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JOHN ESSIGMANN: Let's take a look at storyboard number 10.
Back in earlier sessions, we talked
about the detail of glycolysis.
One of the points that I emphasized
is the fact that it's necessary to maintain
redox neutrality in the cytoplasm of a mammalian cell.
It's also necessary to maintain redox neutrality
and prokaryotic cells, not just eukaryotic cells.
At the glyceraldehyde 3-phosphate dehydrogenase step
of glycolysis, NAD+ was consumed and converted to NADH.
That means we have to find a way to convert NADH back to NAD+
in order to make glycolysis a continuous process.
Back in lectures three and four, I said that there were three
ways to convert the NADH back to NAD+ These were alcoholic
fermentation in anaerobic cells.
Homolactic fermentation, again, in an anaerobic environment.
And the third is respiration, which occurs
in the presence of oxygen.
I'm going to loop back now and revisit this topic.
I want to spotlight three general strategies
that cells use to achieve redox neutrality in the cytoplasm.
The first is lactate dehydrogenase.
The second is called the glycerol-3-phosphate shuttle.
And the third is called the malate-aspartate shuttle.
Panel A of this figure shows the cytoplasm working in concert
with the mitochondrion.
You can see depicted on the left,
the pathway of glycolysis.
In the middle, pyruvate dehydrogenase.
And to the right, the citric acid cycle or TCA cycle,
which we just covered.
There are two important boundary conditions for the discussion
we're about to have.
The first is that oxaloacetate is,
as I mentioned earlier, present only in very small
concentrations within the cell and especially
in the mitochondrion.
As a consequence, the mitochondrion
does not have a transporter to allow it to escape.
In other words, its concentration
is preserved at about one micromolar
inside the mitochondrion.
The second boundary condition concerns the fact that NAD+
and NADH, as well as NADP+ and NADPH cannot go directly across
the mitochondrial membrane.
So in other words, there are two separate pools
of this nucleotide co-factor.
One in the cytoplasm.
One in the mitochondrion.
I'll come back to the importance of the two
pools in just a few minutes.
Let's look first, here in panel A,
at the mechanism by which lactate dehydrogenase achieves
redox neutrality in the cytoplasm.
We've already covered this, so this is a bit of a review.
Note that you see NAD+ getting converted to NADH
in the cytoplasm.
That's at that GAPDH or glyceraldehyde
3-phosphate dehydrogenase step.
The hatched lines that you see represent
the flow of electrons.
In other words, electrons flow from glucose
and they end up in NADH.
Then the lactate dehydrogenase enzyme
transfers the electrons from NADH into lactate.
So these electrons from glucose are
involved in the reduction of the ketone functionality
of pyruvate into the alcohol functionality of lactate.
And that's where the electrons stay.
The other product of this reaction, as you'll see,
NAD+ which is now available to enable the oxidation
of the next molecule of glucose.
What happens to the lactate that's produced?
In a working muscle cell, that lactate
will escape from the cell, go into the blood,
and then go to the liver or another organ that's
capable of doing the pathway of gluconeogenesis.
As I've mentioned in the past, gluconeogenesis
is a pathway by which non carbohydrate
precursors, such as lactate, are built back up into glucose.
Keep that working muscle scenario in mind,
because I'm going to come back to it later
when I talk about physiological responses
to stress, such as what I'll call the fight and flight
scenario.
That's all I'm going to say for now about the LDH shuttle.
That is, lactate dehydrogenase shuttle
in panel A, which is the first of the three pathways
by which redox neutrality is maintained in the cytoplasm.
The second pathway to retain redox
neutrality is the glycerol-3-phosphate shuttle.
This pathway is particularly active in the brain
and in skeletal muscle.
Once again, follow the hatched lines
to follow the path of electrons as they go from glucose.
And ultimately, in this case, they're
going to end up being deposited into oxygen to form water.
Starting at the top, you see in NAD+ being reduced to NADH
at the glyceraldehyde 3-phosphate dehydrogenase step,
GAPDH.
Next, we're going to temporarily borrow
a molecule of dihydroxyacetone phosphate, DHAP.
DHAP is a ketone.
And what we're going to do is deposit the electrons from NADH
into the ketone functionality to make the alcohol,
glycerol-3-phosphate.
The source of the electrons was NADH.
And now you've accomplished your chemical goal,
which was to restore the NAD+ pool -- the cytoplasm,
but we borrowed a molecule of dihydroxyacetone phosphate.
And we've somehow got to get that back.
Let me point out, at this point, that the reduction of DHAP
to glycerol-3-phosphate was accomplished
by the cytoplasmic form of the enzyme glycerol-3-phosphate
dehydrogenase, which catalyzed step 2 on the storyboarded.
We're going to deal more with coenzyme q in the next lecture.
But for now, it's a molecule, specifically
a quinone, that's easily reduced to its hydroquinone form,
called QH2.
The structures of q, in QH2, are shown in the box.
QH2 is in the mitochondrial membrane.
In glycerol-3-phosphate dehydrogenase
the mitochondrial version of it is
present in the outer part of the mitochondrial inner membrane.
Like NADH and FADH2, QH2, the hydroquinone,
is what I've called a mobile electron carrier.
QH2 is going to allow the electrons that started out
in glucose or in any LDH of the gap DH step,
to flow through the electron transport chain, which we'll
come to in the next lecture.
And flow into oxygen, which is reduced to form water.
This terminal reduction is shown in step five.
Effectively, in the glycerol-3-phosphate shuttle
we're using oxygen in order to oxidize NADH back to NAD+ And
once again, maintaining a constant supply of NAD+ is
necessary in order to make glycolysis a continuous
process.
Panel C shows a third strategy for maintaining redox
neutrality in the cytoplasm.
This is called the malate-aspartate shuttle.
and this pathway is operative in heart, liver, and kidney.
To the left we see the production of NADH,
just as we did in the previous two small pathways.
At step one, let's assume that there's
a molecule of oxaloacetate present as part
of the cytoplasmic pool of organic acids.
Oxaloacetate or OA is ketone.
And the cytoplasmic form of the enzyme malate dehydrogenase,
working in the reverse direction from the one
that we see operative in the TCA cycle
is able to reduce the oxaloacetate to malate.
We just reduce to ketone OA to an alcohol malate.
In step three, the accumulating malate
is transported by a malate transporter
into the mitochondrial matrix, which is, of course,
the location of the TCA cycle.
At this point, we're going to be using one
of the steps of the TCA cycle.
Specifically, we're going to use malate dehydrogenase,
the mitochondrial version of the enzyme
this time, to convert malate to oxaloacetate.
That reaction is an oxidation.
We use the mitochondrial pool of NAD+ to carry out that
oxidation.
In effect, we're using the electrons that came in from
malate to reduce NAD+ to end NADH in the mitochondrion.
Now, take a careful look at step four.
Looking to the left you see the hatched lines go all the way
back to glucose, which was the source of the electrons.
To the right, the hatched lines by step five,
go to the electron transport chain all the way to oxygen.
We haven't done the electron transport chain as yet.
So you're just going to have to trust me for a little while.
There's an enzyme, NADH dehydrogenase,
in the mitochondrial inner membrane that will take
the electrons from NADH and eventually regenerate the NAD+
in the mitochondrial matrix.
In step five, we're taking the electrons
from the NADH produced by malate dehydrogenase
and entering those electrons into the electron transport
chain.
Then, in a manner that's quite similar to what
we did in the previous shuttle, the glycerol-3-phosphate
shuttle, those electrons are going
to be transferred to oxygen to make water.
Before I go on, let's review a little bit.
Between step one and step two in the cytoplasm,
we deposited electrons into oxaloacetate to make malate.
That step restored in NAD+ levels in the cytoplasm,
which is what we wanted to accomplish.
However, we've consumed a molecule of oxaloacetate.
And as I've mentioned before, the cell
has to try to preserve the concentration of this very
precious molecule.
We now have to find a way to restore oxaloacetate
that we borrowed in step one.
Now let's look back at step four,
where malate was converted to oxaloacetate
in the mitochondrial matrix.
Because the molecule of malate came from the cytoplasm,
this is a net increase in the mitochondrial matrix
of one unit of malate and, ultimately,
one unit of oxaloacetate.
We need to find a way to get that molecule of oxaloacetate
back out into the cytoplasm, in order to make
the shuttle a continuous one.
In the co-factor section of 5.07,
JoAnne taught us about the ways that
pyridoxal phosphate and pyridoxamine work, in order
to put amino groups into organic acids, such as oxaloacetate.
And that's what's going to happen in this case.
Oxaloacetate is converted into its amino acid homolog,
aspartic acid.
Why did we do this emanation reaction?
Well, there's no way to get oxaloacetate directly out
of the mitochondria because there's no transporter for it.
But there is a good transporter, the apartheid transporter,
that will take aspartic acid out into the cytoplasm.
So the oxaloacetate is converted, temporarily,
into aspartic acid in the mitochondrion.
And that aspartic acid then slips out
through its transporter to the cytoplasm.
Once in the cytoplasm, there's a similar pyridoxal mediated
mechanism to deaminate the aspartate
to regenerate the cytoplasmic molecule of acetate
that we borrowed at step one a few minutes ago.
In panel D I summarize.
That we've looked at three different small pathways that
enable the cytoplasm of the cell to always have enough NAD+
to oxidize glucose to pyruvate.
These pathways are first, the lactate dehydrogenase system.
Second, the glycerol-3-phosphate shuttle.
And third, the malate departed shuttle.