Articles, Blog

13. Kevin Ahern’s Biochemistry – Enzyme Mechanisms & Regulation

October 21, 2019


Professor Kevin Ahern: How was the exam? Better than what I
thought, okay. Student: You tell me. Professor Kevin Ahern: [laughing] Well I don’t even know at this point, they’re still being graded, so that’s why I’m kind of curious to hear
your perspective. What did you think about it? I see a lot of people shaking their heads
yes, that’s always a good sign, or it means like you’re an idiot Ahern. A couple through you off a little bit, okay. Okay. I don’t take things personally, so if you have things you like or don’t like I always like to hear them. Any comments? Student: Clear. Professor Kevin Ahern: Clear. Fair, okay. Unfair? As I said, I don’t take things personally. I think that one of the reasons I ask this- I always ask after an exam- is that I learn a lot about giving exams from
year to year, and one of the things you always hope for
in writing an exam is that it’s clear, and that students understand what you’re asking, and that it’s fair. I think that’s two things that are really
important. And I will tell you that having written many, many exams over my career there are no perfect exams. Much as you would like to have a perfect exam that is absolutely great at assessing ability, there are no perfect exams. Exams can be good, and exams can do a pretty good job, and I hope my exams do a pretty good job of
that, but there are no perfect exams. So I think it’s important as instructors that we listen to you a little bit. Not even a little bit, a l lot, okay. That we listen to you and understand what
you don’t understand and understand where maybe we can make exams
better, because it’s important that exams be fairly testing people’s ability, and I think that’s a very important consideration. So your feedback, even though you’re not giving
me any, is important. Yes sir? Okay, so didn’t like the question on the protein folding problem, okay. Yeah? More time, yeah. How many people felt time was a factor? Okay, yeah, this exam it tends to happen with
that because people tend to take a little bit too
long on the calculation, and I think that’s a factor. It’s usually not a factor on the second exam, it’s never a factor in the final, and the good news is that next term it’s a completely different format of exam, and it’s never a factor there either. So this one exam that you had is the place where it does tend to be a factor. We tried a few years ago to get an evening
exam with this, but evening exams are still only supposed
to go for 50 minutes, So it doesn’t bias out of the problem that
we have of the length. so it’s an issue that I’ve wrestled with a
little bit, and I don’t make it long for the sake of long, but here’s what I always ask students- If I say, ‘would you like to have it shorter?,’ almost everybody would say, ‘yes,’ and I said, ‘okay then that means each question’s worth
more points,’ ‘do you want that?,’ and almost everybody goes, ‘no.’ So, it’s a tough call, and so writing that means that you have to find that right balance. I originally wrote the very last question to have three questions instead of two, and when I looked at the exam I thought, that’s probably a little bit too much and based on what I’m seeing here that probably would’ve been a little bit too
much. I’m glad I left it out, so that was good. Yeah? How do I do the curve? Well I look at where the distribution of grades
are, and I just decide based on that distribution of grades that
people- on the first exam it’s not quite so clear, but as we go further along we’ll see groups of things that’ll happen, and so I try to assign things on the basis of groups that I see there. I don’t have any fixed numbers of A’s, B’s,
C’s, D’s, or F’s that I give, and I do try to make it as best as I can with
those groups, and that’s as good of an answer as I can give
you because that’s really literally what I do. I do look at the overall average in considering where those breaks might happen
and so forth, and so students always say well how can I
improve my grade, and that’ll happen obviously after this exam, and the answer to that is always by improving against the average. So if you improve your score against the average, then you’re going to move up. So as I tell students, if the average on an
exam is ten, and you make it 90, well you just really moved yourself up a lot. Students will always say, ‘well I want an easy exam.’ In fact the best exam for you to move up on
is a hard exam because the lower the average, and the better you do against that low average, the better off you will be. Does that makes sense? So, and no I don’t make them hard, one of the most difficult things I have in
writing an exam is I can never predict what the average is
going to be. I’ve tired and I’ve tried and I have tried, and I’ve written exams and thought, oh that’s a real easy exam, and then you’ll see the scores and it’ll be
down in the toilet. Or you’ll write an exam and think that’s going to challenge them a little bit and you’ll see the highest score ever. So I’m the worst person at guessing those, so I just write what I think are appropriate
questions. I don’t try to steer it one way or another, because I just can’t do it, I’ve tried. Okay, if you feedback feel free to send me
an email. I’m always happy as I said to take that, and it does help me, I think, to write better
exams. so I like to hear that. Well we are well ahead of where we need to
be, and I like to stay that way, so being ahead is better than being behind. So we’ll finish the catalytic strategies today
a little early. Then we’ll start the new material as well. But we have some things we have to finish
in catalytic strategy, so let’s go and do those first. The carbonic anhydrase I talked about last
time is an example of an enzyme that uses nucleophilic
attack to accomplish what it does, which is converting carbon dioxide into carbonic
acid with the help of water, and we talked last time fairly quickly about
this mechanism. So this mechanism that you see on the screen is what’s occurring in- so the mechanism- what’s happening here, we’re looking at the
active site of the enzyme, and in the active site of the enzyme we have a zinc that is held in place by three
histadines. The zinc is a positively charged ion, and that positively charged ion helps to hold
water into place. It’s helping to hold water into place. We can see that coordination right there. We remember, of course, in a water molecule
that it’s asymmetric and that it’s asymmetric for
charge as well. Meaning that the oxygen’s is partially negatively
charged and it’s attracted to the positive ion of
the zinc. The most critical step in the process actually occurs in this very first step, which is the ionization of the water. And as I noted last time the ionization of
the water that is the loss of that proton occurs much
more readily at a higher pH that it occurs at a lower pH and is for this reason that the enzyme works
more rapidly at a pH of 9 that it does at a pH of 7. It still does very well at a pH of 7. You remember that the turnover number at pH
7 was still 600,000 per molecule of enzyme per
second. So it still working very rapidly but it will work even more rapidly at a pH
of 9 because this first step happens. Well the creation of this hydroxide ion in
the active site is a necessary step for the reaction to occur because that hydroxide ion like we saw before with the alkoxide ion and also the hydroxide
ion in the chymotrypsin acts as a nucleophile
attacking this carbon inside of this carbon dioxide. The attack of that carbon results in a bond,
covalent bond, between the carbon and the oxygen that you
can see right here and that is actually the step that’s creating the bicarbonate that is released here. So bicarbonate has been formed by this point. Water comes in and releases the bicarbonate and we’re back right where we started. So that’s what’s happening in carbonic anhydrase and as I said it’s a very rapid process that
this goes through. So that’s the basic process that’s occurring in the catalytic action of a carbonic anhydrase. Well I want to turn our attention now from carbonic anhydrase to yet another group
of enzymes that are interesting enzymes. I’m going to say a little bit about them and in general before I talk about their mechanism. But we’ll see that the mechanism that they
use is what we’ve been seeing the themes of so far. We are going to see an activated water acting as a nucleophile attacking, in this case of phosphodiester bond. So let me just first give you some general
information about restriction enzymes. Restriction enzymes are enzymes that are found in prokaryotic organisms. That is bacteria. So bacteria contain restriction enzymes. Restriction enzymes are also called restriction
endonucleases and restriction endonucleases have a very, very interesting and useful property. The interesting and useful property is that they recognize specific sequences of
nucleotides in DNA. They recognize specific sequences of nucleotides
in DNA. They bind to those sequences the specific
sequences and they cut at those specific sequences. So they recognize specific sequence. They bind to the specific sequence and they cut at the specific sequence. Now you might wonder why they do this? DNA is pretty important and the reason that
they do this is because this is a defense mechanism for
the bacteria. A defense mechanism for the bacteria. How does that work? Well bacteria get infected by viruses just like we get infected by virus. Many of you are infect with a cold virus right now. We have an immune system that goes out there and though it takes a while to get operational and to actually do its job. The immune system eventually takes over and we get better because the immune system is protecting us from invading viruses. For the most part. It’s not a perfect system but it’s a pretty
good system. We can get a vaccine for example and our immune system is what gives us the
protection against infection again by that same of virus
or organism. Well bacteria are single celled organisms. They’re lone rangers, right? They don’t have an immune system. They don’t have the ability to have immune
system because an immune system takes a multicellular
organism and it takes a tremendous amount of cellular
energy to do that. Working together coordinating to make antibodies. Bacteria don’t have that option. So every bacterium is for itself. They can’t go out there and coordinate an
effort against a virus or even another bacterium. Well what they have is since it’s every bacterium for itself is that they have these restriction
endonucleases. So if a virus comes along and it tries to
infect the bacterium that has a restriction endonuclease, restriction
enzyme. Then this system kicks into place. So the virus inserts its DNA into the bacterium. That DNA goes in the bacterium if there’s
nothing present the DNA of the virus will take over and start making copies of the virus and kill
the cell. If the bacterium makes a restriction enzyme that recognizes a specific sequence in the
DNA of the virus then it will cut it. Well cutting it means that you are interrupting the genetic information of the invading virus. Invading viruses DNA is no longer functional and you have just stopped the virus in its
tracks. That’s a pretty cool system. Well you might sit here and think ‘well that’s really great about how in the
world ‘does it keep from cutting its own DNA?’ Because if the restriction enzyme is recognizing a specific sequence won’t it cut that same sequence in its own
DNA? And the answer is if there weren’t other protection
it would. So a very commonly, a very common restriction endonuclease is called HindIII H-I-N-D and then the Roman numeral 3. HindIII. HindIII recognizes the sequence AA GC TT on
one strand. You don’t need to know that sequence, it doesn’t
really matter. But that’s what it recognize. That is a 6 base sequence. And if we say that sequences occur in DNA
relatively randomly that sequence will occur on a random basis once every 4,096 nucleotides. So on average that sequence is going to occur once in about every 4,000 nucleotides. If a virus, some viruses have a DNA sequence that might be 30,000 nucleotides long. 40,000 nucleotides long. Even 150,000 nucleotides long. Okay on average it’s going to have multiple of those HindIII sequences within
it. So if the bacterium has this enzyme and it can cut that sequence then it is very effective at stopping that virus
from doing its thing. So how in the world then does the cell protect
itself from this-from cutting up its own DNA and cutting only the viral DNA? Well the bacterial cells have a very interesting protection for this so when we talk about restriction endonuclease we also have to talk
about a protecting enzyme called methylase. M-E-T-H-Y-L-A-S-E. A methylase. The whole system I am going to describe to
you is called restriction/modification. So restriction/modification. So I’ve told you what the restriction is. That it’s recognizing and cutting specific
sequences. What the modification? The modification is made possible by the enzymes known as the methylases. A methylase recognizes the same sequence as the restriction enzyme and instead of cutting
it, it puts a methyl group in it. Somewhere in that sequence, it varies from
enzyme to enzyme, but somewhere in that sequence it puts a methyl
group in there. Why is that important? Well once the methyl group is in there, the enzyme can’t cut that DNA if it has a
methyl group in it. So the methylase methylates the host DNA at
every place there’s an A-A-G-C-T-T. So the host DNA doesn’t get cut by the restriction
enzyme. The invading viral DNA unless it gets methylated
first, which can happen, unless it gets methylated
first its DNA is naked, its unprotected, the restriction enzyme is going to cut it, and the virus is going to stopped. So it’s the combination of the methylase and the restriction enzyme that give the cell
protection, yes? So the question is: does it insert the methyl
group into the sequence or on to part of sequence? It’s actually modifying a base within the
sequence. So it’s putting onto one of the nucleotides
in the sequence. Very good question. So her question is if the viral DNA gets methylated does that inactivate any part of its functionality? Methylation actually can have some effects
on gene expression but the biggest effect that would happen here is that the restriction enzyme wouldn’t work
on it and the virus would actually kill the cell. So if the methylase wins the race, if the methylase gets to the viral DNA before the restriction enzyme gets there, that cell is toast. Okay? Because that viral DNA is now going
to be protected. It’s going to make copies. It’s going to basically kill the cell. Down here, Okay? Okay good question also. Once the viral DNA gets methylated and then starts copying itself are the copies
all methylated? The answer is probably not, alright? So there will be some protection that’s there
because, perhaps these new copies are being made, that new enzyme is going to have a shot at
getting it though. So it’s a complicated system and it’s not
a perfect system. I find that we always tend to want to think about a perfect system, right? That is that the methylase never wins the
race and restriction enzyme always does the cutting. But we don’t have that. We don’t have that with our own immune system. We’ve seen HIV as a prime example. We don’t have perfect protection but we have
a lot of protection. And the bacterial cells that have restriction
enzymes with a modification system in fact have an awful lot protection as well, yes? Student: Are the methylase and restriction
enzymes concentrations in the cells relatively equal or is there a lot of- Professor Kevin Ahern: Another good question. Are the restriction enzymes and the methylations in the cell relatively
equal? That will very a lot between restriction enzyme and modification enzymes, but for the most part I think that the restriction
enzyme is probably a little bit more abundant. What determines which one reaches the DNA
first? You are going to like this. The process of diffusion. So it’s a chance, it’s a chance thing. So again when we think about this in the overall
scheme of things an individual bacterium may die because it
doesn’t win the race it doesn’t have the right one get their first. But overall you’re going to have bacteria
that will survive that otherwise wouldn’t survive and that’s why the system persists. Does that make sense? I saw another hand, yes? So his question is, is it possible for the
virus to kill the bacterium even though the enzyme is present? Yes Okay. So there’s a variety of scenarios that we
can imagine will happen with this and as I said it’s not a perfect
system but it does ensure a greater likelihood that bacteria that have this will survive. Okay good questions. Remember what I just, what I said when I started
this. This is a system present in bacteria. We don’t have this in our cells. We don’t need this in our cells. We have an immune system for that purpose. Eukaryotic organisms do not have restriction modification systems. Only bacteria have restriction modification
systems. Well now that I’ve given you some background
on what it does, lets look and see how it does it. We’re going to focus on one enzyme called
EcoR Five. I’ve talked about HindIII, here’s another
one. EcoR Five. I don’t like this figure. Well I’ll leave it there for the moment. EcoR Five is an enzyme that recognizes a sequence, we don’t even need to know the sequence at
this point. But if there’s a methyl group in the middle
of this sequence that EcoR Five recognizes, EcoR Five will not cut. And there’s a reason why it doesn’t work and
why it doesn’t cut, and it relates to the mechanism of action
of the enzyme. We’re going to see that in the action of this
enzyme there’s two things that play very important
roles. The ion magnesium is one and the other is
the molecule water. And like we saw with zinc where we saw a zinc
activating water-holding water in place and then having
ionization and the ion attacking the carbonyl carbon
of the carbon dioxide. Aright, so too does the magnesium help to hold water in place so the same ionization
can happen. So the same nucleophilic attack can happen. Only this case it on a on a phosphodiester
bond. This is simply showing the magnesium ion that plays role in this and it’s some higgledy-piggledy
figure. But magnesium is playing an important role in ultimately helping to hold water. This actually shows the sequence that’s recognized, as I said you don’t need to know the sequence, but the sequence that this enzyme recognizes
the G-A-T A-T-C. And most restriction enzymes by the way recognize a symmetric sequence. And when we say symmetric, if we read it 5
prime to 3 prime on the top strand it reads the same way, 5 prime to 3 prime on the bottom strand. Molecular biologist call that a palindrome. It’s technically not a palindrome because
radar is a palindrome because it reads the same way backwards and
forwards. But this reads the same way backwards and
forwards if you read one strand and then you read the
other strand. The methylation that’s happening in this enzyme is happening right in the middle of this sequence
right here. One of the things that happens during the
catalytic action of an enzyme is there’s a couple of steps. The enzyme actually finds its sequence on
a DNA in an interesting fashion. It grabs a hold of any old part of the DNA. As the restriction enzyme grabs a hold of
any old part of the DNA and then it starts sliding down
the DNA sequence. It’s sliding down that DNA sequence. And when it finally hits the sequence that it recognizes, it stops. That’s kind of a cool way of finding the thing that it’s going to cut at. When it gets to that sequence that it recognizes, and it stops, it not only stops but it undergoes a conformational change, because why? The proper substrate has a bound to the enzyme. Just like you saw with the cerium protease. The proper substrate is now this case the
sequence GAC ATC. The binding of that proper sequence in the active site of the enzyme causes the enzymes undergo a conformational
change and the conformational change causes the DNA
to bend right in the middle of where it’s bound. Right square in the middle. Everybody with me? It’s bound the DNA it slid to its proper sequence it’s bound to the proper sequence, it stopped, it changed shape and now it’s holding on this bit DNA in the middle of it. That’s what this guys doing. This bending turns out to be critical for
the cutting. The restriction enzyme, remember, is going
to cut. We’re talking about the restriction enzyme
here, we’re not talking about the methylaids. We’re talking about the cutting by the restriction
enzyme. It’s bendiness. If we were look at this bend inside of this
DNA molecule, what we would see is that there is a nice
little position here, about right here where my pointer is, underneath the middle of this thing where a magnesium ion will be held in place and right above that magnesium ion, there’s a water ion will be held in place and this will only be created, this little pocket will only be created if the DNA is bent and the DNA only bends
if the proper sequence is bound by the enzyme. Well, the water, like we saw before with the
zink and the in the carbonic anhydrase, the water gets ionized, magnesium is holding
in place, the ion attacks the phosphodiesterase bond and cause it to fall apart. Because it is done that, we’ve broken a phosphodiesterase
bond and phosphodiesterase bonds are what hold
DNA together. Now what you’ve done is you broken the bond, the strands come apart. You’ve just cut DNA. So thats how the cutting actually occurs. That’s the mechanism of the cutting. I’ll talk about the methylades in just a second, but the cutting occurs by that mechanism. Questions about the cutting, yes. Very good question, there are two strands
here does this happen twice? And the answer is yes, it does and yes it
must. We have to cut both strands to completely
cut a DNA. Is it possible for ligase to come in after
this? Ligase is an enzyme that will join phosphodiesterase
bonds, it is possible for that to happen, yes. Kimberly? The enzyme has no effect on the rest of the
DNA. It’s only going to have an effect at the place where binds the proper sequence and causes that bend to occur. That bend is necessary for the action that were talking about here. Okay, we’re getting into things we will talk
about next term, but next of it is question is does a topoisomerase, which is an enzyme that unwinds a twist DNA, does it work in a similar fashion to this
involving a bend? The answer is no it does not. Yeah Okay so she’s asked a question here about- there are different enzymes that recognize things in different ways. Let me go back to the sequence and show you what she’s asking about here
first. If we go back to the sequence, the sequence this recognizes G-A-T A-T-C,
alright? And this particular enzyme cuts between the
T and the A, and between the A and the T. So it makes a perfectly symmetrical cut in the middle of a perfectly symmetrical sequence. Which results in what we call blunt ends. The ends are exactly the same sticking out
here. Some restriction enzymes don’t do that. Some restriction enzymes will come between
the G and A here, and the G and A over here. Her question, I think, was why that happens. Was that the question? Student: Yeah. The answer is that’s just simply the way that
they evolved. So it has to do with the way that the other
strand gets cut. So some enzymes that they cut here and they
cut here because they are both cutting between a G
and an A. Leave this end has a A-T-A-T hanging over and this has A-T-A-T hanging over but they’re not blunt like we had before. So don’t get too hung up on the recognition
sites themselves. But let’s suffice it to say that enzymes have
different places within a sequence that they will cut and implications of that are what the overhangs
look like. We’re not going to get to too much more into
that, yes? This is all still bacteria. Everything is bacteria. Okay other questions? Let’s go back then and talked about the effective
methylation. So here’s what happens in the case of the
methylase that protects EcoR Five sites. It modifies the adenine right there. So it’s putting a methyl group on the adenine within that sequence, as you can see, and that methylation of the adenine stops the enzyme from working. And what the methylation is actually doing
is getting in the way of this coordination of the magnesium
and the water. So this very important structure that we need
to have for cutting is not stable when the methyl
group is present there. The methyl group interferes with it. It’s a very effective way of stopping an enzyme
from cutting. And by stopping enzyme from cutting the cell’s
DNA is protected. I see a little bit of nodding, that’s good. Hopefully not nodding off. The last step before you fall asleep. OKAY other questions about that? It’s a cool system, yeah? That’s a very good question. What happens when an enzyme when it encounters
a methyl group? It won’t bend. It doesn’t recognize it as a proper sequence, so you don’t get bending, you don’t get any of the things that we saw
here. So EcoR Five goes along and it’s just as if
it didn’t see it in the first place. Very good. Well those are restriction enzymes. Let’s see. Maybe we should sing about that, I think- I have a song about this we have never sung
this song in class. So it’s a relatively new song it’s the first
time, you guys are going to be the first people, ever to sing this song in class. It’s about what we just heard about. It’s about the enzyme in the HindIII. It’s to the tune of an old song called Chim
Chim Cheree. Everybody know that song? Okay, here we go. I’m obsessed with A-A-G- Let me think about it. I’m obsessed with- [laughing] Now I can’t get the tune in my head. Someone want to get me started. I’m obsessed with A-A-G-C-T-T because it is the binding site of Hin-d-III cutting up DNA most readily The bonds are not blunt when they’re cut up
you see [laughing] That’s the wrong song! [humming] I’m obsessed with A-A-G-C-T-T because it’s the binding site of Hin-d-III cutting up DNA most readily the ends are not blunt when they’re cut up
you see Five prime overhands of A-G-T-C This is awful. Bacteria don’t have an immune system so They must fight off phages or they will not
grow Protection by chopping is their strategy And one of the cutters we call Hin-d-III On binding to A-A-G-C-T-T The site recognition site’s bent easily Phosphodiester attacking meanwhile Has water behaving as nucleophile To stave off the phage for a little while Why don’t these enzymes cut cell DNAs? The answer’s provided by methylase Adding a methyl group on top of what The sequence these enzymes would otherwise
cut So cells get protected in this simple way From nuclease chewing of their DNA The phage is not lucky in most every case Unless methylases win the enzyme race If that happens then, the cell gets erased That was bad. You can tell I’ve never sung that song before. God, that was awful. You know the worst thing is, is that you videotape this and it will be
on YouTube forever. Oh well. I’ve done stupider things I think on YouTube. The last thing I’m just going to mention briefly, I’m not going to go through any mechanism because I think it’s really more than we need to be going through and doing. But they’re just a group of proteins that are very important in what they do. And I’m going to just going to say a couple brief words about them. Myosins are proteins that are involved in translating ATP energy into movement. That’s what they are there for. I’m moving around up here because of myosins that are translating ATP energy into movement. My legs muscles are contracting thanks to
myosin. And that’s a very, very important consideration
for, obviously for animals. And we see myosins occurring throughout all
of biology. Even single celled organisms like slime molds
for example have myosin within them. I’m not going to say anything about them because I later will talk, and it will actually
be next term, but I’ll talk about enzymes called ATP binding
enzymes that have specific structures that enable
them to work. These enzymes have what are called P-loops and they’re very specific structures for holding and manipulating ATP. So we are going to save that until next time. So what I’m going to do at this point then
is move forward and talk about catalytic strategies. So were getting deeper and deeper, as you
can see, with the reactions that we’ve been talking
about. We first talked about general things about
protein structure. Then we talked about mechanisms by which enzymes
have acted. That’s been the past couple of lectures. And now we’re going to turn our attention to controlling enzymes. Because as I’ve alluded to earlier enzymes are able to catalyze things so rapidly that cells need to make sure that they don’t
let too much product or too much substrate be used up. So they have to control those and so they have some very interesting elaborate
schemes that will see for that regulation. If you want to understand how cells do what
they do. This is going to be the most important thing. The control of enzymes. Whether it’s the way enzymes work after their
made or the determination about whether or not
they are made, really are how cells function. This is where it’s all at. So this term we talk about the ways in which the enzyme is made after it’s been
synthesized. Next term we will talk about the ways in which the enzyme synthesis itself is controlled. That’s called gene expression and we’ll talk about how that’s done. So for right now we are going to talk about how the enzymes are controlled once they’ve
been made. Well last-a couple lectures ago I gave you
a term that I’m going to give you a lot more detail
here. It was called allosterism. Anyone remember what allosterism meant? Anyone memorize that definition for the exam? It’s one of my most common exam questions. I didn’t ask you but I almost did. Is that you? Oh, you’re not doing it, okay. So they are-allosterism itself, your on the
right track, enzymes have binding sites on them for molecules. We can think about these occurring in two
ways. One we can think about binding sites for the molecules enzyme acts on. And the other is we can think about binding
sites on the enzyme for molecules that bind that
affect the enzyme. That it doesn’t act on. So allosterism is actually the latter. That is that allosterism, I’m going to give you a definition again because I think it is a very important definition. Allosterism is a process whereby a small molecule binds to an enzyme and affects
its activity. Process whereby a small molecule binds to
an enzyme and affects its activity. That effect can be positive. That effect can be negative. Yes, question? What’s the difference between cooperativity
and allosterism? Good question. Cooperativity is what we refer to in the case
of hemoglobin where we refer simply to the binding of what the protein binds to. They’re different colors of the same thing. But we’ll see allosterism can actually work
with molecules besides what the enzyme binds to. That’s what allosterism-how it really differs. We will see that in just a minute. Allosterism is pretty critical. Now as I go through this I’m going to say
some things that are going to be inaccurate. You like this? Before I get started I’m going to tell you something that’s inaccurate something that’s
wrong. That inaccuracy is that these this mechanism I am talking about allosterism is not an on-off
switch. But I’ll talk about it like it is. I try not to do that, but I want you to remember when we talk about allosterism controlling an enzyme we think about it in simple terms. The enzyme is turned off or the enzyme is
turned on. It’s much more like a radio whose volume is
turned loud or whose volume is turned soft. It’s more a matter of degree when we talk
about allosterism, yes? His question is: are the small molecules that
bind to enzymes that cause the enzyme to be more active known as coenzymes, and answer is no they
are not. Coenzymes are separate things. The classic enzyme we use to describe allosterism is called ACTase. We will talk a lot more about it next term but I’m going to introduce it to you this
term. ATCase I’ll give you the long name for it, which you won’t need to know, but ATCase stands for aspartate transcarbamoylase. For what that’s worth. Yeah don’t you like that? What does it do? That is the most important thing to start
with. Well aspartate transcarbamoylase or ATCase- I’ll call it ATCase because it makes you laugh if I say the long way. ATCase combines two small molecules in together to make a bigger molecule. One of those molecules is aspartate enzyme- I mean the amino acid aspartate. That’s where the enzyme-the A part of the
enzyme gets its name. There is aspartate. Aspartate is-now I’m going to embarrass myself, let’s see which one it is. It’s this guy here. Aspartate is one of the substrates for the
enzyme. The enzyme takes these two guys, puts them
together, and makes a bigger molecule. That’s what ATCase is doing. Well why is this important? Well this enzyme is catalyzing the first reaction
in a pathway that leads to the synthesis of the nucleotide
CTP. Now notice what I just said I said it catalyzes the first reaction in
a pathway that leads to the synthesis of CTP. It is only the first reaction and notice it does not catalyze the synthesis
of CTP. CTP happens only after several more steps. So if I ask you on an exam does ATCase synthesize
CTP? I hope you will tell me no it does not because
it does not. It synthesizes a molecule that later, after many steps can be converted into CTP but it does not catalyze the synthesis of
CTP. Now why am I being picky about that? Well I’m being picky about that because you’re looking for the very first
time what a metabolic pathway is like. When we talk about a metabolic pathway we talk about a series of steps that lead
to a final product. All the things we talked about so far have been this goes to that. We saw a serine protease took a protein and
it cut it in half. We saw a restriction enzyme took a DNA and
it cut it in half. We saw carbonic anhydrase took water and CO2 and put them together. Each of those was once step. A metabolic pathway would take the product
of one step and use it as the substrate for the next step, and then the next step, and then the next
step. So the product of one reaction becomes the substrate for the next reaction, which creates a new product, which now is the new substrate for the next
one etcetera, etcetera, etcetera. Well that turns out to be very, very convenient and controllable. Very convenient and controllable, how so? Well it turns out that the end product of
this pathway is the nucleotide CTP this is the last thing that’s made at the end of this pathway. Cells are very careful in how much of any given nucleotide they make. We will see this next term. I’ll tell you next term repeatedly that cells have greater controls on how much
of each nucleotide they make than anything else they have inside
of them. Why do they care about how much of any given nucleotide they make? The answer is that if they make too much of
a given nucleotide they are much more prone to having mutations
happen. They are much more prone to having mutations
happen. So the best defense against mutation is keep everything balanced properly. Cells don’t want to make too much CTP, just like they don’t want to make too much
GTP, and they don’t’ want to make too much ATP. Making too much or too little of something is going to favor mutation. Look at what the CTP does, you see this little
red arrow, this little red arrow up here is to indicate that CTP binds to ATCase and turns it off. Which means it turns its volume down. It slows the enzyme way, way down effectively
turning off. That’s brilliant. Why is that brilliant? Because by controlling this one enzyme this product will be made in very tiny amounts. Which means that this product is going to
be made in very tiny amounts etcetera, etcetera, etcetera. I control one enzyme, I control the whole
pathway. Control one enzyme I control the whole pathway. That’s really powerful. What I’ve just described to you is the phenomenon we call in biochemistry feedback inhibition. Feedback inhibition. Feedback inhibition occurs when the end product
of pathway inhibits an enzyme at the beginning of the
pathway. Molecule made at the end of pathway inhibits
an enzyme that catalyzes a reaction at the beginning
of a pathway. So if CTP concentrations start getting high, it turns its own synthesis off. That’s pretty cool. It’s a very nice but simple balance. Too much CTP pathway stops too little CTP
pathway starts, yes? The amount of CTP will control whether the
ATCase is active or inactive or loud volume or low volume. There is one thing controls this enzyme. This enzyme is interesting because there’s actually three things that
control it. Second thing that controls it is another nucleotide
ATP. Those of you who remember your nucleic acids
CTP is a pyrimidine. What is ATP? It’s a purine. When we think about the structure of DNA how
on a paired? Purine to pyrimidine, right? If we too much CTP, we have too many pyrimidine’s,
right? Turns off its own synthesis. What if we have to much ATP? It means we don’t have enough pyrimidine’s. ATP turns on the synthesis of pyrimidines. That’s cool. We start to see the balance that cells can
exhibit. We start to see the balance our cells can
exhibit. That’s really important. ATP turns it on CTP turns it off. I said three things that affect this enzyme. The first to bind to sites that are apart
from the active site. They are not anywhere near the active site, we’ll see that next time. The third molecule actually is found at the
active site. It’s found at the active site. What do you supposed it is? To be found at the active site- this is a
good exam question- to be found active site what would it have
to be? It would have to be a substrate right? What’s the only substrate I’ve given a name
to so far? Aspartate, bingo! I always give you enough information answer
a question. You guys had enough information, you didn’t
realize it. Aspartate will activate the enzyme also. Aspartate activates the enzyme. Alight that’s a good place to stop. I’ll talk more about that next time. See you guys on Friday. We have a surprise Friday by the way. [END]

4 Comments

  • Reply Jose Alfonso February 20, 2014 at 8:15 am

    Awesome Vids!! Thank you for posting them on youtube,,they're pure gold

  • Reply kevinyang101 February 26, 2014 at 9:11 am

    I love the song Kevin.
    It acts as a really good summary to the lesson.

  • Reply You Lv October 16, 2014 at 10:51 am

    Thanks Kevin! Your lecture is great!

  • Reply Dominique Cutler September 16, 2017 at 7:45 pm

    Awesome lecture, information passed on very well!

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