Review Session for Exam #2 for Kevin Ahern's BB 450/550


Uploaded by oharow on 15.11.2011

Transcript:
Kevin Ahern: Good evening, everyone.
Student: Good evening.
Kevin Ahern: [high voice] Good evening!
Student: Are you going to put another example Exam 2 up?
Kevin Ahern: I am not putting an example Exam 2 up, no.
I think people study those too much.
There was accidentally one up there and I found it
and I yanked it because I think people waste
their time doing that.
It's way better to spend the time studying the material than
I know you've heard this before,
but I'll say it againóthan studying old exams.
I spend more time dealing with questions that I haven't
talked about this term on old exams than
I spend on questions that would be relevant.
So it's not worth your time.
The format will be exactly the same as the first exam.
The only reason I show you the first one is to give you
format things and that's basically it.
Blah, blah.
How are you guys doing?
Student: Pretty good.
Kevin Ahern: How's the studying coming along, it's a little early?
Kevin Ahern: Studying, what are your approaches?
How are you studying for this material?
Student: Note cards.
Kevin Ahern: Note cards, I really recommend note cards.
Writing down note cards really is a good way to go, and,
especially the further you get into metabolism,
you're going to find that note cards
are really your best friend.
Putting things on note cards helps you because
writing things down really helps put it in your brain.
I discovered that when I was in graduate school.
I waited until I was in graduate school before
I discovered it, but I discovered it and it was really good.
Writing it down really is a very,
very useful thing for you to do.
I haven't written the exam yet.
I haven't even thought about the exam yet,
to be honest with you, so I'm pretty much an open mind.
You guys can convince me what to put on this exam, I suppose.
The format, as I said, will be exactly like the last exam.
The point distribution may be slightly different.
They vary sometimes from one exam to the next.
But for the most part, the exam will be,
not "for the most part," the format will be the same.
Like I said, point values may change slightly,
but other than that, there shouldn't be any change.
Content will change, of course.
But I do, as I've said before,
work from my highlights as a way of writing my exam.
So look at my highlights.
Look at my lectures.
If I talk about it in class, it's fair game.
If I don't talk about it in class,
then I'm not going to ask you.
I will challenge anybody to find a single question
on my exams where I have not talked about it in class.
I try to be very careful to do that.
I don't set out to trick you.
I really have no intention of tricking you.
My end aim is in determining your level of knowledge,
so I really want to make sure that I do that.
All right, so this is going to be like the last review session.
I will be available for questions and let you guys have at it.
So what are your questions?
Neil?
Student: In glycolysis, you talked about aldolase,
the reaction that it catalyzes,
as having a very positive Delta G zero prime.
So I was wondering, just to clarify,
the way that it still makes a forward reaction is because
the enzymes that precede it vary the concentrations up?
Kevin Ahern: So Neil's question has to do with
the aldolase reaction, and basically,
how does the cell manage to make that reaction go forward,
in view of the fact that it has a very positive
Delta G zero prime?
Let's take a look at that reaction.
Here's the reaction that's relevant.
The Delta G zero prime for this reaction is something
like plus 20 kilojoules per mole,
and that's a very large positive energy barrier
and yet this reaction goes forwards.
I think that when we think about Delta G,
it's always important to remember that Delta G consists
of two components: a constant, which is Delta G zero prime,
which in this case is plus 20, and a log term relating
to the concentrations of reactants and products.
So the log term is the concentration of products
divided by the concentration of reactants.
For Delta G to be negative, and given the fact
that we have a Delta G zero prime that's very positive,
the only way Delta G can be negative
is if we have that log term be negative.
For that log term to be negative, the only way that
can happen is if we have small amounts of products
and we have large amounts of reactants.
Somebody in class said, "Well, those Delta G zero primes
of the earlier reactions help to push that."
In a sense, they do, because they do favor making
a lot more of reactant, which is this guy, right here,
and the reactions after this are very
efficient at taking away products.
So when we decrease the numerator and we increase
the denominator, that makes that ratio become much smaller.
The smaller that ratio is, the more negative that log term is,
and we get it negative enough and the reaction goes forwards.
Now, I didn't get a chance to talk to you in class about
the tricks that cells use to accomplish that.
You're not responsible for it on this exam.
You are responsible for the general nature
of making that term be negative.
After, in fact, probably during the lecture tomorrow,
I will talk actually about how that happens,
but you're not responsible for it on the exam.
The exam only covers through Reaction 8 that I talked about.
Somebody said, "Well, you showed Reaction
"9 and 10 on the screen.
"Are we responsible for those?"
The answer is, "No, only the material through which
I talked about and the material through
which I gave you highlights about."
So I'm not sure if that answers your question,
but I hope it does.
Okay, let's go home then, right?
Other questions, yes Connie?
Student: For sugars, what type of ring structures
do we need to know?
Kevin Ahern: For sugars, what type of ring
structures do we need to know?
Any ring structures that I told you in class
you're responsible for.
So you're responsible for the straight-chain structures
of glucose, fructose, ribose, galactose.
You're also responsible for the ring structures of those.
Specifically, you're responsible for the six-membered
ring of glucose, the five-membered ring of fructose,
the six-membered ring of galactose,
and the five-membered ring of ribose.
Student: So we don't need to know like the furanose
form of glucose, then?
Kevin Ahern: You do not need to know the furanose
form of glucose, nor do you need to know
the pyranose form of fructose.
Yes, sir?
Student: What about sucrose?
Kevin Ahern: And you should also know sucrose, that's correct.
Sucrose only exists in the ring form...
but, yes, sucrose, you're right.
Student: To clarify, you said "six-membered ring of galactose"?
Kevin Ahern: Six of galactose, yep.
Yes, sir?
Student: In recitation, in the first week of recitation
after the first exam, they were talking about
[unintelligible]
Do we have to know how to calculate that or get that?
Kevin Ahern: That was on the last exam.
That was material on the last exam.
Student: Right, but [unintelligible].
Kevin Ahern: It'll be on the final,
but it's not comprehensive since the last.
That was material we covered for the last exam.
Unless you want me to put it on there?
Student: Could you speak about proto-oncogenes and oncogenes?
Kevin Ahern: Yes, certainly.
The question has to do with what's
the difference between proto-oncogenes and oncogenes?
The proto-oncogene is the one I always like to describe first.
The proto-oncogene is a normal gene that exists in our cells
and it plays a very critical role in controlling cells'
decisions about division, for example.
And I say, A, that it turns out that there are about several
hundred of these that play very, very critical roles.
Epidermal growth factor receptor,
for example, is a proto-oncogene.
Now, if the epidermal growth factor receptor
doesn't communicate information properly
let's say it gets left in the "on" state,
where it's constantly telling the cell to divide
then that proto-oncogene is no longer functioning normally
and that proto-oncogene that does not function normally
is known as an oncogene.
And the reason it's known as an oncogene is the term
"oncogene" means "cancer gene."
By far, the most common way in which a proto-oncogene
is converted into an oncogene is by mutation.
So it takes mutation to convert a proto-oncogene
into an oncogene, and there are hundreds of examples
where that can happen.
It's because there are so many oncogenes
that there are so many different kinds of cancer.
There are so many different ways that we can screw up
the system that we don't have one type of cancer.
We don't have one cure for cancer because there
are just so many ways in which the signal
or the information can be screwed up.
Does that help?
Student: Yeah, thanks.
Kevin Ahern: Neil?
Student: So when you speak of a mutation, you're saying that,
once the cell manufactures the receptor,
it's a messed up receptor, basically the genetic
material that it's made from is mutated?
Kevin Ahern: This is related to the proto-oncogene
that you're talking about?
Kevin Ahern: Okay, so what I said was,
if the proto-oncogene is mutated
such that it's not communicating the signal properly,
and the example I gave was where it was communicating
a constant signal to divide, like constantly, for example,
the receptor might mutate to the point where it is always acting
as if it's bound to epidermal growth factor.
So if it's always in that same conformation that
it would be in as if it had a epidermal growth factor,
then that's part of that signal that normally would tell
the cell, "Now is the time to divide."
But if it's stuck in that mode,
it's always telling the cell to divide,
and I'm telling you that will happen as a result of mutation.
So I'm not sure if that's answering your question again,
but I hope that's what..
Student: In the aspartyl proteases,
what causes the water to detach and act as a nucleophile?
Kevin Ahern: What causes, in the aspartyl proteases,
what causes the water to detach and act as a nucleophile?
Okay, so let's go back and take a look at those,
and I'll show you the aspartyl proteases.
There's the aspartylóoh, that wasn't what I was looking for.
The mechanism that they use to do their strategy is here.
So his question is, right here, this water,
how does it become a nucleophile?
The answer is that it has to be activated,
just like every other nucleophile that we saw.
So when I showed you the serine protease,
we had that hydroxyl group hanging off of serine.
The hydroxyl group of serine is not active.
Only when that proton gets pulled off
and we make an alkoxide ion, then it's a nucleophile,
and then it attacks the carbonyl group.
We saw a similar thing when I had the cysteine proteases.
We had the SH and that H got pulled off by
the histidine and made an S-minus, and that was a nucleophile,
and that attacked the carbonyl bond.
Well, here, water itself is not a nucleophile.
It has to be activated, and the way it gets activated
is exactly what's happening right here.
This carboxyl side chain of one of the carboxyl groups
is abstracting or taking this proton away from the water.
That leaves a negatively charged hydroxide behind,
and that negatively charged hydroxide is a nucleophile.
It goes right straight for the carbonyl group,
just as the other ones did, and attacks it.
One of the big differences between this and
the serine proteases and the cysteine proteases is that
this OH is not attached to anything else.
It's not attached to the enzyme.
In the case of the alkoxide ion,
the oxygen was attached to the enzyme.
In the case of the cysteine proteases,
the sulfur was attached to the enzyme.
This guy is just floating freely in space.
So this was the example I gave you where I said
this activation and this attack does not take
a fast step and a slow step.
The slow step in the other ones required water
to come in and release that thing.
This water is actually doing the whole thing right here.
So basically it's a one-step process
that is breaking that peptide bond.
Make sense?
But activation, that's the key to your answer...
the answer to your question, I should say.
Connie?
Student: So it breaks the peptide bond by
the oxygen's electrons come back and just destroy
that bond itself?
Kevin Ahern: The oxygen here is a nucleophile,
meaning it has extra electrons and it's seeking the nucleus.
The nucleus is right there.
That causes electronic rearrangement that breaks
that peptide bond.
Do we need to know the further mechanisms
of how exactly that bond is broken?
Kevin Ahern: Well, I haven't shown you in class anything
about that, beyond the fact that it attacks that bond.
If you recall, when I talked about the serine proteases,
I talked about making the unstable intermediate
that gets stabilized by the oxyanion hole?
Kevin Ahern: That's as much as I've said about the mechanism.
Student: Is activation always from removing a proton?
Kevin Ahern: Is activation what?
Student: Is it always caused by removing a proton?
Kevin Ahern: For all the processes I've talked
about in the proteases, yes.
There are other ways of activating molecules,
but for the ones that I've talked about here,
in each case it has been removal of a proton,
whether it was from a serine side chain, a cysteine side chain,
in this case, removal from a water.
You've got a good stack of note cards there, Jerrod.
Student: Yeah, I do.
I do have a question.
I'm just trying to remember all the points before
I embarrass myself.
Kevin Ahern: I won't embarrass you.
The ATCase, on your high lights it says
you want us to know how ATP, CTP and aspartate affect it?
Student: Okay, So how does aspartate affect it?
Because I was reading that to say
it starts to favor the relaxed state?
Is that just when it's by itself?
Kevin Ahern: His question is a point that I made
in the highlights about, for ATCase you should understand
the effects that ATP, CTP and aspartate have on the enzyme.
Basically, what I talked about in class was that,
first of all, this enzyme was a great example
of allosterically regulated enzyme,
and this allosterically regulated enzyme is regulated
by those three compounds in the cell.
So when we look at the structure of ATCase,
we discover it has 12 subunits 6 regulatory
and 6 catalytic subunits.
And we describe two possible structures that the enzyme
can exist in: an R, or relaxed state; or a T, or a tight state.
These R and T forms that we talk about here correspond
exactly to the R and T we saw with hemoglobin.
In the case of hemoglobin, the R state
favored the binding of oxygen.
The T state favored the release of oxygen.
In the case of this enzyme, the R state favors the binding
of substrate, which is necessary for the reaction to occur.
The T state disfavors the binding of the substrate
which is necessary for the reaction to occur.
Now, getting back more specifically to your question,
ATP and CTP both affect the enzyme by binding
to the regulatory subunits.
ATP favors the formation of the R, or the relaxed, state,
which will favor the activation of the enzyme.
CTP favors the T state, which is the tight state,
which disfavors the binding of substrate.
Remember, again, these are not on/off switches,
but up or down.
And then the more specific question you had was aspartate,
how does aspartate affect it?
Aspartate does not bind to the regulatory subunits.
Aspartate is a substrate of the enzyme,
and it also favors the formation of the R state.
So sufficient aspartate will flip it into the R state.
It's independent of anything else that's there.
It's independent of anything else that's there.
It doesn't require anything else to be there
to flip it into the R state.
Student: Can you go over PALA?
Kevin Ahern: PALA, yeah, P-A-L-A.
PALA is another molecule I talked about relative
to aspartic acid... I'm sorry... relative to ATCase.
PALA is basically a suicide inhibitor of the enzyme
that looks like aspartate.
It sort of looks like aspartate.
The enzyme will bind it, but in the process of binding it,
it becomes covalently linked to the enzyme.
So PALA, as I emphasized in class,
is not a natural substrate of the enzyme.
It's a man-made molecule, and this man-made molecule,
when it binds to the enzyme
will lock the enzyme in the R state.
Now, that's interesting because it's covalently bound.
It's locked there.
You say, "Well, if it's in the R state, and it's bound,
and it's suicide, the enzyme's not active,
what does that mean?"
Remember that R state and T state refer to structures,
R being the relaxed, T being the tight.
So I can still talk about the R state structure,
even if the enzyme is dead in the water.
Let's imagine the R state's relaxed, it's all nice and big.
the T state, it's all compact.
So if I have a PALA that binds to the enzyme
and flips it into the R state,
the enzyme is in this big, relaxed form.
It just can't do anything.
So I can distinguish the structure of the enzyme
from the thing that the enzyme is actually doing.
It turns out that PALA actually was very valuable
for distinguishing these R states and T states.
When we see PALA working the way that it does, it tells us,
"Ah, now I understand why aspartate has
the effect on the enzyme that it does."
Does that answer your question?
Other questions?
Student: In the insulin receptor, what has the SH2 domains?
Does IRS-1 have an SH2 domain or...?
Kevin Ahern: Let's go to the figure and
we'll answer that directly.
You can actually see it in the figure, but I will show you that.
So, let's see, insulin, it's right here.
Signaling, right here.
Anything that's bound to a phosphotyrosine,
as you see on here, has an SH2 domain.
That's an SH2 domain, right there,
because that's a phosphotyrosine.
That's also a phosphotyrosine and that's
an SH2 domain, right there.
Your book confuses things a little bit because
they talk about an SH3 domain.
An SH3 domain is just another binding domain,
but it doesn't bind to phosphotyrosines.
It binds to something else.
Student: I was under the impression [unintelligible].
Kevin Ahern: I'm sorry, I can't hear you.
Student: I was under the impression [inaudible]
PIP-2 to PIP-3.
Why is IRS-1 also bound to PIP-2?
Kevin Ahern: Why is IRS-1 bound to PIP-2?
It just happens to bind to it.
Student: It doesn't [unintelligible]?
Kevin Ahern: And there's plenty of PIP-2 here in the membrane.
So you can think about it as an anchoring thing.
You can think about it as anchoring, helping to anchor it.
Student: Can you talk about calcium's role...?
Kevin Ahern: In signaling?
In signaling, you're talking about?
Student: I guess with the prothrombins and how they're related?
Kevin Ahern: Okay, in prothrombin.
That's the other place I've talked about calcium.
So, yes, I'll be happy to.
I think it was right here.
Student: [unintelligible]
Kevin Ahern: I'm sorry?
Student: [unintelligible]
Kevin Ahern: I can't hear you.
Student: [unintelligible]
Kevin Ahern: Say it again.
I still can't hear what you're saying.
Allostery in regulation?
if we go to the blood clotting scheme
and I talk about prothrombin,
prothrombinóokay, let's look at
the big picture and then we'll come to prothrombin.
The bigger picture is that we have this scheme
of blood clotting that I showed.
People asked me, "Do I have to know what the intrinsic
pathway is versus extrinsic pathway?"
No, I haven't talked about that in class.
What I focused on was everything down here.
Basically, these two pathways can be activated
by different processes that are happening
inside of our body...
damage, bruising, cutting, whatever.
So these processes get activated.
We have two ways of starting this cascade,
and the cascade terminates down, in here.
So the aim of these two pathways is to, first of all,
convert prothrombin to thrombin, which, in turn,
converts fibrinogen to fibrin.
Now, the relevance of prothrombin to calcium is that,
in order for prothrombin to be held at the site
of the woundóremember, we've got prothrombin floating
through our bloodstream all the time.
We cut ourselves, that's where we want
our prothrombin to certainly be.
We want it to accumulate.
We want it to do its thing at that place because
that's the place where we want the clot to occur.
So we want to have something that
ó kind of like the 2, 3 - BPG tells the body
where the metabolism is happening rapidly.
we want to have a signal
to tell the prothrombin where to go.
In order to do that, we have to modify prothrombin.
So prothrombin gets modified by an enzyme
that uses Vitamin K.
That enzyme that uses Vitamin K causes prothrombin
to get an extra carboxyl group on it.
I'm going to come back to this figure in a second,
but first I want to show you what's happening
in this modification.
In this modification, here is the side chain.
Here's prothrombin.
Here's a side chain of a glutamate.
Prothrombin has several glutamates
and they can each be modified with
this addition of a carboxyl group on the end of the side chain.
The normal carboxyl of glutamate is just the stuff in black.
The additional molecule is this guy, right here.
So the addition of this guy
requires an enzyme that uses Vitamin K.
So Vitamin K is going toóit's called a "pro clotter"
because of this modification.
We'll see the importance of this modification in just a second.
Now, the addition of this second carboxyl group causes
this end of prothrombin to recognize and bind to calcium.
Calcium is abundant at the site of the wound.
Now we've got a way of attracting and holding onto
prothrombin at the site of the wound.
So when we've got prothrombin at the site of the wound,
the place where this conversion is going to occur
is at the site of the wound.
So now we've got thrombin active.
To get thrombin active, we convert fibrinogen into fibrin,
and fibrin, of course, is the material that makes the clot.
So that activation of prothrombin is a very necessary step.
If we inhibit the action of Vitamin K by using a blood thinner
like warfarin or something like that,
then prothrombin doesn't gain a carboxyl group.
Therefore it never gets attracted to the site
of the wound and our blood has a hard time clotting.
So that's how a blood thinner works,
or how one blood thinner works, anyway.
Does that answer your question?
Student: Are both hard and soft clots watertight?
Kevin Ahern: Are both hard and soft clots watertight?
I've never tried to blow water through one, so I don't know.
I would guess that a soft clot would probably
be less watertight than a hard clot would,
but I honestly don't know the answer to your question.
They're fairly watertight.
I mean, they've got to stop that flow fairly quickly,
so I would say yes,
but they're not going to be as good as a hard one.
Student: How are hard clots formed, again?
Kevin Ahern: How are hard clots formed, again?
Hard clots, okay, the difference between a hard clot
and a soft clot,
I'm going to have to show you the figure for the polymerization.
the difference between a hard clot and a soft clot is that
the soft clot simply involves inserting these betas into the Bs
and the alphas into the gamma sites on the fibrin.
So we see this network of polymers that form.
But these are simply quaternary interactions.
They're not covalent.
Remember, quaternary interactions are things
that generally involve hydrogen bonds.
They may involve hydrophobic interactions and so forth.
But in this case, there's nothing here
that involves covalent interaction.
Covalent bond formation will nail those guys together.
So to put these guys together, there's an enzyme
called glutaminase that will covalently link the side
excuse me, the side chains of glutamine and
I'm going to forget it off the top of my headóand lysine.
I thought it was lysine.
Put glutamine together with lysine to make a covalent bond.
Now, these are happening
in all kinds of places throughout this network.
So it's not just here, it's not just here.
But anytime these guys are in close proximity,
if there's a lysine next to a glutamine,
they're going to get covalently bonded together,
and that covalent bonding of them together
now forms the hard clot.
Clear as mud?
Clear as mud.
Student: Just a question about the format of this exam.
Will there be more questions on this exam than the last one?
Kevin Ahern: So the question is, on the format of the exam,
will there be more questions on this exam than the last exam?
I would say, probably not.
What are your thoughts?
Student: It just seems like there's a lot more
material for this exam.
Kevin Ahern: It seems like more material than the last exam?
Do you want more questions?
Kevin Ahern: No, I mean, seriously, because if I have
more questions, then each one's worth fewer points.
So, I mean, I'm not asking the question to be silly.
Student: We still have only 50 minutes.
Kevin Ahern: You still have 50 minutes.
What I was very pleased about on the last exam
was it's one of the few first exams where I had very few,
relatively few people, saying they had too little time.
Usually the first exam in 450 is the only one of the exams
I give in 450 and 451 where people complain about the time.
So I don't think time will be a factor for you on this exam.
I hope not.
Student: So back to [unintelligible] maybe I just missed it.
Student: But does the calcium activate the prothrombin or..?
Kevin Ahern: Does calcium activate the prothrombin?
No, It's just there to attract it to
the site of the wound.
Student: And it just activates itself?
Kevin Ahern: No, remember those other pathways are converging
to activate the prothrombin.
So that's what's activating the prothrombin.
Student: So the converging of the pathways
activates prothrombin?
Kevin Ahern: Thats right, it's not the calcium.
Calcium doesn't activate the prothrombin, no.
And keep in mind, when I was talking about this material
and the blood clotting, we're talking about activation
of zymogens, and every zymogen I've shown you is activated
by breaking peptide bonds.
That's what's happening all the way down that scheme.
Yes, Connie?
Student: For the catalytic triad,
how does aspartate make histidine more negative
[unintelligible] to tear that proton off the serine?
Kevin Ahern: So She's asking about the catalytic triad
and the role of aspartic acid in making
histidine more negative, is the way I described it.
So let me show you what happens with that.
if I can pull it down here.
So, catalytic mechanisms, and catalytic triad.
Here's the catalytic triad in the active site
of chymotrypsin or as it would look in the active site
of any of the serine proteases.
How is this guy over here affecting things
that are happening all the way over here,
is basically what you're asking.
As I noted in class, what happens is the binding
of the proper substrate...
I'm having more trouble with this thing.
That's too bad, i like this.
There we go.
The binding of the proper substrate to the enzyme
causes a slight conformational change.
Everything we've talked about with respect to proteins
this term has been slight changes that happen,
and these slight changes are very subtle.
You saw in the case of hemoglobin how that very subtle change
caused the oxygen-binding affinity to go way up or way down,
depending upon which direction it went.
The slight change in shape that happens here
is seen in the active site.
In the case of the catalytic triad,
this aspartic acid is moved closer to the histidine.
Aspartic acid is negatively charged.
Histidine is what I like to describe as
a "sink" of electrons.
These electrons are resonant, almost.
And so you put something negatively charged on
this side of the sink, what's going to happen to the electrons?
We're going to be much more likely to find
them on this side than we are on this side.
Negatives repel negatives.
So when that happens, this side of the histidine
becomes relatively more negative.
That makes it much easier to pull off this positively
charged proton from the side chain of serine.
That's what's happening in this process.
It's actually the proximity, the closeness of this guy
that's affecting this overall movement of electrons.
Student: Can you go over, just you talked about why
this is affected when you remove,
when you disable all three instead of just one?
Ahern: Okay, sure.
Let me answer this question, I'll come back to that.
So did that answer your question okay?
Yeah, that experiment's an interesting one I think.
And it may be a little hard for students to understand.
I'm happy to explain that to you.
Here, so this is an enzyme know as subtilisin.
Someone asked me the other day, 'do I have to know subtilisin?'.
No, it's just another example.
It could be just serine protease in general.
So this would be true for essentially any serine protease
that we happen to examine.
What this experiment concerned was trying
to determine the relative importance of each
of the members in the catalytic triad.
Serine, histidine, aspartic acid.
So using genetic techniques,
it's possible to make mutations that affect
only one of those amino acids.
Then you can collect that protein and see,
'hey, how active is this protein?'
Alright, so you start with wild type protein, unmutated.
You measure a certain Kcat.
So this guy's got a pretty good Kcat over here.
We see there is the Kcat of this wild type protein.
We get over here and we examine the first mutant protein.
This is a mutant protein where the serine
has been mutated to aspartic acid.
We noticed that in every mutation they make,
the amino acid got changed to aspartic acid.
So again, we're reducing the number of variables,
we're always making the same relative change.
We're making everyone into an aspartic acid.
If we examine the activity of this enzyme,
and the only mutation that's happened is we converted
the serine in the active site to an aspartic acid,
we see the activity go down by one, two, three,
four, five, six, almost seven orders of magnitude.
That's a log scale.
That means that this guy right here
is one ten millionth as active as this guy.
That tells us that aspartic acid is pretty darn important.
The same thing happens if we mutate
a histidine to an aspartic acid.
The only mutation in this enzyme is this histidine
to this aspartic acid.
We see it also goes down about 7 orders of magnitude.
The serine and histidine play essentially
equal roles in that catalytic process.
If we mutate only aspartic acid,
we still see a pretty good drop,
but we don't see a drop as deep as this.
And the only point of this one is to show us that,
while aspartic acid is important,
certainly we see a difference from here to here,
that's about 1, 2, 3, 4, 5 orders of magnitude maybe,
maybe 100,000 fold reduction in activity,
but not as much as these guys.
It says that aspartic acid isn't as critical in that process
or in that catalytic action as these two are.
Now we look at over here and we say okay,
here's the mutation of all three of these,
how come it's not even any lower than this?
And the answer is that these guys, any one of these is deadly,
essentially as far as the enzyme is concerned.
It doesn't take it all the way down to the uncatalyzed.
And why did I say that happens in class?
Anybody remember?
What else are factors in this catalytic process?
Student: I mean the overall structure is...
Ahern: The overall structure of the enzyme
is still pretty much there.
That tells us there's something in the structure
that's still playing a bit of a role in that catalytic process.
We're not destroying the structure of the enzyme in doing this.
But essentially, these are all pretty much dead in the water.
This is one ten millionth of activity of the wild type.
Does that answer your question?
Student: Yeah, I guess I just thought it would
be a little bit lower, serine and histidine are the same.
Ahern: Well it may in fact be a bit lower.
So it's a little hard to look on a bar graph like that and see,
and remember we're looking at a log scale,
so a little bit lower would be hard to see on there.
We're not going to see 7 orders of magnitude again, no.
Neil?
Student: Does that mean that the most important aspect
of the catalytic role is serine?
Ahern: I'm saying this tells us that serine
and histidine have equal roles.
Right?
Either one knocks it down as low as the other, as all three do.
Yes, Elliot?
Student: Are all of these changed to alanine?
Ahern: In this case, each one is being changed to alanine.
Keep in mind this is one enzyme,
this is a different enzyme, this is a different enzyme.
So only in this enzyme do we change all of them to alanine.
Yes?
Student: I have a question about epidermal growth factor.
Ahern: Epidermal growth factor, okay.
Student: [inaudible]
Ahern: Okay, let me pull that up before
I get going to your question.
So, epidermal growth factor.
Right here.
And EGF is, oh, wrong one.
I do that every time.
How about...no.
How about, where did I do that?
Is it here?
No.
Well now, come on.
Not there.
It will be the last one I get.
Da Dum.
All right.
Back to your question.
Student: [inaudible]
Ahern: I'm sorry, the SOS you're talking about?
Student: The SOS, yeah, is that attracted to Grb-2
or does Grb-2 have an attachment
to tyrosine or [inaudible]?
Ahern: I'm not sure I understand your question.
Are you asking if this is coming in as a group into this?
Student: Yeah, is it coming in as one big group
or does Grb-2 come first and [inaudible]?
Ahern: I can't answer that question.
I don't know the order.
It would be safe to assume for our purposes it's sequential.
But I mean it's possible you could get 2 coming in at once.
I don't know the answer to that question.
Student: And the other question was [inaudible],
how does that cause the internal section to become [inaudible]?
Ahern: Okay, so the question is the dimerization of this,
how does this favor the activation of the tyrosine kinase
activity I think is what you're asking, right?
So this is very much like what we saw
in the case of the insulin receptor.
The only difference was the insulin receptors start out
as a dimer and the two ends were basically stuck
in each other's active site, blocking them.
The slight shape change allowed one of them
to get a little bit further out and get phosphorylated.
It's a very similar mechanism that's happening here.
When these two guys come together, this guy's face
gets stuck in over here and it gets phosphorylated
which in turn causes it to start phosphorylating this.
So it's very similar what's happening with the insulin receptor.
One activating the other and then multiple
phosphorylations happening as a result of that.
Okay?
Student: How is the Grb-2 attaching to a [inaudible]
changing SOS [inaudible]?
Ahern: Yeah, okay, as you can imagine,
there's a lot of complexity in here.
So his question is how can I explain how this binding
of this thing causes this thing to change shape
which causes this thing to dump its GDP and get to GTP?
It's really, it's a schematic diagram is what it is.
It's not unlike the schematic diagram we used to show
the activation of the GDP with the adrenergic receptor.
I showed you the sort of black box and I said then
this dumps its GDP and puts a GTP in there.
The same thing is happening here.
That's really beyond what we can cover here.
But it is, all these things are happening
as a result of these slight shape changes.
And that's the theme we come back to over, and over, and over.
Slight changes in the shape of the protein really affect there.
Yeah?
Student: Other than the dimerization,
is there any significance in the fact
that two EGF have to bind?
Ahern: Her question is, she said other than the dimerization,
is there any significance to the fact that 2 EGFs have to bind?
The significance is these don't exist as a dimer
in the cell so obviously in order to make that dimer,
we have to have EGFs bind.
One to each one.
Student: I was just wondering if concentration mattered.
Instead of together, one molecule binds?
Ahern: Her question has to do if concentration matters.
It turns out it's actually a very good question.
Since you asked it, I'll very briefly tell you a story.
Your book doesn't cover it this time
which is why I didn't go into it.
You're not responsible for this, I'll just tell you,
but it's kind of a cool story.
There's a related receptor that's very similar
to the epidermal growth factor receptor called HER, H-E-R.
And it resembles this EGF.
And in fact, it resembles it enough that HER
can bind to EGF without needing epidermal growth factor.
Normally in the cell, probably at a low rate,
HER occasionally binds to EGF,
it stimulates the response and the cell divides
and everybody's fine and happy.
Mutation of the promoter of HER
causes HER to be overproduced in some cells.
So when this happens, you've got way too much HER
and HER goes around grabbing all the epidermal
growth factors and starts stimulating all of them
and starts stimulating cells to divide uncontrollably.
So this is an example of an oncogene.
HER is an oncogene that will cause that to happen.
So we're just making too much HER.
It's kind of like when me made too much bcr-abl.
Making too much HER and the cell is stimulated to divide.
Well it turns out HER is commonly stimulated
to divide in breast cancer.
And there is a very interesting and a very good treatment
for HER related tumors and there's
a monoclonal antibody called Herceptin that is targeted
specifically to bind to HER and prevent
that stimulation from happening.
Herceptin is a very, very effective anti-cancer drug.
It has very, very few side effects and for HER related tumors,
it's very effective at knocking it down
and in fact knocking it out.
And that relates directly to what you're asking
about which is concentration.
Concentration of these could be a factor.
So if we had too much epidermal growth factor for example,
we would probably have a similar problem.
Let's see, Neil?
Student: To clarify, you said there was a difference
between the structure of the insulin receptor
and the EGF receptor?
Ahern: Yes.
Student: They're both dimers in effect, correct?
Ahern: The EGF normally does not exist as a dimer.
When it doesn't find to EGF, it's a monomer.
It's floating around in the membrane,
not bound to the other one.
That's the thing I talked about when this guy binds to EGF,
this little loop flips out.
And so if it doesn't bind EGF, then there's no loop
and there's nothing to basically attract to the other one.
Student: Do you call it induced dimer?
Ahern: I'm sorry?
Student: Do you call it induced dimer?
Ahern: Okay.
I can call it Kevin.
[laughing]
Yeah.
Student: I'm kind of confused about the rad's GDP,
how it goes to GTP.
Do the Rads drop the GDP?
Ahern: Yes.
It's just like we saw in the beta adrenergic receptor
when we had the G protein that dropped the GDP
and replaced it with GTP.
Exactly the same thing is happening.
That's why it's showing you that GTP is going
in and GDP is going out.
So we're not putting a phosphate in there,
we're actually kicking out the GDP and putting a GTP in there.
Okay?
Connie.
Student: How come it's not a G protein?
Ahern: It's a G like protein.
So it's different enough from a G protein
we don't call it a G protein but we do call it a G like protein.
It's just nomenclature.
Yeah?
Liz.
Student: G proteins always have those three units
like the alpha unit, the beta unit and the gamma unit?
Ahern: Liz's question has to do with
do G proteins have an alpha, beta and gamma sub unit?
As far as I know, they do have all three.
This one as you notice, Ras doesn't have the other
two so that's why we call it a G like protein.
This would be more like the alpha unit that we saw on the G protein.
Other questions?
Yes?
Student: Can you explain restriction modification system again?
Ahern: Certainly.
Restriction modification systems.
I think restriction modification systems
are pretty cool, pretty interesting stuff.
I talked about them relative to catalytic mechanisms
and they are, most of what I had to say about
the enzymes themselves were more the importance
to them relative to bacteria than about mechanism.
I remember very briefly talking about mechanism.
But I think they're most interesting
from the perspective of what they do.
So it was related to the mechanism that I talked
about this because when I talked about mechanisms
of actions of enzymes, I pointed out to the need
to activate various things.
We saw in the case of the proteases,
we had to activate hydroxyls or serines or waters
and those activated nucleophiles then attacked specific,
in the case of protein, carbonyl groups,
and caused peptide bonds to break.
In the case of restriction enzymes, we also see activation.
We have activation of water again.
And that activated hydroxyl that arises,
attacks the phosphodiester bond.
It's not attacking a peptide bond.
The phosphodiester bond is the bond
that's between adjection nucleotides in DNA.
So a restriction enzyme has a catalytic site
that favors the activation of water
to break phosphodiester bonds.
And restriction enzymes themselves have the ability
to recognize specific nucleotide sequences in DNA,
just the same way that a protease has the ability
to recognize specific sequences of amino acids in a protein.
So in the case of the restriction enzyme,
what's happening is when the restriction enzyme
finds the proper sequence, it changes shape,
and the change of shape causes the DNA
it's holding onto to bend.
And the bending of that DNA allows a nice little pocket
for magnesium and water to be located
for water to be activated by the enzyme.
So that's the activation part of that process.
To answer the bigger question about restriction enzymes
relative to bacteria, restriction enzymes
are important as a protected mechanism for bacteria
against invasions of viruses known as bacteriophages.
So restriction enzymes are always paired in a cell
with what's called a methylase.
So they're called restriction modification systems.
The restriction part is the restriction enzyme.
The modification part is the methylase.
And what a methylase does is it recognizes
exactly the same sequence as the restriction enzyme does.
But instead of cutting it, it favors the addition
of a methyl group at a specific place in that site.
And what that single methyl group does is it prevents
the restriction enzyme from recognizing that,
therefore it doesn't bend it,
it doesn't allow that site to be cut.
So the modification system is there to protect
the cellular DNA from destruction by the restriction enzyme,
and the restriction enzyme is there
to attack invading viral DNA.
As I noted in class, it's not a perfect system.
It's not a perfect system because yes,
sometimes a virus will get methylated first
before the restriction enzyme gets there.
You can imagine situations where the restriction
enzyme might occasionally cut the bacterial DNA
and that can happen, too.
But the system is better than no protection at all.
That's what we see throughout biology.
Yeah?
Student: Does this mean the modification goes
in front of the restriction?
Ahern: His question is the modification going
in front of the restriction?
The modification is on a separate enzyme.
So it's a chance thing.
It's really a chance thing.
Connie.
Student: Does each bacterium have its own restriction enzymes?
Ahern: Good question.
Her question was does each bacterium
have its own specific restriction enzymes?
We see a lot of variation in restriction enzymes
from one bacterium to another.
Within a given species, we see limited numbers.
So in the case of the one I showed you in class,
that was EcoR5, that is found in e coli,
and e coli, across all e coli,
we find maybe 3 or 4, 5 restriction enzymes.
But if I went to something like salmonella
or I went to pseudomonas or something like that,
I would find restriction enzymes
that recognize different things.
Student: But if I just isolated one e coli bacterium,
would it just have one restriction enzyme?
Ahern: Her question is if I isolated an e coli bacterium,
would it only have one enzyme in it?
It might have as many as 2 or 3.
So it could have several within a given bacterium.
It's a small number, it's not a giant number.
Jerry?
Student: Are restriction enzymes only on the DNA
or will they be out in the cytoplasm...?
Ahern: His question is basically where
are restriction enzymes located.
So remember that we don't have a nucleus in bacteria
so the DNA is in the cytoplasm all the time,
but I think more specifically your question
is "is the restriction enzyme found only on the DNA?"
And the answer is no it's not.
It's soluble in the cytoplasm and it's floating all around.
And that's important.
Because we don't want it just sitting
there on the cellular DNA.
Because if it were when a virus comes in,
the restriction enzyme would never see
it if it were staying only with the DNA.
Connie.
Student: For feedback inhibition, do you still call
it that if the end product doesn't knock
out the first pathway, like the second or third?
Ahern: Connie's asking me a semantic question here.
The question is, I describe feedback inhibition
as the end product of a pathway inhibiting
the first enzyme in a pathway,
what if it knocks out the second enzyme in the pathway?
Well it's really a semantic argument.
The answer is in a sense, I suppose it is.
Because remember pathways,
what we define as a pathway is random.
I'm not going to split hairs like that.
Because again, what's the last molecule in a pathway?
That's also random what I define as that, right?
Yes, sir?
Student: In recitation today, it was mentioned that
they have anabolic and catabolic [inaudible].
Ahern: Yes.
Student: [inaudible]
Ahern: So his question has to do with anabolic
and catabolic electron carriers.
That's not something I talked about yet in class
but since you asked about it, I will answer it for you.
So catabolic processes of course we remember
are breaking down of large molecules into smaller molecules.
Anabolic processes take small molecules
and build them into larger molecules.
So cells get energy be catabolism,
they use the energy in anabolism, making things that they want.
So catabolic processes,
the one you've seen so far is glycolysis.
We'll talk later in the term about glycogen breakdown.
We'll talk next term about the citric acid cycle.
We'll talk about fatty acid breakdown.
And all of these are catabolic processes.
Larger molecules being broken into smaller ones.
And when that happens, there's frequently
oxidation that has to occur.
Oxidation, you recall, involves a loss of electrons
and loss of electrons, when those electrons are lost,
as I said, they don't just disappear into thin air.
Something has got to happen to them
and cells use electron carriers.
There are two electron carriers we commonly
see in catabolic processes.
NAD and FAD both accept electrons
commonly in catabolic processes.
In anabolic processes, that it making things,
like the best example I can give you
is in making fatty acids that we'll talk about next term,
we see a different electron carrier that is used primarily,
not exclusively, but primarily in anabolic processes.
And this is NADPH.
So that's what we see NADP vs. NADPH.
The NADP carriers tend to be more
involved in anabolic processes.
The NAD and FAD carrier's more involved
in catabolic process that's what they're referring to.
Yes?
Student: Can you define allostery?
Ahern: Yeah.
Can I define allostery?
I'm glad that you asked.
Allostery is the, when a small molecule binds to a protein.
Actually, I defined it in class as an enzyme
where a small molecule binds to an enzyme
and affects the enzyme's activity.
That effect can be positive.
That effect can be negative.
Depends on the molecule, depends on the enzyme.
Okay?
Connie.
Student: A follow up to that question,
does allostery ever tell you where it binds on the enzyme?
Like a regulatory sub unit or active site?
Ahern: Connie's question is actually a complicated one.
It is, "does allostery ever tell us where it binds?"
The answer is just by the kinetic data itself,
we don't really get that much information out of it, no.
We need other structural changes and other structural
information to know are there regulatory sub units,
are they separate from catalytic sub units,
and that's a more involved process.
Yes?
Student: What's the first messenger in the angiotensin system?
Ahern: What's the first messenger in the angiotensin system?
Angiotensin.
Yep.
And you're not responsible for that.
I just give that as an example.
So I just showed you that phospholipase C pathway
saying we've got this going through here.
And I give angiotensin of an example of a type
of pathway that does that but I didn't talk
specifically about the first messenger in that.
Yes?
Student: Can you go over the synthesis N link,
like glycoprotein?
Ahern: The synthesis of the N linked glycoprotein.
Certainly.
Let's go to carbohydrates.
Okay.
When we go to synthesize N linked glycoproteins,
first of all, N linked glycoprotein,
their synthesis starts endoplasmic reticulum.
And in the endoplasmic reticulum,
some of the common things are added
that you see on the screen here.
So all N linked glycoproteins will have a common core
of 5 modified sugar residues.
I'm not asking to know which ones are there
but you should know that there's
a common core that's there.
I talked about how the synthesis
of N linked glycoproteins starts.
I'll talk to you about that in a second.
N linked glycoproteins, their synthesis starts
in the endoplasmic reticulum but it's completed
in the Golgi apparatus.
So N linked are made in both places in essence.
The O linked glycoproteins are made
only in the Golgi apparatus.
Now, to answer your question I think,
which is how do these guys get made and...
that's not what I wanted.
Dolichol phosphate, okay.
I talked in class about the role of this molecule
in the initial synthesis of that carbohydrate
Christmas tree on glycoproteins.
I talked about how that's made.
So dolichol phosphate is a molecule that plays
an important role in that process.
Dolichol phosphate is a membrane lipid.
It's found in the membranes of the endoplasm reticulum.
And to start, it looks like this.
So we can think of the lipid bilayer,
this non polar portion is stuck in the lipid bilayer,
and this phosphate is sticking out because the lipid bilayer
on the inner portion is very non-polar.
This guy doesn't fit very well.
So this is sticking out into the cytoplasm.
The phosphate sticking out into the cytoplasm.
And when we go to make an N linked glycoprotein,
we've got to start making that core.
And we start making it on here.
So there are enzymes that start putting those phosphates
on this phosphate sticking out on the cytoplasm.
And then, so I talked about magic,
and then something magical happens and this molecule inverts.
So the thing that was on the phosphate on the outside
flips and now it becomes on the inside
of the endoplasmic reticulum.
It's crossing that lipid bilayer in doing this
and carrying with it those modified sugar residues
on there that will ultimately get put onto
that N linked glycoprotein.
Once it gets inside, additional residues may be added
and then that beginning of the Christmas tree
will be transfered to a target protein,
making it a glycol protein.
Yes?
Student: I'm sorry if you already said this,
but the ones that are added to it prior to it flipping,
is it just the 5 core...?
Ahern: I haven't specifically said that.
I said we start with the 5.
It doesn't matter for our purposes
if there are 3 or 4 or 6.
The important thing is that the process is starting
out there in the cytoplasm.
So anyway, once it gets inside,
once that flip has happened, then as I say,
additional residues may be added to that,
and then it'll be transferred off of this phosphate
onto the target protein and then this guy will turn
around and flip back out and be ready to do another one.
I forget who asked me the question.
Does that answer your question?
Student: Yeah, so it ends up flipping it
out to the other side?
Ahern: Yeah.
Student: And it just goes to the Golgi?
Ahern: Well, the protein goes to the Golgi.
Remember this molecule is stuck in the membrane.
This is not attached to the protein.
Student: Which membrane is that located in?
Ahern: This is in the membrane of the endoplasmic reticulum.
Yeah?
Student: Going back to the EGF notes,
the table on there that has
like the definitions of like ras growth, raf,
we haven't talked about that in class.
Are we supposed to know...?
Ahern: As I said in class, you're only responsible
for what I've talked about in class.
So I haven't talked about that in class.
You're talking about this guy here, right?
I like arf, it's my favorite.
Yes, sir?
Student: [inaudible]
Ahern: About what?
Student: [inaudible]
Ahern: His question has to do with the three different
pathways that can happen after pyruvate
and how much detail do I want you to know.
Whatever I said in class.
Yes?
Student: Does protein kinase A phosphorylate phosphodiesterase?
Ahern: Does protein kinase A phosphorylate phosphodiesterase?
No.
Student: [inaudible]
Ahern: So let me answer your question.
You're wondering how phosphodiesterase itself
is controlled basically.
So phosphodiesterase just to remind everybody,
phosphodiesterase is an enzyme that breaks down cyclic AMP.
It converts cyclic AMP to AMP.
And when it's converted to AMP,
it no longer behaves like cyclic AMP.
It can't affect protein kinase A anymore at all.
So it's generally on.
So more importantly, we're concerned with phosphodiesterase,
how do we turn it off?
And that's where caffeine comes in.
Caffeine is an allosteric inhibitor of phosphodiesterase
and stops the enzyme from breaking down cyclic AMP.
So when the enzyme stops breaking down cyclic AMP,
cellular concentrations of cyclic AMP increase,
and that then favors that cascade
that ultimately breaks down glycogen to make glucose.
But no, it does not phosphorylate that, no.
Connie?
Student: What's the difference between suicide inhibition
and competitive inhibition besides the covalent bonding?
Is there anything...?
Ahern: What's the difference between suicide inhibition
and competitive inhibition besides the covalent bonding?
None.
Student: None, okay.
Ahern: Alright, so you guys look like
you're a little worn out here.
What do you say we call it an evening?
I will be available.
My schedule as I noted is lightening up this week
so I should be around if you have questions.
Please feel free to come by and see me.
I probably will announce in class tomorrow
that I will allow you to suggest a question for the exam.
So if you want to send me, email me a question
for consideration for the exam,
I will use one student submitted question for the exam.
Student: I was going to ask you a question
but I was busy writing.
Do we need to know how bicarbonate comes
off of the carbonic anhydrase?
Because I have an idea of how carbonic anhydrase
creates carbonic acid from carbon dioxide.
I'm not sure how exactly it comes off.
Ahern: It's just released just like any other product
of an enzyme would be released.
Student: Okay.
[END]