#14 Biochemistry Enzyme Regulation I Lecture for Kevin Ahern's BB 450/550


Uploaded by oharow on 28.10.2011

Transcript:
Captioning provided by Disability Access Services
at Oregon State University.
Kevin Ahern: Okay, folks, let's get started!
As you can see on the screen,
I do not have grades posted.
I will have an announcement about the exam
at the end of the lecture today, however.
So, that'll keep you tantalized.
I spent some time last time
going through most of the mechanisms.
I finished talking a little bit
about restriction endonucleases
and restriction enzymes, which we also call them.
I want to just say a few words about those
and then we're going to move on
to catalytic, regulation and control mechanisms.
So, as I indicated last time,
restriction enzymes are similar in mechanism
to what we saw with proteases.
We remember, of course,
that restriction endonucleases are cutting DNA, not protein,
and DNA, of course, has nucleotide building blocks,
not amino acid building blocks.
And further, we remember that the bonds
between nucleotides are phosphodiester bonds, not peptide bonds.
So there are some different terms,
there are some different things there,
but the similarities are that we're using
an activated water molecule as a nucleophile
to break phosphodiester bonds.
That's a very important consideration.
Students frequently find interesting
the sort of story of restriction enzymes,
so I'll spend a couple of minutes talking about that.
Restriction enzymes are, as I said,
enzymes that specifically cut DNA at specific places,
and you might wonder why such enzymes exist,
because we think of the genome
as being something that we want to protect,
we don't want to damage it, et cetera, et cetera.
It turns out that restriction enzymes
are produced in bacteria.
They're not produced in human beings.
Bacteria use them as a sort of primitive immune system.
It's a very simple, primitive immune system.
In the immune system,
we have antibodies that attack things
that are recognized as foreign.
In bacteria, bacteria get infected by viruses
just as we get infected by viruses.
The viruses that infect bacteria
are known as bacteriophages.
And these bacteriophages typically have a genome
of DNA very much like other viruses have genomes of DNA.
They can have genomes of RNA, but most of them have DNA.
And part of the infectious cycle of any virus
is that the virus must attach to a cell
and inject its nucleic acid into the cell.
So in the case of a bacterium that gets
infected by a bacteriophage,
the bacteriophage has grabbed hold of the cell
and it will inject its DNA,
if it's a DNA virus, into the bacterial cell.
The bacteria make restriction endonucleases as a protection.
So they recognize and cut specific sequences,
and if that invading virus has those sequences,
which it typically will,
because the recognition sequences are relatively short,
typically four to six nucleotides in length,
then the invader will basically get chopped to bits.
And one of the questions that arises then is,
"Well, why doesn't the bacterial DNA
"get chopped to bits as well?"
And that's the last part of the story I need to tell
you about restriction enzymes.
Restriction enzymes are part of a system
that we refer to as "restriction/modification."
The restriction part is the enzyme
that does the cutting, as I described to you.
The modification system I haven't told you about.
The modification system is comprised
of another enzyme that recognizes exactly
the same sequence that the restriction enzyme recognizes.
That is, if I have something that's an EcoRV,
then that EcoRV restriction/modification
system will have a second enzyme
that recognizes GATATC,
just like the restriction enzyme
that does the cutting does that.
Well, what does the second enzyme do?
The second enzyme doesn't cut that sequence.
Instead, it puts a methyl group
in the middle of that sequence, a methyl group.
So the modification enzyme does that.
It's called a "methylase" and when we examine that,
we see in the case of this particular sequence,
there's the unmodified enzyme sequence there.
There is the modified sequence,
and we see that a methyl group
has been put onto the A of the GATATC.
That means that one will go on the bottom A, obviously, as well.
And the significance of that
is that single methyl group prevents water
from binding in the right place to make the cleavage.
So even though the enzyme can recognize that sequence,
water can't get in there and do the attack
and cause the bond to be broken.
So when a DNA has been modified by the methylase,
it will no longer be cut
by that same restriction enzyme
that recognizes that sequence.
So, for example, this guy has been modified.
This could be in the cellular genome,
and it's protected from cleavage
by the restriction enzyme.
The question then arises,
"Well, can the methylase get to the invader phage
"before the restriction enzyme does?"
and the answer is, yes, it can.
And if that happens,
then the phage will survive
the being cut and go on and infect the cell.
You say, "Well, why does that happen?"
Well, it happens because there's no perfect system.
Your immune system's not perfect.
The bacterial system's not perfect.
But, suffice it to say,
this protection system that bacteria
have is pretty darn good.
If you've ever worked in a laboratory
and you've tried to put plasma DNA sequences
into a bacterium that has a restriction/modification system,
you'll find it's very inefficient to do.
Most of the things you try to put in
get chopped up and you don't get
any plasma in it, at all.
But, at a very low frequency,
you do get some plasmas in and then
they get methylated regularly and survive that process.
Okay, so that's the restriction/modification system.
As I said, it exists only in bacteria.
It does not exist in human beings
and we don't need such a system
because we have an immune system that, in theory,
is protecting us from invaders as much as possible.
Okay, questions about that?
Yeah, back there.
Student: Is it possible for the [inaudible] virus genome?
Kevin Ahern: Yeah, so, that was the question I was saying.
Is it possible that it'll get there
before the cutting enzyme can?
And the answer is, yes, it can.
So if that happens, then the invader will actually be protected.
It will replicate and it'll kill the cell.
So, as I said, it's not a perfect system,
but it's a pretty darn good system.
Shannon?
Student: Did you just say that we don't have modification?
Kevin Ahern: We don't have restriction or modification.
So we have neither.
And again, we have an immune system
that's protecting us extracellularly, not intracellularly.
Yeah?
Student: What happens to that methyl group
after [inaudible] got chopped off?
Kevin Ahern: What happens to the methyl group?
Student: Yeah, after the enzyme kills the invader.
Kevin Ahern: I'm sorry, after what?
Student: After the enzyme kills the invader?
Kevin Ahern: After the enzyme kills the invader.
Oh, I see, so if the enzyme kills the invader,
then that means that it didn't get methylated, right?
If it gets methylated,
then it's just going to stay on there.
It's not going to do anything.
It's going to be protected, okay?
Yes, sir?
Student: Doesn't that methylation enzyme
also get assistance from the actual,
during the reproduction of the DNA itself,
when there'll be another reader
that comes along and matches methylation states
from the parent strand to the new strand?
Kevin Ahern: I'm sorry, say it again, now?
Student: If you're copying the DNA...
Kevin Ahern: Uh-huh?
Student: does that methyl get copied, as well?
Kevin Ahern: Okay, if you're copying the DNA,
does the methyl get copied as well?
The methylate still has to come in and do its thing.
So is it possible that the restriction enzyme
may cut that at a low frequency?
Again, the answer is, it's possible, yes.
So the methyl doesn't get copied.
The methyl has to be put on after the DNA is made.
Connie?
Student: When you say we don't have
a restriction/modification system,
does that mean we don't have any restriction enzymes
or modification enzymes, at all?
Kevin Ahern: We have no restriction enzymes,
no modification enzymes, at all.
That's correct.
I'm not sure if I answered your question properly,
back over here, so let me say it one more time.
If the methyl group gets put on,
then the invader will replicate and kill the cell,
basically, because it won't be able
to be cut by the enzymes.
So if that happens, then you just have plenty of viral DNA.
The methyl group doesn't do anything, at all,
because as far as the rest
of the proteins of the cell are concerned,
it's just a GATATC sequence, okay?
If we're talking about how we recycle nucleotides,
we'll talk about that a little bit
when we talk about nucleotide metabolism next term,
so I'll save that for that point.
I hope that better answers the question for you.
Student: Are these all found in, like, the nucleus?
Kevin Ahern: Well, bacteria don't have nucleus, yeah.
Okay, the one last thing I want to say,
and this one doesn't really relate to mechanism so much,
but it does remind us of the importance
of shape changes in proteins.
So the last group of proteins that
are of considerable interest
with respect to catalysis [inaudible]
are known as the myosins,
and myosins are part of the actin-myosin pair.
Actin is one protein, myosin being another,
and these proteins are very, very important for,
in fact, they're essential for—muscular contraction.
So these proteins, together,
produce the contraction that occurs inside of muscles.
There's a whole bunch of stuff with respect to mechanism,
and I just don't think it really tells
us much about mechanism that we haven't already seen.
No surprise, you're going to see an activated
intermediate that's going to cleave ATP.
But the point that I want to make
about actin and myosin is this.
Contraction happens because myosin
literally crawls along the actin,
and that crawling requires molecular
change in a protein, this protein being myosin.
I want you to look and see this protein.
Here is myosin, and when ATP,
in fact, what this protein does
is it hydrolyzes ATP,
and that hydrolysis at ATP induces
a significant shape change in this molecule.
You can see that the unhydrolyzed ATP,
and afterATP hydrolysis has happened,
this motion has happened inside of this protein.
This motion, folks, is what allows you to move.
It allows you to have a heart.
It allows you to have all kinds of motion necessary to function,
and it's happening because of the shape change of a protein.
This motion, right here,
we can think of as like a claw
that is allowing the myosin to crawl
its way along an actin filament.
A really cool thing,
and that happens as a result of this shape
change in this protein.
The shape changes require the hydrolysis of ATP.
So a very cool application of shape changes
that we see in proteins happening as a result of catalysis.
Most of the rest of the protein,
you'll notice, doesn't really change much.
It's only this section out here.
A picture of myosin is actually on here.
You can see these two little heads that are out here,
and these heads actually crawl
their way along an actin filament.
Okay, that's what I want to say about mechanisms of catalysis.
With that, I'd like to turn our attention
to discussing the allostery and regulation.
So allostery and regulation, or control,
as I often times describe it,
is very, very important with respect
to the needs of a cell get harnest on enzymes.
Earlier in the term,
I mentioned that enzymes can be extraordinarily efficient,
extraordinarily fast and I gave the analogy
of driving a Maserati to Fred Meyer at 110 miles an hour.
You could imagine there's going to be some
problems with that if you don't regulate in some way.
We regulate with speed limits.
Cells regulate enzymes by a variety of mechanisms.
One of these mechanisms is known as allostery.
I've mentioned it briefly before,
but I will say it again
and also give you the definition again.
Allostery, or allosterism, is the mechanism
by which the binding of a small molecule
to an enzyme affects the enzyme's activity.
So it's the binding of a small molecule
to an enzyme that affects the enzyme's activity.
When I mentioned this before,
I pointed out that not all enzymes are regulated.
Cells are very efficient in regulating things.
They regulate the most important,
or I'll say the first enzyme,
in a metabolic pathway,
and by controlling the first enzyme,
they control all of the things that flow through it.
Just like if I put a tollbooth out
in front of I-5 in Albany
and I stop all the cars there,
there aren't going to be many cars getting through,
only the ones that the tollbooth allows through.
So it's the same thing that happens in metabolic pathways.
If we control that first enzyme
that catalyzes the first reaction in the pathway,
we can essentially control the whole pathway very easily.
It's very efficient, and so that's a very useful thing.
There are, in the cell,
three main mechanisms that cells use to control enzymes.
One of them is allosterism,
what I've already described to you.
I'm going to show you some details
of allosterism later today.
A second control means that cells
have over enzymes is covalent modification.
They can covalently modify enzymes.
We'll see some examples later which involve
the addition or removal of phosphates from enzymes.
These covalent modifications can activate
or inactivate enzymes,
depending upon the enzyme.
Student: Allostery [inaudible] also?
Kevin Ahern: Allostery can be positive or negative.
That's correct.
The third mechanism that cells use to control enzymes
is controlling whether or not they're synthesized,
that is, whether or not the protein is even made.
And that seems like, well, duh!
It turns out that's one of the most important
considerations for many control systems.
Is the protein being made by the cell or not?
And that control is exhibited in several ways.
It could be transcriptional.
It could be translational.
We'll talk about some of those next term.
What I want to do now is talk for a bit
about allostery and this very interesting
enzyme called ATCase.
So let me show you a little bit of this.
ATCase catalyzes a reaction that,
to be honest with you,
we're not going to pay much attention
to the reaction itself until next term,
but it catalyzes a reaction
that is a very, very important reaction,
because it's the first step in making pyrimidine nucleotides,
the first step.
That is, the very first reaction in making
a pyrimidine nucleotide is catalyzed by the enzyme ATCase.
Now, the enzyme ATCase has a much longer name.
It's known as aspartate transcarbamoylase.
I'm not going to spell that here.
You can get it out of the book if you want to get the word.
We commonly abbreviate it ATCase.
But please note that when you use an abbreviation,
you have to get it right.
You can't call it ACTase, for example.
ATCase is the proper name.
Now, here's the reaction that it catalyzes.
You can see it on the screen.
It involves an aspartic acid.
One of the things that we'll see that's of interest
next term is that all of the nucleotides
that are made by cells have building blocks
that start with amino acids.
So we can start with very simple things
and make fairly complex molecules,
like nucleotides, using various enzymatic systems.
The pyrimidine nucleotides include CTP,
UTP, and TTP, if we're talking about DNA.
To make these nucleotides,
it's more than one reaction
and this is why I refer to these things as "pathways."
To go from these simple molecules here
all the way down to the final product,
which in this case here is CTP,
takes about ten steps.
About ten different reactions are necessary...
Student: What are the pyrimidine nucleotides, again?
Kevin Ahern: What are the pyrimidine nucleotides?
That would be CTP, UTP and TTP.
It takes about ten steps to get to this final product here.
Well, as I said, cells have efficient means
of controlling pathways,
and cells really don't want to make
too much of any given nucleotide.
We know from the study of cells
that cells that have aberrations in them
that cause them to have too much
or too little of a given nucleotide
cause those cells to have higher rates of mutation.
Mutation is generally not a good career
move for cells, certainly not in the short term.
Over evolutionary history,
yes, but over the short term,
most mutations are very detrimental to cells.
So cells are very much what I describe as control freaks.
They put a lot of energy and a lot of controls
in the way of preventing the nucleotides
from getting too high or too low.
Well, what does this all mean?
Let's imagine that I'm a cell.
I'm sitting around and I'm making pyrimidine nucleotides.
I produce CTP and the CTP concentration starts to increase.
Well, if the CTP concentration starts to increase too much,
I don't want to make any more CTP,
so I want to turn off the synthesis.
I want to turn off that pathway.
It turns out that the enzyme ATCase
is an allosteric enzyme and it will bind
to the end product of this pathway.
So ATCase will bind to CTP,
and that's what this little red thing is showing here,
and when it binds to CTP the enzyme is turned off.
So when the enzyme binds to the end product of the pathway,
the CTP accumulation starts to get high,
the enzyme gets turned off,
then that shuts off essentially all the reactions
leading up to CTP,
and, until that CTP starts getting used,
that pathway will essentially be turned off.
Notice I said "essentially."
If you remember, I said we think of these mechanisms
in terms of on and off,
but in reality, they're more like turning
the volume down or turning the volume higher,
but we still have some things coming through.
So allosteric mechanisms do not have on/off switches,
but they have turn-down/turn-up situations.
Well, the beauty of this is, the end product helps control itself.
It can control its own synthesis through this enzyme.
That's a very, very useful thing.
It's a very simple mechanism.
As a result, CTP concentrations inside
of cells don't get too high.
You say, "What about UTP?
"What about TTP?"
Well, it turns out UTP and TTP
are both ultimately made from CTP.
So by controlling CTP,
you're controlling all of the pyrimidine nucleotides.
One step, one enzyme, one molecule,
it doesn't get any more efficient than that.
So that's really cool how cells are able to do that.
This mechanism I've just described to you has a name.
It's called "feedback inhibition."
So feedback inhibition occurs when the end
product of a pathway inhibits
the first enzyme in the pathway.
Feedback inhibition occurs when the end product
of a pathway inhibits the first enzyme in that pathway.
We'll see other examples of feedback inhibition,
primarily next term,
but there are many examples that cells
use of feedback inhibition
because it is so simple and easy to control things.
Well, that's one interesting aspect of ATCase.
There's the actual reaction that is catalyzed,
and, no, you don't need to know all
this stuff that's on here.
I'm just showing you.
There's the carbamoyl phosphate.
There's aspartic acid.
There's the intermediate that it makes.
There's a whole bunch of steps,
and there's the final product of CTP, right there.
I find it really cool and remarkable
that the nucleotides can be made from amino acids.
We know that amino acids exist in space
We know that amino acids can combine in space
and people have actually found nucleotide precursors
in meteorites floating out there.
So this idea of the chemical evolution
of life is pretty cool,
and we use the materials that are available
to us to make us and to make life possible.
If I study this enzyme, ATCase,
and I study the reaction that it catalyzed,
and, remember, ATCase does not make CTP.
It's making this aspartyl carbamic molecule.
CTP is only made way down the line.
That's not made by the ATCase enzyme.
So if I take and I study the reaction
that ATCase is catalyzing and I study it
in the presence of increasing concentrations of CTP,
what I see is that the rate of formation,
this is the product,
we're seeing that the rate of formation is falling,
and this is a log scale,
so it's a fairly significant drop.
This rate is falling as the CTP concentration increases.
This graph is showing you visually
what I've told you in words.
The more CTP there is in the cell,
the more the enzyme will be inhibited,
but, as I noted, we don't have a complete off switch.
We're turning the volume way down,
but we haven't turned it off.
Now, interestingly, if we examine the catalytic activity,
and this is not a very good representation of this,
of this enzyme we discover something else very interesting.
The last figure I showed you showed
the effect of increasing concentration of CTP.
CTP is not a substrate.
Remember, CTP is a product of that ten-steps-away pathway.
It's simply a molecule that binds to ATCase.
So it's not a substrate for ATCase.
If I take one of the substrates of ATCase
and I measure that same rate of formation of product,
I see a sigmoidal plot.
This is the thing that we referred to on the last exam.
That sigmoidal plot is happening
because something else is going on in this enzyme.
You saw the effect that CTP had.
Now you see that a substrate is also having an effect.
How do I know it's having an effect?
Well, I see a sigmoidal plot.
That's not a very good "S"
but that's actually a sigmoidal plot.
It tells us that,
not only can CTP affect the enzyme's activity,
but so too can aspartic acid,
one of the substrates.
So a substrate can affect this enzyme's activity.
We see, at this point,
two regulators of the enzyme.
This regulator is doing to ATCase
a very similar thing to what oxygen
was doing to hemoglobin.
The more of it there was, the more activity we see.
That means that aspartate is activating the enzyme.
So the substrate, in this case, is activating the enzyme.
Now, this actually has biological significance.
I want you to think.
I'm going to tell you a little bit
about that biological significance right now.
Cells have to make nucleotides in order
to make nucleic acid.
They need to make RNA.
They need to make protein.
They need to make nucleotides.
If a cell is getting ready to divide,
the cell darn sure better be able
to make enough nucleotides.
If it doesn't have enough raw materials
to make nucleotides,
then that cell should not be preparing to divide.
Just like if your bank account is broken,
you should not be buying beer for a party on Friday.
It's not a good career move.
You may really regret it on Saturday,
and I can guarantee you the cell
will really regret it if it tries
to go through the division process
without having the resources it needs
to make the nucleotides for the RNA and DNA
necessary to do replication.
Well, how does the cell tell if it's got enough?
One of the ways is right here.
If the cell has a lot of aspartate,
what happens to the activity of this enzyme?
It goes up.
And what is aspartate?
Aspartate is an essential building block,
not only for nucleotides but also for proteins,
and cells need proteins to divide, as well.
When the aspartate concentration is high,
this is one of the signals to the cell that,
"Hey, we can go and have some whoopie!
"We can divide!"
That's cellular whoopie, by the way.
[laughing]
"We can divide!"
You guys are slow today.
So by actually having a little barometer,
which is what this is, on the cellular nutrients,
the cell is able to make an intelligent decision
about to divide or not to divide.
That's really cool.
So not only are we regulating an enzyme,
we're also testing the water.
"Do I have enough materials to go ahead and divide?"
Student: Is it only making this decision based
on the aspartate concentration?
Kevin Ahern: Is it only making this decision
based on aspartate concentration?
The answer is, no, it's not.
But this is one very useful piece of information.
If cells didn't have enough aspartate,
then you could see what would happen to this enzyme.
The enzyme wouldn't go and it would stop everything else.
So it's a good "no" switch,
it's not the only "yes" switch.
But, a very good question.
Student: If it didn't have enough aspartate...?
How is aspartate made?
Kevin Ahern: How is aspartate made?
Well, aspartate can be made by several mechanisms.
There are metabolic pathways that can produce it.
I'll mention a couple briefly next term.
Also, cells can ingest it.
So if they're floating around in a medium
that's rich in amino acids or rich in proteins,
they have sources of aspartate.
So that's really cool.
Now, what did I want to say here?
I don't want to say anything there.
I want to say a little bit of interesting
things about the enzyme, itself.
I'm focusing now on the protein structure of this enzyme.
When we study the protein structure of this enzyme,
something interesting happens.
Under certain conditions,
and what we're looking at here is a centrifugal
analysis of this ATCase enzyme,
it turns out ATCase has 12 subunits to it.
So it's even more complicated than hemoglobin.
It has 12 subunits,
and if I am careful in how I manipulate those subunits,
I can take that 12-unit piece and discover
that it's composed of three pieces of an r2 dimer
and two pieces of a c3 dimer.
I'll show you what those are in a second.
Now, in terms of the appearance of this protein,
this is what it looks like.
We can exclude all the ribbons.
I want you to focus on here.
You see that the enzyme consists of six units
called "c" or "catalytic."
These are subunits where reactions are catalyzed.
And it has six subunits that are called "r" or "regulatory."
Now, you can't see all six of the catalytic
because they're underneath.
So here's the top three and then there's three
underneath there, as well.
So we look at it from the side,
there's two of the three
and there's two of the three, there.
So this is like a double decker of trimers right here.
These are the catalytic subunits.
And the regulatory subunits,
you see three pairs of them here.
Here's a pair, Here's a pair, Here's a pair.
Three sets of pairs of the regulatory subunits,
and they are sort of hugging those catalytic subunits.
What we're going to see is that
the allosteric effectors are going to change
how these guys are all arranged.
The allosteric effectors,
in this case, aspartate or CTP,
are going to change the way
that the regulatory subunits arrange themselves
around the catalytic subunits,
and these changes in structure
will affect the catalytic activity.
Just as we've talked about R and T state with hemoglobin,
so, too, do we talk about R and T state with an enzyme.
When the enzyme is in the very low activity state,
it's in what we call the "T state"
or the "tight state."
When it's in the high activity state,
it's in what we call the "R state" or "relaxed."
I'll show you some more figures of that in just a second.
Now, before I do that,
I need to introduce what will seem to you,
at first, like a sort of a curve ball.
The curve ball is, I want to spend
a few minutes talking about an artificial substrate.
An artificial substrate.
It's, in fact, a suicide substrate,
and though your book doesn't call it that,
that's what it is.
It's a suicide substrate.
So what's a suicide molecule?
What was the definition of that, before?
It resembles a natural substrate,
the enzyme binds it,
and it becomes covalently linked to it, right?
So this molecule I'm getting ready
to describe to you is a man-made molecule.
It's not a natural substrate.
It's something that we've made to study an enzyme.
It resembles the natural substrate.
It's not unlike aspartic acid,
not unlike aspartic acid.
Here's what it looks like.
And, we can see that when this guys
comes into the enzyme it gets bound covalently to it.
This is the synthesis of the molecule.
Here is the artificial substrate.
This artificial substrate will covalently
link to the enzyme itself.
It's called PALA,
P-A-L-A, and I don't even know
the name of it myself,
so I always think of it as PALA.
Well, what's the significance of that?
Why do I tell you about that?
Well, it turns out that if I take the enzyme,
ATCase, and I add PALA to it,
PALA binds, as I expected before,
but something unexpected happens.
Study of the enzyme linked to PALA
first indicated that this enzyme could have two states.
It could have a T state and an R state.
When you take the enzyme all by itself,
and you study it in a centrifuge,
you basically see about one form.
You don't see two forms.
But when you treat it with PALA,
you discover that the ones that haven't bound
to PALA have one form,
but the forms that have bound to PALA
have a very different form.
These correspond to the T state,
on the left, and the R state, on the right.
Now you're sitting here very confused.
"You said the R state was high activity,
"but this is a suicide inhibitor.
"If this is suicide inhibited, it has no activity."
And that's correct.
It turns out that what PALA does,
it's catching the enzyme in that high activity
state and freezing it there.
Why didn't people see this before?
The reason people didn't see this before
is that when the enzyme binds to the normal substrate,
it catalyzes the reaction,
it flips into R, it catalyzes the reaction,
it flips back out,
and you don't see it.
Didn't see that state.
There's a factor of,
I think it's about a couple of hundred
to one that the T state is favored.
So you don't even see that very tiny percentage
that's the R state unless you lock things in it.
And when they locked things in it with PALA,
they discovered, "Wow, the R state exists for this enzyme."
If you look at what this R state looks like,
you can see it's very different than the T state.
In the R state, the enzyme is relaxed,
it's opened up.
Access to the catalytic units
for the normal substrate is high.
The normal substrate,
if PALA weren't here,
could get in here very easily
and cause the enzyme to be able to bind it very readily.
On the other hand, the T state,
you see everything is up tight, it's up close.
It's much more difficult for substrate
to get into the catalytic subunit,
and that's why we see the T state
having less activity than the R state.
So the T state and R state are very, very different
states of these two enzymes.
PALA allows us to see that.
You'll notice the arrows here,
indicating that the T state is way to the left,
the R state is way to the right.
You'll also notice something else on this screen,
and that is the effect of CTP.
Based on what you know about activities
of enzymes and the allosteric inhibitors
I've described to you,
it's not surprising to think,
then, that CTP,
which reduces the enzymatic activity,
is going to favor the T state,
whereas aspartic acid is going to favor the R state.
Now what's of interest here is that
they bind to different places on the protein.
R stands for "regulatory subunit."
The regulatory subunit is where
a regulator would bind, like CTP.
You might say, "Oh, well, then that means
"aspartate must bind the regulator, as well,"
and it turns out it doesn't.
Why? Because aspartate is a substrate
and substrates bind at the catalytic subunit,
where the catalysis will occur.
So aspartic acid binds to the catalytic subunit
and CTP binds to the regulatory subunit.
There's CTP locking in the T state.
and we basically see that there.
And there's the kinetic effect.
If we measure the reaction
in the presence or absence of CTP,
you've seen this earlier,
that you see a reduced activity.
There it is in the presence of CTP.
There it is in the absence of CTP.
So, not surprisingly, CTP is turning down that activity.
The last thing I want to say about this
is something that is even more interesting.
This enzyme is playing a critical role
in regulating CTP and in measuring the barometer
of do we have enough nutrients,
in this case, aspartic acid,
to go through and do replication.
It turns out the enzyme responds to something else, yet.
Well, let's think about this before I describe it to you.
We think about
that when we go to make RNA or we go to make DNA,
if we have a lot of pyrimidines,
then we need purines, right?
Because C pairs with G.
G's a purine.
C's a pyrimidine.
T pairs with A.
T's a pyrimidine.
A's a purine.
We want to have the right balance
of nucleotides present in the cell.
I told you if we make too much CTP we've got trouble.
But what if we have too many purines?
What if we have a high level of ATP and GTP?
What's going to happen?
Well, we may have the same problem with mutation,
and if we have that happen,
one of the things we would like to do
would be to increase the amount of pyrimidines, right?
Because if we have a high level of purines
and we raise pyrimidines,
we're going to have roughly the same balance.
So it turns out that ATCase
has the ability to sense this, as well.
ATCase is allosterically activated by ATP.
It's allosterically activated by ATP.
Here's the same curve you saw before,
now in the presence of ATP,
and you see we get increased activity in the presence of ATP.
This serves as yet another barometer for the cell.
So remember that I said ATCase is telling the cell,
"Do you have enough amino acids in the form
"of aspartic acid to go through with division?"
ATP is an indication of a cell's energy.
High ATP, high energy.
If the cell is full of energy,
the cell is full of purine nucleotides,
and the cell has plenty of aspartic acid,
this is a sign it's prime time to divide,
let's start making some pyrimidines.
And that's what this is doing.
So this enzyme is performing
some very, very important functions
in terms of helping cells to make intelligent
decisions about dividing.
ATP is an allosteric activator
of the enzyme and, like CTP,
it binds to the regulatory subunits.
It binds to the regulatory subunits.
That causes the enzyme to shift
from T to R.
So ATP is going to favor the R state.
Aspartic acid is going to favor the R state.
CTP is going to favor the T state.
This really interesting control system
that we see here is one of the reasons
why ATCase has been one of the most
studied enzymes in biochemistry.
It's a classic enzyme
for understanding allosteric regulation,
and it's not the only enzyme
that responds to more than one thing.
This ability to respond to different molecules
in different ways is really key to having
elaborate controls over metabolic pathways,
very, very important.
Now I'm doing a lot of talking here.
Let me ask for questions,
and then I'll finish with a couple of things.
Shannon?
Student: How is it that ATP indicates purine concentration?
Kevin Ahern: ATP is a purine.
ATP is a purine, so when ATP is high,
purine concentration is high.
Student: So CTP binding at an R site...
Kevin Ahern: Yes?
Student: [inaudible] causes the molecular [inaudible].
Kevin Ahern: His question is actually
leading to my very next topic.
It's a very good question.
His question is,
do CTP and ATP cause these changes?
Or is there something else that's involved?
I know it's not exactly what you asked,
but that's what the implications of the question are.
It turns out that neither one causes this to happen.
We thought of cause and effect with hemoglobin.
We thought of the first oxygen
caused the second one to be favored,
caused the third one to be favored,
caused the fourth one to be favored.
So we saw cooperativity.
And we talked about that as a cause and effect.
Oxygen caused that to happen.
Well, there are other models that are consistent
with changes that are not cause and effect,
and that's what I'm getting ready to tell you about.
Actually, maybe we'll save that for next time,
but then I have two things for you.
So let me save the answer to that question for next time.
I'm running out of time.
One, I thought we would sing a song,
and then, two, I'll make an announcement about the exam.
That way, you'll sing loud.
This is a song about taking exams.
[laughing]
[singing "The Mellow Woes of Testing"]
Lyrics: The term is almost at an end,
ten weeks since it began.
I worried how my grade was
'cause I did not have a plan.
The first exam went not so well,
I got a 63.
'Twas just about the average score
in biochemistry.
I buckled down the second time,
did not sow my wild oats.
I downloaded the videos
and took a ton of notes.
I learned about free energy
and Delta Gee Naught Prime.
My score increased by seven points,
a C-plus grade was mine.
I sang the songs I memorized,
I played the mp3s.
I learned the citrate cycle
and I counted ATPs.
I had electron transport down
and all of complex vee.
I gasped when I saw my exam, it was a 93.
So heading to the final stretch,
I crammed my memory,
and came to class on sunny days for quizzing comedy.
I packed a card with info
and my brain almost burned out.
'Twas much to my delight
I got the "A" I'd dreamed about.
So here's the moral of the song,
it doesn't pay to stew,
if scores are not quite what
you want and you don't have a clue.
The answers get into your head
when you know what to do.
Watch videos, read highlights
and review, review, review.
Ahern: Now, the exams are graded.
The average on the exam was 65.5,
not a bad one for the first exam.
They're available for pickup
in the BB office, ALS 2011.
I just literally got them done
just before I came to class.
There's a key posted outside my office.
You can look at the key outside my office
and I will post a grade distribution later this evening.
Student: What was the high score?
Kevin Ahern: High score on the exam.
This is really interesting.
The high score on the exam was 107,
perfect, and the low was in the 20's.
I want to say about 21.
[END]