#40 Biochemistry Nucleotide Metabolism II Lecture for Kevin Ahern's BB 451/551

Uploaded by oharow on 13.02.2012

Kevin Ahern: Welcome back.
Enjoy that three-day weekend?
No, there wasn't one, that's why.
Pop quiz.
I'm just testing to see if you were paying attention or not.
I could go for a three-day weekend right now.
Student: Agreed.
Kevin Ahern: Agreed, Yes.
Should we just call off class today?
Class: Yes.
Kevin Ahern: And do twice as fast on Wednesday?
Student: Give us all A's.
Kevin Ahern: All A's yeah.
Sure, if we're going to dream we might as well dream big, right?
All F's too, right?
I mean that goes both ways.
Alright, so we're in very good shape with nucleotide metabolism
and I hope one of the things that I communicated to you
in talking about it on Friday was the importance of balance.
As I said when I started the lecture about nucleotide metabolism
I mentioned the importance of maintaining that balance.
If the nucleotides get out of whack-we make too much A
or make too much G or too much C or too little U or whatever,
then we're much more likely to have mutation inside of cells.
So its very important that cells maintain this balance
and we are going to see even more balance today.
So you saw balance last time between purines and pyrimidines.
You saw balance within pyrimidines
and you saw balance within purines.
Today you're going to see balance
of deoxyribonucleotides as well,
and they're interesting schemes by which cells do these things.
Well I'm going to start today by talking about dNTP synthesis.
You'll notice on the outline that I've got "Salvage" up here,
and I will save that until I talk about some things at the end.
So salvage actually does become significant
for some components of human disease as we shall see.
But before we do that, I do want
to talk about deoxyribonucleotide synthesis or dNTP synthesis.
It turns out that dNTP synthesis is derived,
not surprisingly, from ribonucleotide synthesis.
And there's an interesting hook with it.
The interesting hook is that the deoxyribonucleotides
that you see down here,
and by the way you can put a "d" on there.
A lot of people don't.
I happen to like putting a "d" on there.
The reason they don't put a "d" on there was originally
it was felt that thymidine nucleotides don't appear in RNA
so there's no reason to put a "d"
because it's automatically "d."
But in fact some RNAs have ribothymidine
so I think we really should ignore that old thing
and put it as a dTTP.
I'll take either, but in any event...
Well as we look at the scheme,
what we see is that the starting materials
for making the deoxyribonucleotides
are nucleoside diphosphates.
And those individual nucleoside diphosphates
are converted into deoxynucleoside diphosphates.
By the way, a nucleoside diphosphate,
people say, "Why don't you say nucleotide diphosphate?"
Well it would be redundant.
A nucleotide has a phosphate in it
so a nucleoside phosphate equals a nucleotide.
A nucleoside has no phosphate,
put a phosphate on a nucleoside and you've got a nucleotide.
So nucleoside diphosphate is a starting material for this
and all of the nucleoside diphosphates are acted on
by the same enzyme.
The enzyme in this case is ribonucleotide reductase.
Now I'm going to have a few things
to say about that enzyme in just a bit.
But suffice it to say one enzyme handles all of them.
And this enzyme has a very interesting regulatory scheme
involving allosterism.
Two types of allosterism as we shall see,
and I'm going to give you a simplification for one of them.
The other one's pretty straightforward as it is.
Because of this enzyme we can make
deoxyribonucleoside diphosphates.
Notice we're making diphosphates from diphosphates.
We're not creating phosphates out of thin air.
These deoxyribonucleoside diphosphates
can be converted into deoxyribonucleoside triphosphates
by our favorite enzyme
which acted on all the phosphates which was?
I can't hear you.
Anybody remember from last lecture?
Student: NDPK.
Kevin Ahern: NDPK, thank you.
NDPK works on all of the diphosphates as I mentioned last time.
So NDPK will take us here.
Now this figure is a little misleading
in that there's not one step in going from here to here.
I'm going to talk about this separately
but there's a couple of steps that's necessary
to get to this point.
And they're kind of interesting and they're kind of odd.
So we'll keep this last one in mind for upcoming discussion.
Well if we look at ribonucleotide reductase
it's one of the most studied enzymes
of all nucleotide metabolism.
It consists of a set of two dimers.
There's what's called the large subunit
which is known as the R1 subunit.
It's also called the large subunit.
And there's a small subunit called the R2 subunit
and both of these are present in a dimer as you can see here.
The R1 subunit is the place where the reaction is catalyzed.
So that conversion, and what that enzyme is doing,
remember it's converting ribonucleoside diphosphate
to deoxyribonucleoside diphosphate,
what's happening is its converting the ribose
in that ribonucleotide into a deoxyribose in that nucleotide.
That's how we get the "d."
So we're having to basically convert an OH to an H.
That's what this enzyme is doing.
The reaction is catalyzed in the large subunit
as I mentioned.
The small subunit, with my microphone going off.
The small subunit
has some very interesting features about it, however.
One is that it has within it something called,
it has a side chain of tyrosine that ultimately is responsible
for making this reaction possible,
and I think my thing is giving out on batteries...
And there are no more.
I shall speak loud.
So the side chain-I'm just going to take this thing off.
The side chain of this tyrosine in the small subunit
gets made into a radical.
There's actually a proton that gets pulled off of it
by an unusual reaction.
We don't need to worry about the reaction
but this causes basically an unstable electronic configuration
in the side chain of a tyrosine in the small subunit.
What's interesting is that electronic instability
is communicated all the way back to the large subunit
at the active site.
Now if you want to have really good evidence,
or you're interested in electronic circuits that exist
in biomolecules, this is a prime example
of an electronic circuit
because the instability is created here.
It's communicated all the way through the subunit
all the way up through the other subunit
into the active site.
That's pretty cool.
And because of that the active site catalyzes the reaction.
I'm going to show you a mechanism.
I'm not going to hold you responsible for a mechanism
but I'll show you that in just a little bit.
Yes sir?
Student: Does this enzyme work
with two riboses at a time or just one?
Kevin Ahern: Does this enzyme work on two riboses at a time
or only one?
The subunits as far as I know will act independently
so they can have two at the same time or one at a time,
it really doesn't matter.
Now another interesting feature-yes sir?
Student: What kind of batteries do you need?
Kevin Ahern: I think they're nine volts.
Let's see I'll make sure of that.
Oh actually they're not.
They're double A's.
You have a double A?
Oh look at this.
Student: How many do you need?
Kevin Ahern: Twelve.
[class laughing]
But two would do.
Student: Do I get an A?
Kevin Ahern: What's that?
Does he get an A for that?
I don't know, should I give him an A for the day?
Wait just a second, I'll come get them.
They usually have an extra pack in there
and they don't have an extra pack today.
Alright, so batteries.
Thank you sir.
I will see that you get replacements.
Now we'll see and make sure this works.
We'll see.
Duh du dah.
[class laughing]
You don't want me shouting so much.
That's probably why he said, "Take the batteries.
"I don't want you spitting all over the first three rows here."
[class laughing]
Kevin Ahern: I had that happen to me.
Have I ever told you this story before about going to Ashland?
And I get to Ashland and I didn't have any tickets
so I go and I buy tickets.
True story.
I go and I buy tickets at Ashland and there's this guy there
and he says, "I've got front row seats for you
"in the outdoor theatre,"
and I said, "That's awesome!
"How much do you want?"
I figure he's going to scalp me, right?
"I'll let you have them for what I paid for them," which he did!
And I thought, "Well that was really great."
So I went and sat in the front row
and learned very quickly why front row in Ashland
really isn't a good place to be because the projecting is,
they're spitting all over the first three or four rows.
It was just absolutely disgusting.
I think he had been there the previous night and said,
"I'm not going to do this again."
You guys know this up here, too.
Or you would if I didn't have these batteries.
So the other interesting feature of the small subunit
is it's where the allosteric information is communicated.
So the regulation happens
because of binding of allosteric regulators
to the small subunit.
So the small subunit's pretty critical
in that even though it doesn't catalyze the enzymatic reaction.
There's a structure and inside there we can see
that there's iron that is attached to side chains of amino acids,
not too far away from a tyrosine.
And I mentioned that this tyrosine
gets a proton pulled away from it creating an unpaired electron
and an unstable state.
The reaction mechanism is there.
I'm not going to go through it with you
and I'm not going to hold you responsible for it,
so you can just look at it and admire it.
But you can see in this active site that what's happening,
here is the nucleoside diphosphate that's being held in place.
You see that sulfhydryls are playing roles.
You'll see a carboxyl playing a role.
And the upshot of this is that this unstable intermediate
that was created
in the tyrosine of the small subunit
is communicated up to here.
You see here is this unpaired electron that is in place
because of that instability
that was created in that small subunit.
This starts a series of reactions and process
that ultimately result in the production of a hydrogen
at this point where we had an OH to start with.
So we've created a deoxyribose and as a consequence
we've created a deoxyribonucleoside diphosphate.
The other thing that you notice here is we start with SH's
and we ended up with a disulfide bond.
Now you know that whenever we do an enzymatic reaction
we have to return the enzyme to its active,
I mean to its original state,
which means this disulfide bond has got to be reduced.
And it gets reduced in an interesting reaction
that ultimately involves NADPH.
So that's not shown on here
because it's not part of the reaction mechanism as such,
but that has to be reduced to get the enzyme
back to its active state.
This shows what happens with that NADPH.
And again I'm not holding you responsible for this
but you can see that there's a lot of transfer of electrons
that ultimately have to happen
in order for this overall process to occur.
Here's that last step where we're taking that disulfide bond
and we are ultimately bringing it back up here
to the sulfhydryl state.
We see that this molecule known as thioredoxin
plays a role in this process and that is something
I think that you should know.
Thioredoxin is a very interesting small peptide
that plays very important roles
in redox reactions inside of cells.
Reduction/oxidation reactions inside of cells.
Thioredoxin is a source of electrons to reduce this
and thioredoxin gets its electrons from thioredoxin reductase
which gets its electrons from NADPH.
So we can see that this process happens
ultimately to get those electrons to the enzyme
and regenerate the enzyme to the original state
and then that original state is used to convert ribose
into deoxyribose within a nucleoside diphosphate.
That big mouthful of words there.
Well let's get back to things
that you really are responsible for here
and this includes the synthesis of thymidine.
Now as I said thymidine has an interesting pathway
to becoming thymidine.
It's made from UDP.
So you saw on that scheme that I gave you
that ribonucleotide reductase converted UDP into dUDP.
Well it might think like the logical thing for the cell
to do would be to simply take dUDP
and convert it into dTDP, right?
Well it turns out cells don't do that,
and at first it's going to seem very odd why they don't do that.
Instead of simply converting dUDP to dTDP,
cells convert dUDP into dUMP.
It takes a phosphate off and it takes a dUMP,haha.
It makes a dUMP.
Well why does it do that?
Why does it do that?
It turns out that there's a very important reason
why it does it.
If you recall our friend NDPK does what?
It converts diphosphates into triphosphates.
If we have much dUDP sitting around,
what happens is NDPK will convert it into dUTP
and dUTP can be put into DNA by DNA polymerase.
That's not a good longterm things for cells to do.
It happens at a very low frequency, actually.
But to reduce the likelihood that cells will have that reaction
going on they have this enzyme
that converts dUDP-
you know I'm saying this backwards actually.
As I'm saying it I'm realizing
I'm telling you an incorrect story.
Oh no, Ahern.
So I said it takes it from dUDP down to dUMP.
It doesn't.
It goes from dUDP up to dUTP and then it cleaves that.
Now the consequence of that is that cells
will get a little bit of U in their DNA.
A little bit of U's in their DNA.
The enzyme that converts dUTP into dUMP is known as dUTPase.
It's a very important enzyme in cells
because if dUTPase is not active
then you're going to get much more U inside of your DNAs.
Student: [Inaudible.]
[class laughing]
Kevin Ahern: I'll back up and say it again
since I've already confused you once.
So let's just wipe the slate clean.
We'll just start over, how's that?
We've got dUDP.
The cells make dUDP.
You have an NDPK that will take dUDP and make dUTP out of it.
That's given because NDPK will work on all diphosphates
and make them into triphosphates.
It grabs dUDP, it makes dUTP.
If dUTP hangs around for any period of time
DNA polymerase will use it in place of T
because it looks just like a T
as far as the DNA polymerase is concerned.
Remember that U pairs with A in RNA.
So too will U pair with A in DNA if it's allowed to.
Cells don't want that to happen
so they have an enzyme called dUTPase
that they cleave dUTP into dUMP.
They clip two phosphates off.
dUTPase does that.
If dUTPase is absent or not very active,
cells will therefore get much more U
into their DNA in place of T.
Yes sir?
Student: [Inaudible]
Kevin Ahern: What type of negative effects will come of that?
Chemically U is not stable
over as long of a period of time as T is.
U can deaminate readily and make C problems.
That's why we don't have U
to any significant extent in our DNA.
Yes sir?
Student: The dATPase, does it clip off a pyrophosphate
or individual phosphates?
Kevin Ahern: The dUTPase clips off a pyrophosphate.
It clips off two phosphates with one fell swoop.
One cut.
Why am I showing you this on the screen?
What's that got to do with this?
Well I had to tell you that because your book
doesn't have a figure for that to tell you how we get dUMP.
So dUMP has to be converted into dTMP.
That's the next step.
And yes I will review this,
this whole process as I get through it.
dUMP gains a methyl group
from this monstrosity here,
and we're going to call this THF.
There are several tetrahydrofolates.
I don't think we need to distinguish them.
We're going to call this THF, tetrahydrofolate.
Tetrahydrofolate is a source of the methyl group.
The methyl comes and gets put right here on the U
and that makes it into a T.
And again, this should be a dTMP.
That's cool.
The product of that reaction is a dTMP
and something called dihydrofolate
which we're going to call DHF.
So this is THF.
THF gets converted into DHF in that process.
No I don't care if you have, if you understand the mechanism.
It doesn't matter for our purposes.
You do need to know that in this reaction,
dUMP is being converted into dTMP.
THF is being converted into DHF.
Yes, Jen?
Student: [Inaudible]
Kevin Ahern: I was just getting ready to tell you the enzyme.
So the name of the enzyme that catalyzes all this
is called thymidylate synthase.
Thymidylate synthase.
Well if we have dTMP, how do we convert it into dTDP?
We have a monophosphate kinase that does that, right?
We have a monophosphate kinase that converts it into dTDP.
And how do we convert dTDP into dTTP?
I said it right.
[Professor laughing]
What's the enzyme that converts diphosphates into triphosphates?
Kevin Ahern: NDPK, there we go!
So finally we've got all four deoxyribonucleotides.
Now I said I would summarize and I will.
How do we get from UDP to dTTP?
Let's step through the steps.
UDP goes to dUDP by what enzyme?
Ribonucleotide reductase.
If you want to call that RNR you can.
Student: [Inaudible.]
Kevin Ahern: UDP goes to dUDP catalyzed by RNR,
ribonucleotide reductase.
We've got dUDP.
NDPK takes it up to dUTP.
dUTPase takes dUTP down to dUMP.
dUMP goes to dTMP by thymidylate synthase.
And bang, we're set.
I showed you in the reactions on Friday
that purine nucleotide synthesis requires folates.
Folates I said were the sources of one-carbon sources.
We see here that production of thymidine
also requires folates.
Folates are therefore very very important
for nucleotide metabolism.
Just like NAD and NADH,
cells only have a limited amount of them.
When they use up what they have they have to recycle it.
This folate, DHF, has to be converted back to THF
if cells are to keep nucleotide metabolism going
because we have to have it for purines,
we have to have it for pyrimidines,
or this pyrimidine anyway.
That turns out to be a very important consideration
for cells in a couple of respects.
Here is the enzyme that does that conversion.
It's known as dihydrofolate reductase.
Don't confuse DHFR with DHF.
DHFR is an enzyme, DHF is a molecule.
This enzyme uses electrons from NADPH to do this conversion,
at least partly.
This reaction is essential.
If this reaction does not happen
then cells are not recycling their DHF
and everything's going to accumulate as DHF
and cells are not going to be able
to do nucleotide biosynthesis.
This enzyme is a target for anticancer drugs
and for antibiotics.
Student: So it goes from THF to DHF or DHF to THF?
Kevin Ahern: I'm sorry, this is going from DHF to THF.
Did I say it backwards again?
Hopefully I didn't, yes sir?
Student: The enzyme one more time please?
Kevin Ahern: The enzyme is dihydrofolate reductase, DHFR.
Now this enzyme is really important as I said
because without this enzyme you don't have enough folates
that you need for nucleotide biosynthesis.
Here's the enzyme.
Dihydrofolate going to tetrahydrofolate.
There's some other reactions that have to happen
to get it over to here but without this step we can't get this.
So this enzyme is targeted by some drugs
that are used in chemotherapy to treat cancer.
Aminopterin and methotrexate,
of which methotrexate is probably the more commonly used one,
are folate mimics.
They're competitive inhibitors of the enzyme.
They're competitive inhibitors of the enzyme.
So the strategy of using, for example,
methotrexate in chemotherapy is to first of all
have some cells that are dividing more rapidly
than normal cells.
Some cancers are very aggressive in this respect.
If you treat a person with methotrexate
what's going to happen to their nucleotide synthesis?
Well it's going to stop in regular cells
just like it's going to stop in cancer cells.
The strategy is to give it for a short period of time
and you flush it out of the body basically,
with the hope that in that short period of time
that you've given it the more aggressively growing cells
like cancer cells are much more likely to be killed by it
because they will not have nucleotides to divide.
Now there are some pretty good side effects
with this as you can imagine
because there are other cells in the body besides cancer cells
that are dividing very rapidly.
They include cells in your intestines.
One of the reasons people get sick
with some types of chemotherapy
is its interfering with the division of those cells
in your intestines that are turning over very rapidly.
Your digestive system goes on strike against you.
You lose appetite.
You have a variety of things.
You may lose your hair.
Rapidly dividing cells are targeted by this stuff.
There's also another target.
Fluorouracil, is a, it's actually a suicide inhibitor
of thymidylate synthase.
If you stop this reaction cells have the same problem
as if you stop this reaction.
This is simply a competitive inhibitor.
This is a suicide inhibitor.
It kills the enzyme.
This is what aminopterin looks like.
No, you won't need to know the structure.
And a last consideration here
is there's another folate mimic that's actually used
to kill bacteria.
And it's on the screen here.
It's called trimethoprim.
And trimethoprim inhibits the folate production
and is particularly important in bacteria
because in bacteria they have to synthesize their own folates.
And this will stop actually the synthesis of folates.
It doesn't happen in our cells
because we get folates external to us in our diet
and so forth and so you can kill,
you can selectively kill bacteria with trimethoprim
because they're making their own and they have to make their own
whereas we don't have to make our own
and we're protected by that.
Trimethoprim is used frequently for people
who have bladder infections,
particularly if they have issues
with being penicillin sensitive.
It's very effective against bacteria.
Well that takes care of what I want to say
about deoxyribonucleotide synthesis in general.
Now i'd like to turn our attention to regulation
and remind you of some of the things
that I've already talked about
and then say some new things about ribonucleotide reductase.
I probably don't need to say anything more about ATCase again.
We went through this in some detail last year
and I mentioned it briefly to start nucleotide biosynthesis
but I will say ATCase catalyzes that first step
in the synthesis of pyrimidines.
The end product of that synthesis, CTP,
is a feedback inhibitor of it.
ATP is an activator of it.
This enzyme provides balance between purines and pyrimidines.
Purines will favor its activation thereby
increasing the amount of pyrimidines.
Pyrimidines will disfavor its activation
and thereby reduce the amount of pyrimidines.
The other enzyme I mentioned the other day was this enzyme
known as PRPP amidotransferase.
PRPP amidotransferase.
It catalyzes the reaction that,
in fact this is I think not accurate
indicating that it inhibits here.
The primary inhibition is right here,
the making of the phosphoribosylamine.
I'll take, again if the book has that
I'll take either answer but basically
the reaction right here is the critical one.
And this is where PRPP amidotransferase works.
PRPP amidotransferase is inhibited
by the end products of purine biosynthesis.
These include AMP, GMP,
and the branch point molecule IMP as well.
So it's a feedback inhibition that's turning off this enzyme.
And this was the enzyme that had the very interesting property
that AMP and GMP together will completely inhibit the enzyme.
AMP alone will not completely inhibit the enzyme.
The enzyme will be a little bit active.
GMP alone will allow the enzyme to be a little bit active.
Why is that important?
Well it turns out to be very important.
Imagine my balance of AMP and GMP
is such that everything I have is in the form of GMP.
If only GMP turns off this enzyme I have no way to make AMP.
So if only GMP is present the enzyme is a little bit active.
It continues making some of the stuff through here.
And when it gets to the branch point,
remember that this one is inhibited by GMP,
then synthesis goes up here.
If I only had AMP the enzyme is a little bit active.
It gets up here to the branch point
and is inhibited there and the cell makes GMP.
So indirectly PRPP amidotransferase
is helping to balance A versus G.
A versus G.
Student: [Inaudible]
Kevin Ahern: I'm sorry?
Oh the little ball is bouncing?
Goodbye ball.
Now the last enzyme that I want to talk about-
actually before I do that let me mention one other enzyme
I mentioned as a regulatory capacity.
It's not shown on here.
And that was CTP synthase.
CTP synthase was inhibited by CTP.
CTP synthase converts UTP to CTP
and therefore helps to balance U versus C.
Student: Can you repeat that?
Kevin Ahern: CTP synthase is another regulatory enzyme.
It catalyzes the conversion of UTP into CTP
and it's inhibited by CTP.
So therefore CTP synthase helps to balance U versus C.
So we've got balance for pyrimidine versus purine.
We've got balance for U versus C.
We've got balance for A versus G.
And what I'm getting ready to show you now is we have balance
for the individual deoxyribonucleotides as well.
That happens through ribonucleotide reductase.
Ribonucleotide reductase has an allosteric site.
It has two allosteric sites and it has an active site.
And this can get confusing so I'm going to be very careful here,
Very careful.
Here's the active site.
The active site you recall is the place
where the reaction is catalyzed.
There are two allosteric sites to consider.
Two allosteric sites to consider.
One is called the specificity site
and one is called the activity site.
Notice activity site sounds a lot like active site.
They're not the same thing.
Active site is where the reaction is catalyzed.
Activity site is where the enzyme is controlled.
Let's start with the activity site
because it tells us something very important about the enzyme.
What does the activity site do?
It determines if the enzyme is turned on or turned off.
It's very simple.
What binds to it?
Well if ATP binds to the activity site, the enzyme is turned on.
That's pretty good because ATP indicates high energy.
High energy we want to be making nucleotides.
Bang, that's good.
The second molecule that can bind to the activity site is dATP.
That is dATP.
dATP will turn the enzyme off.
And dATP is an indication
of how many deoxyribonucleotides a cell has.
Too many deoxyribonucleotides, enzyme off.
Abundance of energy, enzyme on.
So we have an enzyme on or off.
That's pretty cool.
It's the next site that I'm going to simplify for you.
The next site is the specificity site
and it's rather complicated how it determines
which things will be catalyzed.
The specificity is controlling
which substrates bind to the active site.
Now, let's imagine the following.
Let's imagine I have an abundance of purines in my body.
I'm short on pyrimidines, deoxy pyrimidines.
How might I control this enzyme?
Well if I had an abundance of purines
I know I need to make more pyrimidines.
So if I had some way of measuring purines
I could turn the enzyme on for pyrimidines only.
And that's what the specificity site does.
Now the specificity site will bind to triphosphates.
The active site will bind to diphosphates.
How does this work?
Let's say I've got a ton of dGTP in my cell.
If I have a ton of dGTP I sure as the heck
don't want to be converting GDP into more dGDP
because I've already got too much.
A ton of dGTP will bind here
and it will disfavor binding
of purines at the active site,
thus GDP and ADP will be disfavored
if a purine binds here.
If a pyrimidine binds here, let's say I have a ton of dCTP,
then pyrimidines will be disfavored here
and purines will be favored.
So if I have dCTP bound here
then this guy is going to prefer to bind to GDP or ADP.
Now I know there's a lot of things here.
There's triphosphates, there's diphosphates,
there's ribonucleotides, there's deoxyribonucleotides,
and all the words I'm going to throw at you with this
are just going to be mush after awhile.
I want you to sit down,
look at the highlights I'm going to give you for this,
and understand what is binding at each of these sites
and how the enzyme is affected.
Now I will stop at this point
and take questions because I'm sure
I've probably raised a few.
Yes, Emily?
Student: Isn't this R1?
Kevin Ahern: This is R1.
I said R2 earlier and that was incorrect.
It's in the R1, I'm sorry.
Oh I'm sorry you're drinking.
I thought that was your hand up there.
Oh yes, Connie?
Student: I have a question actually about the anticancer drug,
the competitive one.
Kevin Ahern: Yes, the anticancer drug.
Competitive one.
Student: How exactly does adding an inhibitor
and then flushing it out slow the cancer cells?
Kevin Ahern: How does adding the inhibitor for,
let's say methotrexate, how does adding methotrexate
and then flushing it out kill the cancer cell?
Well during the period of time that the methotrexate
is there in abundance,
nucleotide synthesis is going to be stopped.
So if cells are needing to divide
and they run out of nucleotides, they die.
Student: Okay.
Kevin Ahern: So the idea is that cancer cells are dividing
more rapidly than regular cells
so they're more susceptible during that period of time
when I've got them, when I have the drug present.
I flush it out so I don't end up calling the organism,
the person taking the anticancer drug,
but preferentially I hopefully kill more cancer cells
than I kill anticancer, or non-cancer cells,
not anticancer cells.
Any other questions on this?
I will summarize these in the highlights.
Please look at that and If you have questions let me know.
Well in the last ten minutes
what I want to do is talk about some of the byproducts
of nucleotide metabolism and their impact on human health.
Their impact on human health.
The first of these that I'll talk about
is a very interesting disease
known as severe combined immunodeficiency.
This is a disease where,
it's a genetic disease where a person,
there are a couple of causes
but one relative to nucleotide metabolism
is lack of the enzyme adenosine deaminase.
Now first of all let me tell you what the disease is.
So it's a complete failure of the immune system.
If you've heard of the Bubble Boy story
of quite a few years ago this was a kid
who was born without an immune system.
They recognized it very quickly and they put him
in a sterile bubble where he lived for most of his life
before he finally died.
He lived most of his life in this sterile bubble
because even the simplest bacterium
that you and I would have ourselves coated with, would kill him.
The immune system completely lacking.
The question is, how and why is his immune system lacking.
Well what you see here is part
of the breakdown of purine nucleotides.
Here's AMP.
Let's say I'm a cell.
I have brought in, I've ingested a nucleic acid
or I've even ingested a bunch of AMP
and I want to break it down to its substituents
so that I can use them as necessary.
Individuals that are lacking this enzyme right here,
adenosine deaminase, are susceptible to developing SCID,
severe combined immunodeficiency.
Well it turns out that if this enzyme
is deficient we back up a whole bunch of processes
and what accumulates is dATP.
Now based on what I just told you,
you should be able to tell me what the consequences
of dATP accumulating would be.
What's going to happen to cells
when they've got a lot of dATP?
They're going to shut down their reductase,
they're not going to divide very well.
And this problem appears to manifest itself
mostly in the immune system, that is the accumulation of dATP.
Other cells aren't as affected by that.
But in the immune system they're very affected by it
and they have no nucleotides to make DNA.
To mount an immune response our immune cells
have to divide considerably.
They can't.
So adenosine demonise a very important enzyme in that respect.
Another enzyme's that's important
was one we saw up here in salvage that I haven't talked
about but I'll just show you here.
This is an enzyme that's important in recycling
some of the purines to be able to use them.
So for example let's say I have a bunch of guanine.
That's a base laying around.
And I need to make nucleotides,
so I take that base that's laying around known as guanine,
I combine it with PRPP, and when I do that I create GMP.
So now I'm on my way to making nucleotides
and I've salvaged a base that was sitting around.
You'll see that this enzyme, it's called HGPRT,
also works on hypoxanthine.
So what's hypoxanthine?
Well hypoxanthine is produced right around here
so it's like a purine.
It's basically a purine as well.
So this enzyme is helping to scavenge
through salvage purine bases and make purine nucleotides.
Why is this important?
Individuals who are deficient in this enzyme
develop a bizarre syndrome.
The bizarre syndrome is known as Lesch-Nyhan syndrome.
And I think I've got it right here.
Lesch-Nyhan syndrome.
That's it right there.
And this syndrome has many problems
but particularly in young males what happens
is that they will literally chew their lips off.
It's a bizarre neurological problem and they have
to be restrained because they will literally,
when they have the chance, do that.
Now this is a relatively minor enzyme it would appear,
and the full link of the neurological phenomenon
to the deficiency of the enzyme is not completely understood,
but suffice it to say sometimes even what appears
to been very minor enzymes can have major effects.
Needless to say retardation and a variety of things
happen as a result of a deficiency of this enzyme.
So salvage is important.
If salvage doesn't work properly we can see real problems.
The last thing I want to say relative
to nucleotide metabolism is something
also that's interesting from a perspective of human health,
and to show you that I need to go back
to my purine breakdown pathway.
So here's the one I showed you earlier.
Purines are being broken down.
AMP to adenosine, blah, blah, blah, blah.
What I'm getting ready to tell you
isn't a deficiency of an enzyme.
It's a sufficiency of something else,
meaning we have too much.
If we have too many purines,
and by the way, the disease that arises
from this is called gout.
Gout is a disease where the first place people usually realize
is they have this excruciating pain in their big toe.
It used to be, they used a lot
for comic effect in the movies.
"Oh my God, my toe," et cetera, et cetera.
It used to be known as rich man's disease
because diets that were rich in red meat,
red wine and so forth were rich in purines
and only the rich ever got this disease.
They got it because they had too many purines,
and when they had too many purines this breakdown
pathway takes over and starts making a lot of uric acid.
Well uric acid is a good and normal thing, okay?
It's on the breakdown pathway to excretion.
Uric acid is actually an excretion
product for birds, for example.
It's a way of getting rid of excess nitrogen.
We don't get rid of nitrogen that way.
We actually get rid of it with urea
but this is a normal breakdown pathway.
The problem is that uric acid doesn't ionize very much.
It's not a very strong acid.
And as a consequence what will happen
is if too much is produced it will crystalize,
and the place where it will crystalize
is the gravitationally lowest place in your body
which ends up being in your big toe.
And when it crystalizes it crystalizes in nerve cells
and that's why the excruciating pain arises.
Now there's a downside to gout but there's also
a good side to gout that was realized a few years ago.
And it too is not fully understood why
but it's about interesting observation.
The observation is that people who tend to have gout
tend to be less likely to have multiple sclerosis.
Uric acid may have some sort of a protective
effect against multiple sclerosis.
The relationship is not understood at this time
but it's an interesting observation that something
that can cause a lot of pain might actually
have a little bit of benefit.
Well if you have the pain, probably you're not thinking
too much of the benefit at the time
because as I said this can be excruciating for some people,
and the way they treat it is they treat
with a compound called allopurinol,
and allopurinol inhibits this enzyme which you notice
catalyzes two separate reactions here
and stops the production of uric acid.
Allopurinol is a very effective treatment for gout.
It really can stop the pain because it stops
the production of uric acid.
Yes sir?
Student: This is only from an excess of purines?
Kevin Ahern: This is only from an excess
of purines, that's correct.
Student: So diet modification would be sufficient?
Kevin Ahern: His question is a good one.
Is diet modification used?
The answer is yes it is to some extent.
In modern times they've gone much more
to using allopurinol as a treatment for it
but diet modification is another approach to doing that.
Alright folks.
I will see you on Wednesday.
[class murmur]