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]

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2 thoughts on “#14 Biochemistry Enzyme Regulation I Lecture for Kevin Ahern’s BB 450/550”

  1. As challenging as this material may come, Ahern always makes me smile regardless 🙂 I feel very lucky to have this professor at my school!

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