.
What if I put something like
let's say a tyrosine here
that might have a lot of electrons
and ability to perhaps influence that proton.
That might have an intermediate effect or some other effect.
Make sense?
Yes, sir?
Student: Why is it just as ineffective
when you replace as three as when you take off one them?
Ahern: Very good question.
Why is this one the same as this?
Why would that be?
It says that essentially once we disable these,
we're pretty much dead in the water.
I mean, one ten millionth is there.
And that the contributing,
these aren't additive effects
but in fact this is any one of these mutations,
well I guess they don't have that.
Any one of these mutations alone is enough
to knock the enzyme out essentially.
You might say well why do you have any activity at all?
And that's also a good question
but remember we have a very little activity
and we have the rest of the structure
of the enzyme intact.
We have the binding site,
we have an environment in there that maybe,
in addition to the specific amino acid
we see in an environment there that's favoring
to some extent these activation
and breaking of peptide bonds.
We're focusing on the nucleophilic attack
and the other components to breaking peptide bonds
that are still present in the structure of the enzyme.
Does that answer your question?
So that's an interesting observation.
What I want to do now is show you some other proteases.
So some related proteases.
Subtilisin is a protease,
it's an S1 protease,
but there are other proteases
that aren't S1 that behave very much like S1,
well like serine proteases, alright?
I keep saying S1, it's in my head.
One of these classes of proteases
is known as cysteine proteases.
And the cysteine proteases,
a good example is papain,
it comes from papaya fruit.
When we look at this active site,
we see something interesting.
We don't see a catalytic triad.
We see, in this case, a catalytic diad.
And the diad consists of a cysteine
residue adjacent to a histidine.
If I were to ask you to predict
what you think happens in this catalytic mechanism,
what do you suppose is going to happen?
How will this enzyme function?
I will give you a hint and tell you
it also performs a nucleophilic attack
very much like S,
like the serine proteases
perform a nucleophilic attack.
Student: The nitrogen on histidine withdraws
the electron from the sulfhydryl group on cysteine,
which is then nucleophilic and attacks the substrate.
Ahern: Basically yes, except you said electron.
It's actually removing the proton.
The histidine is removing the proton
from the sulfhydryl cysteine,
that makes a sulfur left behind with extra electrons.
Those extra electrons are nucleophilic,
they attack the peptide bond and very much like we saw,
and I'll show you a mechanism in a second,
very much like what we saw with the serine proteases,
the cysteine proteases work the same way.
So let's take a look at that mechanism
and that mechanism is here.
It's flipped because now the histidine
is on the left instead of being on the right.
But we see the histidine,
there's exactly that same geometry
that we saw with the serine proteases.
There's that hydrogen out there for the taking.
The binding of the proper substrate
to the active site changes
the orientation between the histidine
and the side chain of the cysteine,
making it favorable for those electrons
in the histidine ring to pull off that proton
from the cysteine side chain.
That leaves behind a negatively charged sulfur ion.
It's very reactive, it attacks the peptide bond
and exactly all the other things
we saw in the serine proteases happen.
We form a covalent intermediate just like we saw before.
We form, we see a fast step,
we see a slow step,
just like we saw before.
So mechanistically, this class of proteases
is essentially identical to that of the serine protease,
at least for our level of understanding.
Yes, Shannon?
Student: When there's an X, does that mean collagen?
Ahern: When there is an X,
that means the rest of the molecule.
Remember these are peptides that we're cutting here, right?
Another class of proteases that is slightly different
but interesting are called the aspartyl proteases.
An example which is are renin.
And the aspartyl proteases, at first glance,
look somewhat different than the serine proteases
but I hope to show you in the mechanisms
some similarities that I think you will agree with.
But those similarities aren't all the way
through like we see with the cysteine proteases.
How does this work?
First of all we have,
as you can see on the screen,
in the active site we have 2 aspartic acid residues
that are very close to each other.
And between them, forming a bridge is a water molecule.
One of the protons of the water molecule
is attracted to the negative charge
that's on the side chain of the aspartic acid
and it doesn't matter for our purposes here which one.
And when we look at the mechanism, we see this.
Here we have one of the aspartic acids on the left,
the other aspartic acids on the right.
Here's the polypeptide chain that's up here.
And here's the water being held in place.
Now what's happening here,
if we think about what happened
with the cysteine proteases.
[Ahern coughs]
Excuse me.
The cysteine proteases and the serine proteases,
we had to extract a proton to make a reactive molecule.
In the case of the serine proteases,
we had an alkoxide ion.
In the case of the cystein proteases,
we had a negatively charged sulfur ion.
What happens with the aspartyl proteases is very similar.
We extract a proton using the electrons
of the aspartic acid residue,
creating a reactive hydroxide ion.
It's got an extra electron,
it's negatively charged and just like we saw
in the other two cases,
this acts as a nucleophile.
The nucleophile attacks the peptide bond
just as we saw before.
The peptide bond falls apart
and we've broken the polypeptide chain.
Now there is one part of this mechanism
I just described to you
that is different from the other
two classes of proteases.
Does anybody recognize one thing
that's going to be different here?
Student: It flips which residue starts
with the water between each one as it switches.
Ahern: He says it flips between
which residue starts the water and the answer is no.
That's not the difference.
Student: [inaudible]
Ahern: What's that?
Student: Is it still the same covalent bond being formed?
Ahern: What covalent bond are we talking about?
You're on the right track, that's why I'm asking you.
Student: The intermediate.
Ahern: She's right.
If we think about the, for example,
the serine proteases,
we made an alkoxide ion, right?
We had that oxygen atom that had an extra electron.
It attacked the carbonyl group
that caused one part to fall off
and what happened to the oxygen?
It became covalently bound to the other change
and that chain is therefore stuck at the enzyme.
That oxygen was attached to the rest of the enzyme, right?
Do you see this water being attached
to the rest of the enzyme?
No. So we're not going to see a fast step
and a slow step with this.
Once we break the bond, we break the bond.
There's nothing holding it to the enzyme
such that the other chain has to be released.
That's what made the slow step in the serine proteases.
Everybody understand that?
In the case of the serine
and the cysteine proteases,
the nucleophile was physically attached to the enzyme.
The oxygen was the side chain of a serine,
the sulfur was the side chain of a cysteine.
Those are covalently attached to the enzyme.
So when they become covalently attached
to half of the polypeptide chain,
that half the polypeptide chain has to get released.
Here, water is not attached to anything.
When water attacks that carbonyl group,
the bond breaks, it's free.
One step, bam.
Everybody got that?
Student: So it's all just fast?
Is it all very fast?
Ahern: I won't say it's fast or faster because remember
we have to still get that proton off.
I'm not trying to compare speeds,
I'm just saying we won't have two steps,
a fast step and a slow step.
We won't have that with an aspartyl protease.
Yes sir?
Student: Does this reset itself by [inaudible]
stealing the hydrogen off...[inaudible]
Ahern: yeah, yeah.
Student: Is that how it resets itself?
Ahern: Yeah your question is a very good one.
How does the enzyme reset itself.
And if we think about the water that was added,
one of the protons gets grabbed here.
And the hydroxyl does the attack,
but to break a peptide bond,
we have to add both an OH and an H.
And so that H can either come from here,
that H can come from here.
And then the enzyme to reset itself
is going have to get back to its original state.
So if it comes from here,
then it's a simple matter.
We've just gone back to the place where we were at.
Let's move on to the 3rd class of proteases.
The 3rd class.
These are called metalloproteases.
Now we're getting further and further away
from serine proteases but I hope you see a common theme.
An example of metalloproteases is thermolysin.
These enzymes derive their name by virtue
of the fact that they use a metal ion
as their way of holding on to a water molecule.
We'll see this theme come up also.
A metal ion to hold onto a water molecule.
We saw the aspartyl proteases that the water was in place
and the water had to get positioned right there.
The water played an important role in that catalytic process.
The metalloproteases have a means of holding water there.
That actually could make the enzyme
more efficient because in the aspartyl proteases,
that water could be bouncing around.
That's why I didn't say they were faster.
In the case of the metalloproteases,
we've got something that's going to hold onto the water.
The most common ion that's used is zinc.
And I'm going to show you that mechanism here.
In the case of metalloproteases,
we're looking at the active site.
And we remember that if we're going
to break that peptide bond,
we have to have a nucleophile.
And that nucleophile and coming from water,
we're going to make a reactive hydroxyl group
just as we did for the aspartyl protease.
But the way in which we make it is slightly different.
First, water is bound by this zinc ion.
The zinc, you'll notice, is positively charged.
The positive charge of the zinc
is attracted to the negative,
or the relatively negative charge,
partial charge, on the oxygen of the water.
That interaction helps to hold water at the right place.
And then, and this is going to vary
from one enzyme to another,
there is a side chain that will help
to remove a proton from water.
That might be a histidine,
that might be another like lysine, or arginine,
or something else capable of pulling a proton.
That's listed on there as a B for base.
So some other side chain is going to help
in the removal of that proton from the water
and again, once we've removed the proton,
we've got a hydroxide behind that
is very electron rich,
attacks the peptide bond,
and bang, we're off and running.
Is this going to have a fast step
and a slow step or just a fast step?
Student: Two step.
Fast and slow.
Ahern: Fast and slow?
What does it take to have a fast and slow?
What's the slow step in the serine protease?
Releasing the polypeptide chain from the enzyme, right?
Do you see the polypeptide chain
getting attached to the enzyme anywhere here?
No, this only has one,
this doesn't have a fast and a slow.
This does not have a fast and a slow
because there's no covalent intermediate with the enzyme.
The water, again, is the attacker.
The hydroxide in this case is the attacker.
Student: Is the water bounded to the zinc?
Ahern: The water is attracted to the zinc.
Student: Okay, so it's not a covalent bond?
Ahern: It's not a covalent bond of any sort, no.
These are partial ionic interactions,
not unlike a hydrogen bond,
except for it's not a hydrogen that's involved.
It's an oxygen.
Hopefully what you see in that is a common theme.
First of all, every single protease
that I've shown you so far has a nucleophile.
Every one.
That nucleophile is what attacks the carbonyl group
and it's the attack on that carbonyl group
that results in the breakage of the peptide bond.
That common theme goes through all of them.
The only ways in which these enzymes
which I've described to you so far
different is the means by which
they generate the nucleophile
and what the nucleophile itself is.
That's the only ways in which they differ.
The basic mechanisms are the same.
We created a nucleophile,
the nucleophile attacks the carbonyl group,
the peptide bond breaks,
and the pieces go their way.
Well this business of creating nucleophiles
is not unique to proteases.
There are other enzymes that use nucleophiles
and generation of nucleophiles
in their catalytic mechanisms.
One of these is an enzyme
we've been talking about some already,
that's the carbonic anhydrase
and that's right here.
You guys look very tired.
Ahern: Would you like a joke?
Students: Yeah.
Ahern: Would you like to stretch before the joke?
Okay, so let's stretch.
So this my, this is my magic genie joke, alright?
My magic genie joke.
This guy's walking along,
down the street, kind of bummed.
He kicks a bottle
and all of a sudden realizes,
"Whoa! What's that?"
He grabs it, you know.
Wipes the dust off of it.
Of course in the process polishes this thing.
And out pops this magic genie.
And the magic genie says,
"oh master, thank you, I will grant you 3 wishes."
and the guy says,
"oh, this is really great' he says,
'first of all, what do I want?
"I guess I want a billion dollars
"so that I can be a very rich man."
and poof, a certificate appears in his hands
and it says he has a billion dollars
in a Swiss bank account.
This is really good, right?
Second wish he says,
"I'd really like to be a very powerful guy."
and poof, a certificate appears in his hands
and he's the president of Apple,
or Microsoft, it doesn't really matter.
This is not a computer joke.
Depends on where you lean, I suppose.
"This is really awesome,
"I'm a really powerful guy.
"Third wish, I've got money, I've got power.
"I want every woman to love me."
and poof, he turns into a box of chocolates.
[class laughing]
And you thought it was going to be a dirty joke, didn't you.
[class laughing]
That's a dumb joke, I know.
But it always gets a big laugh.
I hate telling joke about sex or the other, but...
Carbonic anhydrase, so you've heard
about carbonic anhydrase already.
I hope I've connived you it's a pretty amazing enzyme.
carbonic anhydrase, you recall,
was the enzyme I said when we first talking
about enzymes that had a Kcat of a million per second.
It can catalyze the conversion
of a million molecules of substrate
into product per second, per enzyme.
Remarkable thing.
Here's the reaction that it catalyzes.
We see carbon dioxide plus water going to carbonic acid.
Carbonic acid can then ionize and form bicarbonate.
And that can happen with incredible rapidity
under the right conditions.
Under the right conditions.
Now what does that mean?
Well, there's something very odd that happens
with this enzyme.
Most enzymes have a fairly narrow pH range
where they work that's ideal.
And most of those ranges are set
pretty much to correspond to the physiological
environment in which we find them in the body.
Most of our body tissues are at a pH of 7 to 7.4.
Blood, for example, is about 7.4.
We look at the activity of carbonic anhydrase,
and by the way, you're seen Kcat changing here.
Kcat will be constant for a given set of concentrations.
If we change the concentrations, Kcat itself will change.
We look at the Kcat of this enzyme,
and we discover something very odd.
We see, not surpassingly at a low pH, it's very low.
We get to about pH 7
and we're looking maybe half a million per second.
But this guy tops out at about pH 9,
where we get up to a million per second.
Why is this enzyme odd in this respect?
Why is its Kcat so high
at such a high pH that it really
doesn't encounter in the body.
Well the reason that this is the case
is it gives us a clue in to a very important
consideration in how the enzyme works.
And so that's what I want to show you next.
It's going to relate to the things I've been talking about.
During the catalytic process inside of this enzyme,
we see something happens that happened
very much like what was happening the metalloproteases.
That is we have a zinc ion,
and then zinc ion functions to hold water
so that we can create a nucleophile.
It functions to hold water so we can create a nucleophile.
The rate limiting step in the catalytic action
of this enzyme like that of the proteases
that you saw before,
is the rate of formation of the nucleophile.
The rate of formation of the nucleophile.
The faster the nucleophile can form
or the easier the nucleophile can form,
the faster the enzyme is going to be.
If we go to pH 9,
we could imagine that it's going
to be a lot easier to remove protons
off of water than if we're at pH 7.
We have more basic conditions,
water's going to give up its protons more readily,
and so at pH 9, we see that this enzyme
is far more active than it is at pH 7,
indicating that a very, very important step
is the removal of that proton.
That proton can come off many ways,
but if the buffer is helping that proton to come off,
it does even better.
Does that make sense?
I see a lot of nods, okay.
My next question is do you suppose
if we kept raising the pH,
it would get faster and faster and faster?
I see nos.
They say "it's a trick question.
"I know Ahern.", right?
The answer being no, why?
Within H of the enzyme.
So at pH 9, the enzyme is still holding
its shape enough that it's actually
able to continue catalysis.
When we get above that,
we're going to see the ending drop off precipitously.
So that's a very interesting step.
This actually shows the mechanism
and again, it's nothing like you haven't already seen.
We see the water being bound.
We see something pulling off that proton,
whether it is just ionization of the water itself,
which will happen in water to a limited extent,
or it's like a side chain of a histidine,
or a lysine, or an arginine that is pulling that off.
Once we've got the nucleophile created,
the nucleophile attacks the carbon dioxide
as it defuses into the active site
and that actually creates this,
in this case, bicarbonate ion that is released.
So it's the formation the nucleophile
just as we've been seeing all along
that is the critical step in this process.
Would you product that aspartyl proteases
might work better if we raise the pH on them?
You can stick your neck out, I won't chop it off.
I hear a no.
Do I hear any yesses?
What do aspartyl proteases have to do, folks?
What about the metalloproteases?
They both use water.
How about metalloproteases?
Do they work better at a higher pH?
Probably would.
They probably work a little better at a higher pH.
What's going to determine if they work
better at a higher pH or not?
The stability enzyme structure.
That's going to be the only limitation.
If the enzyme structure is stable at pH 10,
the enzyme will be way better at 10 than it is at 7.
Because again it's easier to make that
nucleophile at pH 10 than it is at pH 7.
Yes, sir?
Student: So that being in consideration,
is there any movement,
like the biotech industry to create man-made enzymes
by taking a natural enzyme,
sticking in a bunch of cysteines and crosslinking
it back and forth like crazy
and making it stable at a higher pH?
Ahern: You should work in the biotech industry, sir.
The answer is yes.
There is a lot of interest in manipulating
enzymes to infact increase their activity
and increase their efficiency.
In the case of carbonic anhydrase,
though, remember what's limiting them
is actually defusing it into the active site.
It's probably not going to get much better
than that million even if we were to improve that.
But for a metalloproteases,
it may very well be useful
because now we can stabilize the enzyme
with disulfite bonds,
we can use it at a higher pH,
that might very well be a strategy.
You bet.
We're slithering along through this.
Let's take a, spend the last 5 or 6 minutes
talking about restriction enzymes.
We'll see some similarities we saw before.
So you really hope,
I hope you're starting to see the themes now.
Restriction enzymes are enzymes
that many of you have used if you've ever worked
in a lab that does DNA work.
And restriction enzymes catalyze
the breakage of DNA double strains
and specific nucleotide sequences.
Restriction enzymes,
they're also called restriction endonucleases,
I'll take either one, that's fine.
They catalyze the breakage of DNA double strands
at specific nucleotide sequences.
Now that's what they do.
That's not how they do it.
How do they do it?
Well I will tell you that they hydrolyze them.
They use water to break the bond.
And that should give you some hint
about the mechanism that they use
and you can start seeing the wheels turn,
thinking 'I'll wager that they're going
"to make an activated water molecule in some way,
"like taking a proton off,
"making a nucleophile,
"nucleophile's going to attack,
"and that attack is going to result
"in breakage of a bond."
and you would be exactly correct.
You would be exactly correct.
Please turn your phone off, whoever has it on.
Now let's take a little bit closer look at this
in terms of how they operate.
Actually that's not a very good figure.
One of the things we see in restriction enzymes
is that they all require magnesium for their action.
They all require magnesium
and magnesium, like zinc,
is a divalent KCat ion and one of the things
that magnesium helps to do is it
does help to position the water
so that it can lose a proton
and make an attack on,
in this case, a phosphodiester bond.
We're not breaking peptide bonds, obviously.
We're breaking phosphodiester bonds
because those are the bonds
between the adjacent nucleotides in a DNA molecule.
Now I'll show you something that's really cool
and interesting.
Actually, I have to explain it to you
because it doesn't show it very well.
Let's take a look at this enzyme.
There are many different restriction enzymes.
This one is called ecoR-five.
EcoRV recognizes the structure, the sequence GATATC
and it cuts right in the middle of it.
Now if you look at this carefully,
you'll see that on the top strain,
we go GATG, or ATC, on the bottom strain,
we go to GATATC.
These are what are called symmetric sequences.
And these symmetric sequences are very common
features of sites that restriction endonucleases recognize.
It means that we can cut right in the middle of this guy
and we've cut both of them.
We just cut this guy right in the middle.
Not all of them work in the middle,
but this particular guy works in the middle.
Now I want to explain to you just physically
how a restriction enzyme works
and then I'll show you very briefly
a little bit of mechanism.
A restriction enzyme is a protein.
That protein grabs a hold of DNA.
So many proteins will grab hold of DNA.
DNA is a negatively charged molecule,
we would expect that to grab a hold of DNA,
perhaps positive or neutral,
we certainly wouldn't expect the protein
to be negatively charged
because it wouldn't interact very well with DNA.
The enzyme grabs a hold the DNA molecule
and what it does is it literally slides down the DNA molecule.
Now most of the time it's going down
that trip down the DNA molecule,
it does not encounter the sequence GATATC.
In fact that sequence will occur randomly,
only about once every 4,000 residues.
So a lot of the time it's just floating along here,
sliding itself down the DNA molecule
and then all of a sudden in the binding side of the enzyme,
GATATC is there.
It's found the right site.
What happened in the serine proteases
or in any of the proteases
or in any of the enzymes when the proper substrate bound?
What happened physically inside those enzymes?
Shape change, right?
We saw a shape change that happened in those.
Those very tiny shape changes
caused the enzyme to have all of its properties.
In the case of the serine proteases,
that shape change resulted
in the creation of the alkoxide ion.
In the case of the restriction endonucleases,
that shape change is more dramatic.
What it does is it actually causes a bend to occur in the DNA.
So we think of the DNA molecules
of being straight and linear,
but when the enzyme is bound to that proper site,
the enzyme goes "oh, whoa!" and it bends.
The DNA molecule is physically bent at that point.
It's physically bent.
Now that bending turns out to be critical
for the catalytic action.
Now, we can see that bending happening right here
and when we analyze the structure
that's present at that bending,
what we see first of all is that
there's a nice little water molecule
that gets positioned right here,
held in place by a magnesium right here.
And that only happens when the bend occurs.
Without the bend,
there's no pocket for that holding to occur.
So when the proper substrate has bound,
the water's in place,
the magnesium is in place,
the proton can get removed
and the nucleophile can be created.
Okay, so we've said a lot today about nucleophiles.
Once that nucleophile is created,
it attacks the phosphodiester bond,
the bond gets broken just like we saw
with the peptide bond and everybody's happy.
I will very briefly go over that next time
and I will see you on Friday.