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MATTHEW VANDER HEIDEN: Hello, everybody.
Last time, I introduced the idea of the TCA cycle,
tricarboxylic acid cycle.
Also known as the citric acid cycle, because citric acid
is a tricarboxylic acid, as you'll see later today.
Also known as the Krebs cycle, named
after Hans Krebs, who discovered it
in the early part of the last century.
The TCA cycle is the series of reactions
that occurs in the mitochondrial matrix
and it allows the complete oxidation of two carbon
units, derived from many things, including pyruvate, derived
from glucose and glycolysis and enables the complete oxidation
of that carbon to CO2.
Now, it's a cycle because those two carbon
units from pyruvate or other sources enter the cycle,
combine with 4-carbon oxaloacetate,
and combine to make 6-carbon citrate.
Hence the citric acid cycle, or TCA
cycle, that is then oxidized back to 4 carbon units,
forming a cycle that allows cells
to release lots of energy.
Complete oxygen enables, ultimately,
complete oxidation of glucose to CO2.
We've talked many times how this releases energy.
And it also generates lots of intermediates
for the cell that can be used to make other stuff.
Last time, I alluded to the fact that citrate can be
used, say, to make fatty acids.
Today, what I want to do is I want to dive
into the details of how this TCA cycle occurs
and the consequences of the way it works,
and how that affects other aspects of metabolism.
You'll see that it actually affects
the ability of different organisms,
whether or not they can make things
from intermediates in this cycle,
because of how the cycle works.
Now, to start.
Of course, if we're going to start from pyruvate.
Pyruvate, of course, has three carbons.
And so if we're going to generate a two-carbon acetate
group, we have to lose CO2.
And as I described a couple lectures ago,
acetate is also the thing that we
can derive from metabolism of ethanol,
showing us two things--
metabolized glucose to pyruvate or ethanol itself--
can both be turned into acetate.
But really, the donor is this molecule, acetyl-CoA.
Which is basically also a carboxylic acid, but rather
than being a carboxylic acid, instead makes this
a thioester bond, which activates this carbon
as a leaving group, such that it can combine with oxaloacetate,
which you'll remember from gluconeogenesis,
releasing that S-CoA molecule to generate six-carbon citric.
So, this is citric.
You can see that we've made a bond from this carbon
on the acetate, losing this S-CoA group to this carbon
oxaloacetate to make this six-carbon citrate molecule,
which is a one, two, three--
three-carboxylic acid, or tricarboxylic acid.
Hence the name citric acid cycle,
tricarboxylic acid cycle.
Now, these six carbons can then--
or this six-carbon citrate molecule
can then be oxidized, generating two CO2 molecules that
are released, and ultimately reforming
oxaloacetate that can pick up another two-carbon acetyl-CoA
to generate another citrate, and around and around the cycle
goes, allowing in the end the net entry
of two carbons, effectively from acetate,
and release of two carbons as CO2.
Now, as I alluded to, this can come from pyruvate.
It can come from acetate itself, vinegar.
It can come from alcohol.
It turns out that when you break down fat,
you also break it into two carbon units.
And so this cycle becomes very useful for cells,
because it allows the oxidation of many different molecules
to completely turn that carbon into CO2.
Now, if we're going to do this from glucose,
however, you'll remember that pyruvate has three carbons.
And so if we're going to turn pyruvate
into acetate, or acetyl-CoA, we have to lose a carbon of CO2.
We have to lose this carbon as CO2.
Now, we saw this before, that we can do this via--
this is exactly how we generated ethanol
when we did fermentation of pyruvate to ethanol,
but that molecule didn't generate acetate,
It generated acetaldehyde.
The difference, of course, being whether or not this carbon gets
oxidized in the case of acetate to the acid,
or in acetaldehyde in ethanol metabolism,
where it remains an aldehyde.
Now obviously, you can make ethanol and then
turn that ethanol into acetate.
And that's a pathway that I guess certainly would work.
However, that's not the way it happens in most organisms.
Most organisms actually directly produce
acetate, or more correctly, acetyl-CoA from pyruvate.
So let's take a look at that reaction.
So here once again is pyruvate.
And if we turn that pyruvate into
this two-carbon acetyl-CoA, let's
look here what has to happen.
Well, the first thing is that we have
to decarboxylate this molecule.
And so that generates a CO2.
We have to lose that one carbon.
Also, as I pointed out, if we do this
as if we did it in ethanol metabolism,
we'd be left with an aldehyde.
But this is an acid, and so this carbon also has to be oxidized.
We know how to do that.
We can donate those electrons to something like NAD,
so the NAD is reduced to NADH.
And we had to add this S-CoA molecule, which
I'll come to in a minute.
And so each of these steps ends up
making a fairly complicated reaction.
Obviously, several co-factors are going to be involved.
You should be able to guess that just by looking at it.
Obviously, I already drew up there NAD.
If we're going to decarboxylate, remember
this is a alpha carboxylic, an alpha
ketoacid-- the ketone group is alpha to the carboxylic acid.
And so if we're doing alpha decarboxylation,
as you might guess, we need a co-factor.
That co-factor, as I told you before,
is the co-factor factor thiamine pyrophosphate.
And finally, there's this S-CoA group.
We need to describe what that is.
However, before delving into those, and there's
actually a couple other factors that are needed as well,
I want to mention one other issue
about this reaction, in that this reaction happens
in the mitochondria, because that's also
where the TCA cycle happens.
So, recall that if we divide the cell into two compartments
like an eukaryotic cell, here we have the cytosol
and the mitochondria in a eukaryotic cell.
As we've already talked about, having different compartments
helps facilitate different metabolic reactions,
because you can have different conditions in the two
compartments.
And so as we said, glycolysis occurs in the cytosol.
Glucose to pyruvate.
And last time I mentioned, the TCA cycle
occurs in the mitochondria.
And so that means if we're going to fully oxidize
the pyruvate carbon to CO2 using the TCA
cycle in the mitochondria, that pyruvate
has to get from the cytosol to the mitochondria, or at least
carbons from the pyruvate have to get there.
It turns out, you'll see in a minute,
acetyl-CoA is a very large group.
And so pyruvate itself is transported
into the mitochondria.
And that's where the reaction occurs
to turn it into acetyl-CoA that can then enter the TCA cycle
and oxidize.
However, what this means is that a transporter is actually
needed to get this across the mitochondrial membranes
and into the matrix of the mitochondria where the TCA
cycle happens.
And I like to mention this because it turns out
the pyruvate carrier--
that is, the transporter-- the way
that it actually gets that pyruvate from the cytosol
into the mitochondria actually was
an unknown thing about metabolism
until 2012, so not that long ago.
Sometimes one can be left with the feeling,
listening to these metabolism classes or reading a textbook,
that everything about metabolism has been known forever,
but it's actually not true.
Here's a very key, central part of the pathway that
was actually just discovered, at least
at the time of this lecture, less than 10 years ago.
It also illustrates that not all metabolism is completely
understood.
Now, let's get back to this reaction, how you turn pyruvate
into acetyl-CoA.
And I want to go through now and point out
that several co-factors are needed.
And so I already mentioned one of them, S-CoA,
which is shorthand for coenzyme A. So,
I need to tell you what that is.
You hopefully are already familiar with TPP plus,
thiamine pyrophosphate.
We talked about that when we talked
about how you do alpha decarboxylation to generate
ethanol from pyruvate, also used here
for the alpha decarboxylation reaction.
Redox reaction happens, and so we
needed NAD plus to get converted into NADH.
We talked a lot about how that serves as an electron carrier,
but it turns out that there's two additional electron
carriers that are involved in this reaction.
One of them is called FAD and the other one
is called lipoic acid.
Now you might say, why do we need
all these electron carriers?
Well, these are just different molecules
that can carry two electrons, similar to NADH.
And effectively, what these can do by having multiple electron
carriers--
one can build chains of oxidation and reduction
reactions.
And it turns out these chains of oxidation reduction reactions
really become central to energy transfers in biology,
because building these chains allows
more easy stepwise release of energy
as one moves across these oxidation reduction reactions.
Which remember, as I alluded to earlier, really
are at the core of bioenergetics and a lot of what allows energy
release from these pathways.
What I mean by this will be more explicit
as we go through what some of these co-factors look like.
So, I'm not going to draw TPP plus or NAD again, but let's
define what some of these other cofactors look like.
So, let's start with coenzyme A. So, coenzyme A, it turns out,
is useful.
It's actually involved in lots of acylation reactions.
What's an acylation reaction?
Well, that's basically if you're making a carbon-carbon bond
by adding a molecule of greater than one carbon, so two carbons
or greater, to something else.
That's an acylation reaction, as we
did with adding the two-carbon acetate to oxaloacetate
to make citrate.
And you'll actually see coenzyme A will come up
in this in many, many lectures throughout the rest
of the course.
Why this becomes useful is because it activates
this basically carbon to the left of, in this case,
the ketone.
Because, by having that thioester there,
ends up activating that carbon and allows it to carry out
these acylation reactions.
Coenzyme A itself is derived from a vitamin
called pantothenic acid.
And as a vitamin, this is something
that you have to get from the diet.
Now, when we abbreviate coenzyme A,
which is often abbreviated S-CoA,
you get the sense that it's this little tiny molecule
that's just stuck on sulfur--
stuck on to the end to make this thioester bond.
But it turns out coenzyme A is actually a giant molecule, one
of the reasons why you actually synthesize acetyl-CoA
in the mitochondria, because it's
hard to transport this giant molecule.
And so this is what pantothenic acid looks like.
So, if I put an acid group here and a hydroxyl group there,
that would be pantothenic acid, the thing
that's in your cereal, on the side of your cereal box.
Then there's these additional pieces.
This is the active end of the molecule,
that's that sulfur from the S-CoA,
and then this side of the molecule is esterified to two
phosphates, which are esterified to--
I'm not going to draw it out, but this
would have an adenine base and a phosphate there.
So basically, this is ADP, with a phosphate
added to the 3 prime position of the ADP molecule,
added to pantothenic acid, added to this short chain
with the sulfur on the end.
And this whole molecule together is coenzyme A.
And so when we say acetyl-CoA, it's
this giant molecule, S-thioester to the carbonyl,
the acid on acetate, to CH3.
And so that would be, basically, acetyl-CoA.
And so one convenient thing about this
is it's much easier than drawing that big molecule,
but it is a little bit misleading in terms
of its size.
So, that's coenzyme A.
Next one I want to talk about is FAD
which stands for flavin adenine dinucleotide.
So, flavin adenine dinucleotide is an electron carrier,
just like NAD plus.
And it's derived from the vitamin riboflavin,
also referred to as vitamin B2.
Another thing from the side of your cereal box, and basically
looks like this.
So, like NAD, it's a dinucleotide.
And so here's ADP, just like we do for CoA, or just
like one end of NAD.
One difference is that the other nucleotide down here
actually isn't technically a sugar.
It's ribitol instead of ribose.
What does that mean?
It doesn't have an aldehyde.
Instead, it is just a five-carbon chain
where all of the carbons are alcohols.
And so since there's no aldehyde,
it doesn't form a ring.
And the base on this end, as a nicotinamide,
is this flavin group, which looks like this.
And so this is FAD, which is in the oxidized form.
Turns out, in the oxidized form, FAD
is yellow, hence riboflavin-- "flavin", yellow.
And the reason it's yellow-- you see
there's a conjugated double bond here across this part.
It turns out this is the active part of the molecule,
and it works as follows.
And so if you have a hydride ion,
remember the way we can transfer two electrons.
Can transfer the two electrons that way,
and that allows it to generate.
And I'll just draw the middle, here.
Active part of the molecule.
So, that would be these two nitrogens.
We've added two electrons to it.
And so this is abbreviated FADH2,
or the reduced form of FAD.
And it is colorless, because now you no longer
have that conjugated double bond system, loses its color.
And so you can follow whether FAD is oxidized or reduced
by just looking at color change.
So, that's FAD.
It's an electron carrier, carries electrons
by a hydride transfer, very similarly
to what we described for NAD, but obviously
a different molecule.
And the last molecule, the last co-factor
that we need for this reaction, is lipoic acid.
Which, unlike most things in metabolism,
doesn't have an abbreviation and also functions
as an electron carrier.
And so lipoic acid looks like this.
So, that is lipoic acid.
This is in the oxidized form.
And so where it's oxidized is here
at this sulfur-sulfur bond.
So, you can think of this as the same as a disulfide bond
that you learned about from Professor Yaffe in proteins.
And so this is the oxidized form of the disulfide bond.
If I take this hydride, transfer two electrons across that bond,
then it goes to-- and I'll just draw here the active end.
Then we go here to the reduced form of lipoic acid,
and there's no abbreviation for oxidized
or reduced lipoic acid.
It's just lipoic acid, oxidized lipoic acid, reduced.
And how they're oxidized and reduced
is basically very similar to the disulfide bonds
that you saw in proteins being oxidized, disulfide bond being
oxidized, or it can be reduced to not be a disulfide bond.
Now, it turns out that these cofactors, our TPP, FAD,
and lipoic acid, are all stably associated
with different subunits of a multi-protein complex
that assembles to catalyze that reaction to convert pyruvate
to acetyl-CoA.
The enzyme or enzyme complex that
carries this out is referred to as PDH,
sometimes abbreviated PDC, which stands
for the pyruvate dehydrogenase complex.
So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex,
PDC.
This complex is basically three different polypeptides
that come together to form a complex
and catalyze that reaction.
Now, where this complex sits--
so, this is the mitochondria.
This is the matrix.
That's where this pyruvate dehydrogenase reaction occurs.
That's where the TCA cycle occurs.
And this PDH complex is basically
sitting here at the membrane on the matrix side
of the membrane.
Now, the three different polypeptides
that come together to form this complex
are creatively named E1, E2, and E3, four.
Enzyme one, two, and three.
And each of these, as I alluded to,
is associated with a different co-factor.
And so E1 is associated with thymine pyrophosphate.
E2 is associated with lipoic acid.
And E3 is associated with FAD.
Now, let's go through the mechanism
for how this pyruvate dehydrogenase reaction works.
So remember, this is the active part of TBP plus.
It is bound in the active site of E1.
And it reacts with pyruvate to catalyze alpha decarboxylation,
just like we described for conversion of pyruvate
to alcohol.
So, you're going to see exactly the same mechanism
that we drew before.
So, that decarboxylates the alpha-keto acid,
just like we saw to generate acetaldehyde.
The difference is rather than resolve this such
that this carbon has the same oxidation state
and make acetaldehyde, instead the next step of this reaction
is going to be oxidized by reducing lipoic acid.
So, the active part of lipoic acid
that is in the active site of the E2 subunit.
So this will now regenerate E1, but what we're left with,
then, is this.
Now, this intermediate bound to E2.
Here's where coenzyme A can come in, which
will then generate acetyl-CoA.
But now we are left with E2 in the reduced state,
rather than being in the oxidized state.
So E2 has to be re-oxidized in order for this complex
to carry out the next catalytic cycle, and the way
that works is as follows.
So you have FAD bound on E3.
And so you have a hydride ion from the oxidation of E3
that can be transferred to FAD.
That will generate FAD from the oxidized to the reduced form.
So re-oxidize lipoic acid on E2, reduce FAD to FADH2,
and then that FADH2 can be re-oxidized back
to FAD via transferring those electrons to NAD
plus to generate NADH.
So in this case, FADH2 re-oxidized the FAD, NAD
plus reduced to NADH.
And so, effectively, what this happens
is that enzyme E1 and E2 cooperate
to call what's referred to as oxidative alpha decarboxylation
of pyruvate, while adding -CoA, so that's
where you get acetyl-CoA, with reduction of lipoic acid in E2,
and then E3 re-oxidizes the lipoic acid in E2
while generating NADH.
This NADH, once it's generated, of course
those electrons have to go somewhere,
so they, like in glycolysis--
it also needs to regenerate to NAD at some point.
This is ultimately the electrons that end up on oxygen,
and it's really this series of electron transfers,
with oxygen being a good electron acceptor, that
ultimately allows controlled energy release during carbon
oxidation.
And cells, you'll see, can use that to make ATP or do
other kinds of work.
So, the net reaction and/or another way
to draw the pyruvate dehydrogenase reaction
would be as follows, and that's taking pyruvate to acetyl-CoA.
And so we're going to take coenzyme A and release CO2.
This is done by TPP plus, as part of E1.
That involves converting the lipoic acid in E2
from the oxidized to the reduced state.
That lipoic acid then has to be re-oxidized.
If something's oxidized, something else
has to be reduced.
That's FAD on E3, which also then cycles
between the oxidized and reduce state.
And ultimately, those electrons ended up
being transferred to NAD plus to generate NADH.
And so the PDH complex is basically a chain
of electron transfer reactions.
And it's the first example of a chain of electron transfer
reactions.
We're going to see that there is the electron transport
chain in the mitochondria, effectively does
the same thing.
And by coupling oxidation and reduction reactions
across chains of molecules like this, effectively as a preview,
allows the stepwise energy release of these oxidation
reactions to occur.
So remember, if we burn glucose, completely oxidize it in one
step, where those electrons are directly transferred
to oxygen in combustion.
Lots of energy released, but all in one step.
By doing these stepwise electron transfers,
we can then basically break up that energy release
in a way that can be captured by cells to do work.
Here, the way energy is captured,
it's not so obvious in this electron transport reaction.
But effectively, you're using oxidation
of the ketone on pyruvate, with decarboxylation to the acid
to generate, rather than just the acid, a thioester bond.
And that's that thioester bond--
as well as NADH, that's energy as well-- but it's
really that thioester bond that then can be recaptured later
to drive synthesis of citrate in the TCA cycle.
Now, next what I want to do is I want to discuss the TCA cycle
reactions.
Now that you see how you can get acetyl-CoA, at least
from pyruvate, I want to discuss how you can now
use that acetyl-CoA and oxidize it back to combine it
with oxaloacetate, and oxidize it back
to make citrate, and then oxidize it back to oxaloacetate
to run the TCA cycle.
OK, great.
So, the first reaction of the TCA cycle.
Here's acetyl-CoA.
It will combine with oxaloacetate.
This reaction is catalyzed by an enzyme called citrate synthase,
and generates the six-carbon tricarboxylic acid citrate.
So, how does this reaction work?
Here's drawing acetyl-CoA in a slightly different way.
If I redraw this as the enol, this
will generate citrate via, effectively, that mechanism.
Next, citrate is converted by adding water
across this carbon-carbon, or by removing water
across this carbon-carbon bond, to generate
this intermediate called cis-aconitate.
So this intermediate is called cis-aconitate.
so all I did was dehydrate across that bond
so there's a double bond there.
And then if I re-add water across that bond,
I generate this molecule called isocitrate.
So effectively, to convert citrate to isocitrate,
I'm moving the hydroxyl group from that carbon
to this carbon.
To do that, I basically dehydrate, make a double bond,
remove water, re-add water across that bond
in the opposite direction to generate this molecule,
isocitrate.
This reaction is carried out by an enzyme called aconitase,
and converts citrate to isocitrate.
I think it's a little easier to see this reaction
if I draw it a slightly different way.
So, this here is just drawing citrate
by just slightly rotating the molecule to look like that.
And so I'm basically removing water here.
And then I'm now just adding water back
in the opposite orientation to generate isocitrate.
Now, you'll notice when I drew this--
if you look at citrate, this is actually a symmetrical
molecule, so the top half and the bottom half of citrate
are identical.
And so what's interesting about nature is
that it treats these carbons--
the green carbons that came from acetyl-CoA--
different from the side of the molecule that
comes from oxaloacetate.
And effectively, nature always moves the hydroxyl group
to this carbon that came from oxaloacetate,
and never moves it to this carbon
that came from acetyl-CoA.
This is an example where enzymes--
nature treats a symmetrical molecule like citrate
in an asymmetrical way.
And this has consequences for how carbon is actually traced
through the entire TCA cycle.
Because even though you might think things could get
scrambled at citrate, they never do.
Meaning an isocitrate-- it's always these green carbons
that came from acetyl-CoA.
You never get those green carbons
on the other side of isocitrate.
And so when we go through the TCA cycle,
I'll keep these carbons green until the point where
you can no longer distinguish which carbon came from
came from which reaction.
The next reaction is we're going to oxidize
this carbon of isocitrate.
So if we're going to oxidize that carbon,
those electrons have to go somewhere.
And so if we oxidize the carbon, we
can use NAD plus as an electron acceptor, reduce it to NADH.
That generates this intermediate.
So hopefully this is clear to everybody at this point,
but just in case.
So, this carbon here and isocitrate.
If I oxidize that alcohol to the ketone,
now I generate a hydride ion.
Those two electrons and the hydrogen can go to NAD plus
and reduce it to NADH.
That generates this intermediate.
It's called oxalosuccinate.
Which then, if you notice, the oxalosuccinate
is now a beta keto acid.
So alpha, beta.
The acid group is beta to the ketone,
so it's a beta keto acid.
Remember, beta decarboxylation is favorable,
and so I can lose that CO2.
And what I'm left with is this molecule, which
is called alpha keto glutarate.
So this whole reaction here, the oxidation
of the alcohol to the ketone, followed
by beta decarboxylation of oxalosuccinate
to alpha ketoglutarate, is carried out
by an enzyme called isocitrate dehydrogenase.
And of course, just to remind you,
here's that beta keto acid in oxalosuccinate.
And so that can decarboxylate, leading this enol.
Which can, of course, rearrange back to the ketone that we see,
an alpha ketoglutarate.
Now, if you look at alpha ketoglutarate,
you'll notice that-- and I will redraw over here.
So, this here is just me redrawing alpha ketoglutarate,
which is often abbreviated alpha kG.
Just drew it as a straight line.
Now, if you'll notice, alpha keto glutarate
is a alpha ketoacid.
And so here the acid group is alpha to the ketone.
So, an alpha keto acid.
It's effectively just like pyruvate,
but has this additional pieces on it.
And it turns out the next step in the TCA cycle
is the exact reaction that we saw
with pyruvate dehydrogenase.
It's alpha decarboxylation, oxidative alpha
keto acid decarboxylation, as follows.
So, we carry out.
This is a molecule called succinyl-CoA.
And so, you'll see what happened there is decarboxylated here,
this carbon, while oxidizing this ketone to the acid
and adding -CoA.
That's a redox reaction.
So NAD, NADH, it turns out this is exactly the same mechanism--
so I don't need to draw it again--
that I just showed you for pyruvate dehydrogenase.
So it needs all the same cofactors--
TPP plus, lipoic acid, FAD.
And in fact, it even shares some of the same enzyme complexes,
subunits, as pyruvate dehydrogenase.
And so this is a reaction that's catalyzed by alpha
ketoglutarate dehydrogenase.
Like pyruvate dehydrogenase, this is a complex,
and so it has a unique E1, which makes sense.
Remember, E1 of pyruvate dehydrogenase
was actually the subunit that bound the pyruvate.
E1 of alpha ketoglutarate dehydrogenase is unique.
It binds alpha ketoglutarate instead of pyruvate.
However, they share the same E2 and E3s,
and so the E2s and E3s would carry out
exactly the same reaction and play the same part
in the mechanism of how you do this alpha oxidative alpha
decarboxylation to turn alpha ketoglutarate
into succinyl-CoA.
Now, remember in pyruvate dehydrogenase,
once we got that acetyl-CoA, we then
use this CoA group to drive condensation with citrate.
Well, in this case, what happens is
you don't want to combine condensation here.
Instead, what is going to happen is
you want to use this CoA group to now generate ATP.
And so this is going to couple release of the CoA
to generate an ATP equivalent.
It's actually GTP that's generated by the TCA cycle.
And so, this is going to generate
this molecule, succinate.
And the enzyme that does this is called succinic bio-kinase.
So, let's go through over here how this enzyme works.
So here's succinyl-CoA, basically using favorable loss
of the CoA breaking the thioester bond to generate
this acid anhydride.
We saw an acid anhydride before in glycolysis.
Remember, we made 1-3-bisphosphoglycerate,
so that's a good phosphate donor.
And then that can be used to generate succinate,
and transferring that phosphate to GDP to make GTP,
just like 1-3-bisphosphoglycerate,
was able to transfer the phosphate
from the acid anhydride to ADP to make ATP in glycolysis.
So, the next step is to oxidize this carbon-carbon bond
in succinate.
So if we're going to oxidize a carbon-carbon bond,
those electrons have to go somewhere.
So there's our carbon-carbon bond.
And so if we generate hydride ion
just like we did in other oxidation reactions,
but this hydride ion is transferred not to NAD,
but instead to FAD, a different electron carrier,
to generate if FADH2.
Now, I should point out succinate, like citrate, is
a symmetrical molecule.
But at this point, nature doesn't tell the difference.
Once it generates succinate, this molecule
now gets scrambled.
And so everything downstream of succinate,
you no longer know which carbons came from acetyl-CoA.
This generates this molecule called fumarate,
and this reaction is carried out by an enzyme called
succinate dehydrogenase, often abbreviated SDH
for succinate dehydrogenase.
Now, the next reaction is, we're going
to add water across this double bond of fumarate.
And that generates this intermediate, malate.
This reaction is carried out by an enzyme
called fumarate hydratase.
And it's simply adding a water molecule
across that double bond.
Once we have malate, if you look what's
the difference between malate and oxaloacetate,
the difference is that in oxaloacetate, this carbon
is a ketone.
Whereas in malate, it's an alcohol.
And so if we want to turn this carbon from the alcohol
into the ketone, that is, of course, a oxidation reaction,
and so those electrons have to go somewhere.
Don't need to draw the mechanism again.
It's basically just the hydride transfer
to oxidize this to an alcohol, to the ketone.
That oxidation couples to a reduction of NAD plus to NADH.
This is carried out by an enzyme called malate dehydrogenase.
And doing this completes the TCA cycle,
regenerating oxaloacetate, that can then
recombine with another acetyl-CoA
to go through another round of the cycle.
You'll notice that going through this cycle,
there's two CO2s lost.
One of them is lost here at the isocitrate to alpha
ketoglutarate reaction.
So, this decarboxylation from oxaloacetate
to alpha ketoglutarate.
That beta decarboxylation, that's the first CO2.
The other one is lost here at the alpha ketoglutarate.
The alpha ketoglutarate dehydrogenase step,
where you have this alpha decarboxylation
to take alpha ketoglutarate to succinyl-CoA
oxidative alpha decarboxylation.
Now, what's cool about this, as you noticed,
we just discussed all the reactions of the TCA cycle.
And I showed you, reminded you, of some chemistry
that you've already seen, but unless you
count the chemistry we showed you earlier for how the PDH
and alpha ketoglutarate dehydrogenase
reactions work with E1, E2 and E3, with lipoic acid, FAD.
That was obviously new for today.
But other than that, everything else
was chemistry that you've already seen.
And this really points out the point
that I made earlier, that metabolism is really variations
on relatively few reactions.
We've just repurposed some of the same tricks, if you will,
that we're used in glycolysis, and allowed
it to now do an entire different pathway, the TCA cycle.
It also points out how Hans Krebs was-- well, is still--
remarkable, able to figure out from chemistry alone,
because there's actually quite a bit of logic to the way
metabolism works.
Now, I want to say this again.
Note there were two carbons that entered acetyl-CoA,
and two carbons that were lost to CO2.
But if you look, the green carbons
remain in the same places until they get to succinate.
and so the two carbons that enter
are not lost on the first turn of the cycle.
It's actually two carbons that came from oxaloacetate
that are converted to CO2 as that acetyl-CoA goes
through the cycle.
And so to oxidize the exact carbons from acetyl-CoA to CO2
requires more than one turn of the cycle.
You'll also notice that the cycle is oxidation.
And so oxidation reactions, of course, release energy.
We've talked about that.
And so it's favorable.
And the products, if you will, are three NADH molecules.
You can say plus one more NADH if we're going all the way
from glucose or from pyruvate.
Glucose derived pyruvate because the pyruvate dehydrogenase
reaction also generates an NADH to make that a acetyl-CoA.
One FADH2, as well as one GTP molecule.
And so lots of oxidation going on here.
We completely oxidized two carbons to CO2,
so that's energy release.
But you notice you only get one GTP from the molecule.
Now, this GTP, of course-- that reaction,
the succinic thiokinase reaction,
like the reactions we saw on glycolysis,
is such that it can generate GTP at a high DGP-GDP
ratio, or ATP-ADP ratio.
Remember, those are equivalent.
Those energy charge are similar, and so that makes sense.
But most of the energy released is actually
reducing NAD and FAD to NADH and FADH2.
And of course, these need to transfer their electrons
somewhere else, and that's the role of oxygen.
Oxygen, remember, is a very good electron donor,
and so it's the ultimate transfer of those electrons
from these molecules to oxygen that also provide energy
that the cell can use to do work, but it does so, in a way,
by charging up different ratios in the cell.
So the NAD-NADH or the FADH2-FAD ratios.
And just like we talked about, the ratio or the energy of ATP
is in the interconversion between ATP and ADP.
It's the ratio that drives the free energy change.
The same thing exists for an NADH and NAD, FADH2 and FAD.
And so charging up these ratios while passing through the TCA
cycle, and the ultimate downstream transfer
of those electrons to oxygen, really
is where most of the energy is captured as carbon
is oxidized through the TCA cycle.
And exactly how that works and how it can be related to ATP
will be something that'll be more explicit in the coming
lectures.
Now, I want to point out apart from the oxidation,
there's actually lots of intermediates made here.
And it turns out, a bunch of these intermediates
are useful for cells to make stuff.
So we talked about gluconeogenesis.
Gluconeogenesis needs electron balance.
We get NADH from the TCA cycle, and so you
can think of gluconeogenesis as an alternative
to fermentation to dispose of electrons.
Well, you can use the NADH from the TCA cycle
to run gluconeogenesis as well.
But beyond the cofactors, the carbon itself.
So citrate, I've alluded to now a few times,
is important as a precursor to make fat.
We'll discuss that in later lectures, too.
But other intermediates in this pathway
are useful for various amino acids and nucleic acids.
And so there's lots of things that
can come from the TCA cycle that cells
can find useful to do, not just catabolism,
but also anabolic processes.
Now, the way the TCA cycle works, though,
is that there's actually an issue
if you want to use the intermediates from the TCA
cycle to make stuff.
And so what is that issue?
Well, the TCA cycle functions at a site as a cycle.
And so, if we're going to take things in and out of it,
that has consequences for how the cycle runs.
Now there's a couple words for this
that I want to just introduce to you.
The first one is cataplerosis and the second one
is anaplerosis.
And so cataplerosis is the act of removing stuff
from a metabolic cycle.
So, we're going to remove citrate
from the cycle to make fat.
That's cataplerosis.
And anaplerosis is adding stuff back to a metabolic cycle
so it can continue to function.
Viewing this is really evident if you think of the TCA cycle
as a chicken and egg problem.
So, the very first time acetyl-CoA
was generated, how do you start the TCA
cycle in the first place?
You can't add it to the TCA cycle
unless you have oxaloacetate to combine
with the acetyl-CoA, which can then
generate another oxaloacetate.
So where does the first oxaloacetate come from?
Well, we already talked about one reaction.
We talked about it in the context of gluconeogenesis.
That can solve this problem.
And so we have pyruvate.
And so we talked earlier today how
we can do oxidative decarboxylation of pyruvate
to give acetyl-CoA.
But we talked to in the gluconeogenesis
lecture how we can add a CO2 pyruvate to generate
oxaloacetate.
So if I do those two reactions, now I
have all the carbon I need to generate a citrate
and start off the TCA cycle.
Now obviously, if I do cataplerosis
and I remove that citrate I made to make fat,
well, now I need two pyruvate again
to generate the next citrate if I'm using this pathway,
because every time I bring an acetyl-CoA into the cycle,
I need an oxaloacetate to combine it with.
And so if I remove something, I have to add something back.
And so pyruvate to oxaloacetate, the pyruvate carboxylase
reaction is an example of an anaplerotic reaction.
Now what this means, though, is that in order
to do an anaplerosis, you have to be
able to generate four-carbon oxaloacetate,
or a four-carbon molecule.
Now, pyruvate carboxylase, pyruvate to oxaloacetate,
allows you to do that.
We can take a three-carbon molecule
and generate four-carbon oxaloacetate.
However, if we start from a two-carbon molecule
like acetate or acetyl-CoA that enters the cycle,
it's actually not so simple to take that two-carbon molecule
and turn it into four-carbon oxaloacetate.
And in fact, humans lack any enzymes
that allow them to take a two-carbon unit,
to take acetate or acetyl-CoA, and turn it
into anything that's longer than net-- turn
it into anything that's longer than two carbons.
And this has important implications
for human physiology, because what it says
is that we can't make glucose from anything
that starts with something less than three carbons long.
So if you drink alcohol and you metabolize that alcohol
to acetate, it turns out you take fat
and you break down fat also to acetyl-CoA,
to acetate two carbons long.
There is no way to turn those molecules into glucose,
because you cannot generate the oxaloacetate to do
the anaplerosis that's necessary to get it there.
What this means is that our body can only
store calories that come from two carbon units,
fat or alcohol, as fat.
We can never turn them back into glucose or make glycogen.
And this is very relevant for those of you that
go to medical school, because it's
relevant to our physiology.
And that is, when our bodies exhaust
all of our stores of glucose, what happens?
Our liver can no longer do gluconeogenesis.
And so what happens?
Now it has to switch over to doing something else.
It has to work with two carbon units.
And ultimately, this is ketone metabolism, which we'll
talk about in a few lectures.
It also said that the body--
that this is also the basis of a very old adage that's
out there that some of you may have heard--
that you need to have some other fuel if you're
going to burn fat.
The basis for that is that fat is turned into two carbon
units, acetyl-CoA units.
And so if you're going to take those acetyl-CoA units
and ultimately burn them away, turn them into CO2,
you need a source of oxaloacetate,
or your TCA cycle won't work.
And you don't need a lot of something, but it is true.
You can't start just with acetyl-CoA as a human and turn
it into CO2, so you need some--
at least a little bit of oxaloacetate
to get your TCA cycle started.
Now, that's a problem that we as humans and other mammals face,
but it turns out there's lots of microbes out there
that grow just fine on acetate or on alcohol,
even if it's only carbon source.
And so those organisms must have some way
to build stuff from two carbon units.
That is a way to use two carbon units and do an anaplerosis.
And it turns out the way they do this is via something
called the glyoxylate cycle.
And so the glyoxylate cycle is an alternative version
of the TCA cycle that effectively uses two enzymes
that we lack as mammals.
And so I'll quickly tell you about it here.
So, this is isocitrate from the TCA cycle.
And some microbes have an enzyme called isocitrate lyase.
And what isocitrate lyase does is basically splits citrate
in half, such that the top portion of the molecule
is another TCA cycle intermediate, succinate.
And the bottom portion of a molecule
is this two carbon aldehyde called glyoxylate.
Glyoxylate can react with acetyl-CoA by another enzyme
that we lack as humans called malate synthase.
And I don't have time to show the mechanism again,
but malate synthase basically adds the two carbons
from acetyl-CoA to the aldehyde, the carbonyl,
the aldehyde carbonyl of glyoxylate in a reaction that
is, for all intents and purposes, exactly what happens
in citrate synthase.
That will generate this molecule, which is malate, also
in the TCA cycle.
And so having these two extra reactions, isocitrate lyase
and malate synthase, gives microbes
the ability to have acetyl-CoA be anaplerotic.
And so how does that work?
Well, that's because if we start with one oxaloacetate, four
carbons, and acetyl-CoA, two carbons, can run the TCA cycle
and make citrate, six carbons.
Turn that citrate into isocitrate.
Use isocitrate lyase to generate glyoxylate, two carbons.
Plus succinate.
That succinate can run through succinate dehydrogenase
to generate malate, which you can
go through malate dehydrogenase to generate oxaloacetate.
This glyoxylate can start with a second acetyl-CoA.
Two carbons come together, generate malate.
That generates a second malate molecule,
which can then exit the cycle as malate or oxaloacetate
or whatever you want.
And so basically, it allows two acetyl-CoAs to net
generate an oxaloacetate, and so net generates a way
to do an anaplerosis from two carbon units
by having this malate synthase reaction
and this isocitrate lyase reaction
and run this alternative version of the TCA cycle,
called the glyoxylate cycle.
And it's a nice way how life-- again, no new chemistry here,
just variations on what we've already shown--
repurposed the similar chemistries
that it's already using as a way to live off
of carbon sources that contain only two carbons, like ethanol
or acetate.
So in closing today, the last thing
I want to talk about, very briefly,
is how the TCA cycle is regulated.
And we don't need to spend a ton of time
on this, because it really follows principles that
make sense, particularly when we think about things that we've
already talk about.
And so, regulation of the TCA cycles is of course important,
and it's really a critical hub, both for anabolic and catabolic
pathways.
So, you needed to get energy to fully oxidize carbon,
but it's also a useful place to get stuff.
And before I talk about regulation,
I just want to point out that many of the enzymes in the TCA
cycle, even though we talk about it in the mitochondria
and we're about to talk about regulation in terms
of catabolism-- that is oxidation,
ways to release energy--
many of these enzymes are also in other locations
in the cell, because there's functions for them in making
stuff that is very different than what goes on in the TCA
cycle.
And the regulation that I'll tell you about,
and that comes up on MCAT exams and stuff
like that, usually talks about this pathway
as a catabolic pathway, as a way to make CO2, to make ATP.
However, recognize that there's also
variations on this pathway that can use in anabolic things,
making stuff.
And that regulation is something that really is not necessarily
what we're going to talk about here
and is a little bit less well-understood.
But at least the regulation, in terms
of catabolism, if we put it in context of glucose metabolism--
so here's glycolysis, turning glucose into pyruvate.
And then that pyruvate can operate
through the pyruvate dehydrogenase reaction
to generate acetyl-CoA.
That acetyl-CoA can combine with oxaloacetate
to generate citrate.
That citrate, of course, can be used to generate fat,
as we've talked about.
That can go to isocitrate.
Isocitrate to alpha ketoglutarate.
That's catalyzed by isocitrate dehydrogenase,
which I will abbreviate IDH.
Alpha ketoglutarate to succinate.
More correctly, to succinyl-CoA by alpha ketoglutarate
dehydrogenase, Alpha KGDH.
And then this back to oxaloacetate.
And so the main enzymes that are typically
discussed as being regulated are alpha ketoglutarate
dehydrogenase, isocitrate dehydrogenase,
and pyruvate dehydrogenase.
I also drew glycolysis.
And I'll just write over here, gluconeogenesis up here,
because we can now see more fully how this plays
in with citrate acting as a positive regulator
with gluconeogenesis and an inhibitor of glycolysis.
Again, we're full in citrate, stop running glycolysis,
start running gluconeogenesis.
Now, the other regulation.
If you have a lot of acetyl-CoA, stop making it
from pyruvate. acetyl-CoA is a negative regulator of PDH.
This is carbon oxidation.
Releases a lot of energy, generates a lot of ATP,
generates a lot of NADH.
If you have lots of those things,
no reason to keep sending carbon into
acetyl-CoA to go into the TCA cycle.
Ultimately, we have to send those electrons somewhere.
So if there's nowhere to put them,
low oxygen also inhibits pyruvate dehydrogenase.
And of course, if you need more energy, if ADP is high,
that activates pyruvate dehydrogenase.
So at least in terms of glucose, complete glucose oxidation,
a lot of regulation happens at pyruvate dehydrogenase.
And a lot of it really makes sense.
High levels of ATP, high levels of NADH,
also inhibit isocitrate dehydrogenase and alpha
ketoglutarate dehydrogenase.
Makes sense.
Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase.
High levels of ADP, need to release more energy,
activate alpha ketoglutarate dehydrogenase.
Again, these are the feedbacks that people talk about
on board exams.
Good to know, but you can almost guess what they
would be from first principles.
Because remember, this is a pathway
that releases a lot of energy.
High energy high ATP, high NADH--
don't run the cycle, don't enter carbon in the cycle.
Low energy, high ADP, put carbon in the cycle,
run the cycle faster.
Makes sense.
Also makes sense of the reciprocal regulation
of glycolysis and gluconeogenesis.
Great.
Next time, we will talk more about
how we can oxidize fatty acids, fat, by accessing acetyl-CoA
and entering it into the cycle.
Thanks.