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JOHN ESSIGMANN: We're now at storyboard 19,
panel B. In the last lecture, I mentioned
that we're going to be looking at several special cases
with regard to the metabolism of fatty acids.
The first special case concerns the metabolism of fatty acids
that already contain double bond.
You remember from last time that a double bond
with a trans configuration forms naturally
during beta oxidation.
In the special case we're going to look at now,
the double bond has a Cis configuration.
Fatty acids with a Cis-double bond
typically come from membranes.
One of the ways nature plasticizes
our membranes is to make them more fluid
by putting in Cis-double bonds that bend the fatty acid
molecule and reduce stacking.
Ultimately, this reduction in stacking
results in a lowering of the overall melting temperature
of that part of the membrane.
The example I'm going to use is oleyl Coenzyme A.
This fatty acyl Coenzyme A comes from Oleic acid, which
also has the shorthand notation (18:1) delta 9.
That is, there is a double bond nine carbons in
from the carboxylate.
The reaction I've shown in panel B
goes through three rounds of beta oxidation
resulting in a Cis delta three enoyl Coenzyme A.
The enzyme oleyl Coenzyme A isomerase
will then remove the acidic hydrogen from the 2 carbon,
repositioning the double bond, and then
putting that double bond into trans configuration, which
is now amenable to further hydration and beta oxidation.
Overall, the isomerase has taken the Cis-double bond
and repositioned it to make it a trans double bond that is now
able to undergo further oxidation
by the classical beta oxidation pathway.
Let's turn now to panel C of storyboard 19.
The second special case that I'd like
to deal with concerns the way that we metabolize
fatty acids that have an uneven, that is odd,
number of carbons in them.
One example would be the C15 fatty acid Pentadecanoic acid,
which we get from milk fat.
In this case, six rounds of beta oxidation will release six
acetyl Coenzyme A's.
But note that we're left with a 3 carbon residue,
or carboxylic acid called propionyl Coenzyme A.
Nature doesn't like to throw anything away.
It's going to take this three-carbon molecule,
propionyl CoA, that doesn't easily
integrate into any other biochemical pathways.
Nature's going to add a carbon to it,
hence making it into a four-carbon molecule.
As we'll see, that four-carbon molecule
will integrate seamlessly into the TCA cycle.
The conversion of the three-carbon compound
into a four-carbon compound begins
with the enzyme propionyl Coenzyme A carboxylase.
In a few minutes, we'll look at the fine details
of how this enzyme works.
For now, however, what it does is use the cofactor
biotin in order to introduce a carbon dioxide, or CO2,
into the 2 carbon of the propionyl Coenzyme A.
The 2 carbon is the middle carbon of the propionyl group.
This is now a branched molecule that's
called methylmalonyl Coenzyme A.
Next, the molecule is subjected to a carbon chain rearrangement
that takes the branched molecule and linearizes
it to form succinyl Coenzyme A. Succinyl Coenzyme A will then
integrate directly into the TCA cycle
to allow its carbons to be metabolized fully to CO2.
In summary, nature puts a CO2 into
the three-carbon propionyl CoA, forming a four-carbon branch
structure.
Then, in an amazing rearrangement
that we'll look at later, the branched molecule
is made linear, forming succinyl CoA, which
is a TCA cycle intermediate.
So the otherwise useless C-3 molecule, propionyl Coenzyme A
is converted to something of value.
Lastly, let's turn to panel D. I'm
going to deal more with the details of this reaction
in a few minutes.
But for now, what I'd like to say
is that there are many sources of propionyl CoA,
not just from odd chain fatty acids in the diet.
Secondly, the introduction of succinate into the TCA cycle
results in an increase in the amount of carbon
going through the TCA cycle.
That is, this is truly an anapleurotic reaction.
This is a reaction that we can use
to increase the overall rate of carbon metabolism
in the TCA cycle.
We're now going to turn to storyboard 20.
Let's look at panel A. In the last lecture
on fatty acid metabolism, I talked about propionyl CoA
carboxylase, which uses biotin to add
a CO2 moiety to the middle carbon of propionyl Coenzyme
A. This lecture is a chemical interlude, in which we're going
to look at propionyl CoA carboxylase
and several other carboxylases that are used in biochemistry.
One of these is acetate CoA carboxylase.
This carboxylase puts the CO2 group
onto acetyl CoA to make malonyl CoA, which is the precursor
to fatty acids during the fatty acid biosynthesis reactions.
Let's also look at panel B. We're also
going to be looking at pyruvate carboxylase, which
adds a carbonyl group to the pyruvic acid molecule in order
to make oxaloacetate.
This is one of the anaplerotic enzymes that
can increase the quantity of carbon going through the TCA
cycle.
That is, it facilitates anapleurosis
in the biological system.
Let's now look at storyboard 21, panel A. The
carboxylases all use carbon dioxide
as their carboxylating reagent.
CO2 itself is not very soluble in water,
so cells use carbonic anhydrase in order
to hydrate it to form carbonic acid.
The biotin carboxylase subunit will then
phosphorylate the carbonic acid in order
to form a chemically-reactive intermediate
known as carbonyl phosphate or carboxy phosphate.
Now, carbon dioxide by virtue of its structure-- that is,
a carbon with two electronegative oxygens pulling
on it--
is pre-activated for nucleophilic attack.
And as I said a few minutes ago, CO2
is just not present at high enough concentration
in the cell for it to be able to add effectively
to nucleophiles.
A nucleophile such as the amide nitrogen of biotin
would be an example.
So what the cell does is use ATP to make
carbonyl phosphate, which is a very water-soluble molecule.
It's a very efficient delivery vehicle for carbon dioxide
into the active site of an enzyme.
Now let's look at panel B. The biotin carboxylase
subunit of pyruvate carboxylase has
a biotin cofactor at the end of a 14-Angstrom-long swinging
arm.
This subunit of the enzyme breaks
the bicarbonate phosphate bond and releases carbon dioxide
in high concentration in the active site.
Biotin on the swinging arm then attacks the high concentration
of carbon dioxide in order to become N-carboxy biotin
as shown in this figure.
The N-carboxy biotin takes advantage of its swinging arm
to move away from the active site at which it acquired
the CO2 and then move to a second site
on the enzyme at which the arm releases carbon dioxide, once
again at high concentration.
But this time CO2 is released at high concentration
in the vicinity of the substrate, pyruvate.
Pyruvate, in order to be a good nucleophile to acquire the CO2,
probably exists as shown in the figure as the pyruvate enolate.
Let me review panel B at this point.
Overall, this reaction has resulted
in putting a carboxylic acid residue
on the 3-carbon of pyruvate.
And this results in the formation of oxaloacetate.
Thus, activation of pyruvate carboxylase
will result in taking a molecule of pyruvate
and converting it to oxaloacetate.
This anaplerotic reaction gives the ability
to increase the volume of carbon that's
cycling in the TCA cycle, thus allowing
you to be able to increase their overall rate of respiration.
And as I've said several times in the past,
oxaloacetate is present at rather limiting concentrations
within the mitochondrial matrix.
So this is a very useful biochemical reaction.
At this point, let's move on to story board 22
and look at panel A. Let me loop back now and give some bigger
picture points with regard to carboxylase chemistry.
Pyruvate carboxylase and the other enzymes of this class
have two active sites.
One of them is the biotin carboxylase domain,
where a biotin moiety covalently attached to the enzyme
will acquire CO2.
Let's call this step 1 of the reaction scheme.
Once the biotin on the swinging arm
has been N-carboxylated as in step 1,
the N-carboxy biotin moves to site 2, where the substrate is.
The second site is the biotin transferase domain.
In the example I just used, the substrate was pyruvate.
But it just as easily could have been
another quote unquote "carboxylation substrate,"
for example propionyl CoA.
In the case of pyruvate, as we just saw,
it's the pyruvate enolate that becomes carboxylated in site 2
in order to form the final product oxaloacetate.
This carboxylation of substrate is the second and final step
of the overall reaction.
Take a look at panel B. Now let me give you a second example
of carboxylase chemistry.
In this case, our substrate will acetyl
Coenzyme A. We're going to use the same chemistry we described
a minute ago for pyruvate, but in this case
it is another carboxylate-- acetyl CoA carboxylate--
that picks up the CO2 residue at its carboxy transferase domain.
In step 2, which occurs in the carboxy transferase domain,
the substrate acetyl CoA picks up CO2 on its 2-carbon--
the one distal to the Coenzyme A group--
to form the three-carbon product malonyl Coenzyme A.
We'll see later that the carbon dioxide that's
been added to acetyl CoA to form malonyl CoA
is an excellent leaving group.
Malonyl CoA is the fundamental precursor to all fatty acids
through the fatty acid biosynthesis pathway.
Again, we'll see the details of this reaction scheme
later, but keep in mind now that this
is a biotin-requiring reaction, just as the one we just looked
at with pyruvate carboxylase.
Let's look now a panel C. My last example
of carboxylase chemistry will be propionyl
Coenzyme A carboxylase.
I mentioned earlier that the breakdown of odd chain number
fatty acids results in the formation
of a 3-carbon linear fatty acid called propionyl CoA.
I also said that nature doesn't want
to waste any of the carbons.
What she is going to do is add a carbon dioxide or a CO2
to the central carbon of the propionyl group.
As well as fatty acids with odd numbers of carbons,
certain amino acids--
such as isoleucine, methionine, valine, and threonine,
for example--
also break down to form propionyl CoA.
So what I'm going to do now is describe
a fairly general method of metabolism
that involves restructuring of the carbon chain
subsequent to the carboxylase reaction.
In this overall reaction, a carboxylase
is going to carboxylate the middle carbon
of the propionyl group.
Then, following the carboxylase reaction,
there's going to be a skeletal rearrangement
to form succinyl CoA, which will then enter the TCA cycle.
As shown in the reaction scheme, propionyl Coenzyme A
carboxylase carboxylates propionyl CoA to form a very
specific stereoisomer, (S)-methylmalonyl Coenzyme A.
Please note the position of the carbonyl group
that's highlighted in blue.
This carboxylate in blue is attached
to a methylene carbon that has an inverted triangle on top
of it.
Epimerization about the carbon with the triangle
results in the blue carboxylate pointed down
the way I've drawn it.
The resulting epimer is (R)-methylmalonyl Coenzyme A.
In the (R)-methylmalonyl Coenzyme A molecule,
I put a little lasso around the hydrogen atom at the methylene
moiety of the molecule.
I've also lassoed an electron in some
of the atoms of the Coenzyme A moiety.
A vitamin B12-dependent enzyme called methylmalonyl Coenzyme
A mutase, which contains an adenosylcobalamin functional
group, will now act on this molecule.
In this enzyme, a cobalt residue is
going to enable the formation of an adenosyl radical.
Look at the book for the detailed mechanism.
But for now, it's sufficient to say that this adenosyl radical
is going to facilitate the homolytic scission of the bond
between the carbon and hydrogen of the (R)-methylmalonyl
Coenzyme A. That is, you're going to get a free radical
formed at the carbon that has the small blue o written over
it.
That molecule will lose its hydrogen atom.
That hydrogen atom will migrate to the carbon that
has the small blue triangle.
Coordinately, the carbon with the small box
that's part of the Coenzyme A carbonyl group
will migrate to the position originally occupied
by the hydrogen atom on the carbon
with the small o over it.
The skeletal re-arrangement is shown here in panel C,
and it results in a linearization
of the original methylmalonyl Coenzyme A.
If we redraw it as in the box, you'll
see that you form succinyl Coenzyme A. Succinyl Coenzyme
A will then feed directly into the TCA cycle.
This is an anaplerotic reaction.
That is, it will increase the volume of carbon that's
rotating in the TCA cycle.
Overall, in the way of a review, the three-carbon compound
propionyl Coenzyme A, by way of the carboxylase reaction,
is converted to a four-carbon branched molecule called
(S)-methylmalonyl Coenzyme A. It's epimerization forms
the (R)-methylmalonyl Coenzyme A,
which is the substrate for a third enzyme,
methylmalonyl Coenzyme A mutase, a vitamin B12 enzyme.
This enzyme catalyzes a free radical-mediated rearrangement
of the side change.
Specifically, one hydrogen and one
Coenzyme A functionality switch positions.
This rearrangement linearizes the molecule
forming, ultimately, succinyl Coenzyme A, which
then flows into the TCA cycle.