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TRACKING CARBONS
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Glucose to Pyruvate
TCA Cycle
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GLUCOSE TO PYRUVATE
Glucose
(O“)CH–CH(OH)–CH(OH)– ƒ –CH(OH)–CH(OH)–CH2(OH)
1
2
Pyruvate
3
5
1,6
2,5
3,4
CH3—C(“O)—CO2
3
Acetyl-CoA
4
2
1
6
Glucose numbers
Pyruvate numbers
1,6
2,5
3,4 Glucose numbers
CH3—C(“O)—ScoA CO2
2
1
Acetyl-CoA numbers
Tracing labeled carbon atoms through metabolic pathways would, at first
glance,1 appear to be a pretty irrelevant thing to make you do. But if
you’ve got to do it, there are a couple of conceptual tricks that make it
somewhat easier.
The first concept is that organic compounds are numbered starting
with the end of the molecule that is closest to the most oxidized carbon.
1
The second and third glances may appear this way too.
236
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Tracking Carbons
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In glucose, C-1 is the carbon of the aldehyde. In fructose
[CH2(OH)–C(“O)–CH(OH)–CH(OH)–CH(OH)–CH2(OH)], C-1 is the
carbon at the end closest to the C“O; the C“O itself is C-2.
Labeling a given molecule at a specific carbon atom means that an
isotopically labeled carbon atom (14C or 13C) has been introduced at a
given position in the starting molecule. For example, the statement that
glucose has been labeled at C-2 means that only carbon number 2 of glucose has been tagged with a 14C or 13C carbon atom. The other carbon
atoms are not labeled (they are 12C). The problem is to determine where
this label ends up after the glucose is metabolized to some other compound.
Tracing the carbons of glucose to pyruvate gets complicated when
fructose 1,6-bisphosphate (FBP) is cleaved into two 3-carbon fragments,
glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate
(DHAP). The numbers of the original carbons of glucose are indicated
by the superscripts next to the carbon.
CHOH
—
5
6
CH2OP
FBP
Pyruvate
Number
CHOH
CH (— O)
G3P
3,4
CO2
1
2,5
C —O
2
1,6
CH3
3
—
3
—
5
CHOH
—
—
CH2OH
3
CHOH
DHAP
Aldolase
–
4
4
CH
(— O)
CHOH
2
—
3
CH2OP
—
C —O
1
TIM
—
C —O
—
2
2
—
—
CH2OP
CH2OP
—
1
1
6
CH2OP
G3P
The guilty party is the triose phosphate isomerase (TIM) reaction that
interconverts DHAP and G3P. To be converted to pyruvate, the DHAP
first has to be converted to G3P. TIM just moves the carbonyl group
between the two carbons that don’t have phosphate attached. TIM doesn’t
touch the phosphate. So, if the DHAP is labeled at the carbon that has
the phosphate attached, the G3P that comes from DHAP will be labeled
at the carbon with the phosphate attached. The carbon with the phosphate
attached in the G3P that was produced directly by the aldolase reaction
came from C-6 of glucose, but the carbon with the phosphate attached in
the G3P that was produced from DHAP came from C-1 of glucose. After
TIM does it stuff, the carbon of G3P that has the phosphate will be
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Basic Concepts in Biochemistry
labeled if either C-1 or C-6 of the original glucose was labeled. This all
makes sense if you remember that pyruvate only has 3 carbons but glucose has 6. Each carbon atom of pyruvate must come from 2 different
carbons of the original glucose molecule.
The carboxylate group of pyruvate, being the most oxidized, is
called C-1, and the CH3 group is called C-3. The carboxylate group of
pyruvate comes from the aldehyde of G3P, so C-4 and C-3 of glucose
end up at C-1 of pyruvate. There is an easy way to remember which carbons of glucose end up on the same carbon of pyruvate—the numbers of
the equivalent carbons sum to 7.
The labeling trick also works backward. You could have to decide
which carbons of glucose become labeled when you use pyruvate labeled on
a given carbon. Labeling pyruvate on C-1 will result in a glucose molecule
that is labeled on both C-3 and C-4, again because of the TIM reaction.
TCA CYCLE
The two carbons entering from acetyl-CoA do not leave as CO2 on
the first cycle.
Carbons 2 and 3 of succinate are equivalent.
Carbons 1 and 4 of succinate are equivalent.
To actually understand how labeled carbons travel through the TCA
cycle, you have to draw out the chemical structures of the cycle members, put a tiny little asterisk by the labeled carbon, and follow it around
and around. There are two concepts, however, that you have to know in
order to do this. First, the two carbons entering the cycle as acetyl-CoA
are not lost as CO2 during the first turn of the TCA cycle.
—
C—O
—
CH2
—
*CH2 — CO2
HO — C — CO2
—
—
*CH3 — C(— O) — SCoA
CO2
CH2 — CO2
Citrate
CO2
Oxaloacetate
The two CH2–CO2 arms of citrate are different (Fig. 22-1). They can be
(and are) distinguished by the aconitase enzyme. To see that they’re dif-
BG McGraw-Hill: Gilbert, Basic Concepts in Biochemistry, JN 5036
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Tracking Carbons
BASE
H H
H
*
CH2CO
2
OH
CO
2
H OH CH2CO
2
H
ACID
BASE
Correct citrate binding
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CO2 H
*
ACID
If citrate binds incorrectly,
the OH and H are in the wrong
orientation for the reaction
Figure 22-1
THE TWO CH2CO
2 ARMS OF CITRATE ARE NOT IDENTICAL, and
the enzyme aconitase can tell them apart. The CH2CO2 arm that was derived
from acetyl-CoA (*) is not metabolized to CO2 during the first turn of the TCA
cycle.
ferent, imagine grabbing the CO
2 group of the central carbon of citrate
in your right hand, and the OH group of the central carbon in your left
hand. The CH2–CO
2 that came from acetyl-CoA will be pointing up
while the CH2–CO
2 that came from oxaloacetate will be pointing down.
Aconitase holds onto citrate in the same way, and when it moves the
hydroxyl group of citrate, it moves it specifically from the central carbon
to the CH2 group on the bottom. Aconitase can move the OH group only
because the catalytic apparatus is in the right place. If we were to try to
bind citrate to aconitase with the CH2–CO
2 that came from acetyl-CoA
in the down position, the CO
2 on the central carbon would be on the left,
and this wouldn’t do. Aconitase is set up to expect the CO
2 group on the
central carbon to be on the right.
As we follow the label from citrate to succinyl-CoA, there are no
problems. But at succinate, it all seems to fall apart.
CH2C(— O)SCoA
*CH2CO2
—
—
*CH2CO2
CH2CO2
Unlike the CH2–CO
2 groups of citrate, the two CH2–CO 2 groups of succinate are indistinguishable. If you pick up succinate by the labeled CH2
group, you will always be able to pick up succinate by the unlabeled CH2
group so that it looks exactly the same (except for the label).
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Basic Concepts in Biochemistry
CO2
H — C— H
H — *C — OH
H — *C — H
H — *C — OH
—
—
—
H — C—H
CO2
—
—
H — *C — H
CO2
—
CO2
—
CO2
—
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CO2
Malate
The consequence of the symmetry of succinate is that the enzyme succinate dehydrogenase has a fifty-fifty chance of picking up succinate in
either of the equivalent orientations. By the time you get to malate, in
which all the carbons are obviously different, one-half of the original
label is found at the CH2 group of malate while the other half of the label
is on the CH–OH group of malate. Each molecule of malate has only one
labeled carbon; however, in the collection of all malate molecules, the
label is equally distributed between the two carbons in the middle [if it
started out on the CH3 group of acetyl-CoA (C-2)]. The same thing happens with the label that comes into the TCA cycle from the C-1 of acetylCoA except that the label ends up equally distributed between the two
carboxyl groups of the malate.
Since neither of the carbons that come in from acetyl-CoA is lost
during the first turn of the TCA cycle, it’s reasonable to wonder when
they are lost. If the label was originally at C-1 (the C“O) of acetyl-CoA,
it ends up in the two carboxylate groups of oxaloacetate. On the next turn
of the TCA cycle (go around again without bringing any more label in
from acetyl-CoA) both of these carboxyl groups are lost as CO2. So when
C-1 of acetyl-CoA is labeled, all the label is lost from the TCA-cycle
intermediates on the second turn of the cycle.
It’s a lot more complicated when C-2 of the acetyl-CoA is labeled.
After the first turn of the cycle, this label ends up on the central carbons
of oxaloacetate, and neither of these is lost during the second turn of the
cycle. However, on the third turn of the cycle, half the label is lost
because half the total label is on the carboxylates of oxaloacetate. On
each subsequent turn of the cycle, half the remaining label is lost. The
only way to sort this out for yourself is to sit down with the TCA cycle
and go round and round. It’s a dizzying experience that leaves you a little bit nauseated when it’s over.
BG McGraw-Hill: Gilbert, Basic Concepts in Biochemistry, JN 5036