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VIII. THE TRICARBOXYLIC ACID CYCLE
THE HISTORICAL BACKGROUND
The further catabolism of acetyl CoA, whether it is the end product of aerobic glycolysis or derived from the
oxidative degradation of fatty acids (this is the major source and will be described after mid-term) is the same;
complete combustion to CO2 and H2O with the biosynthesis of large amounts of ATP. This combustion of acetyl
CoA occurs by way of the Citric Acid Cycle (with the acronym TCA from Tricarboxylic Acid Cycle). As you can
infer from the name this metabolic pathway differs conceptually from glycolysis in that the reaction sequence is
cyclic rather than linear in character; a second striking difference is that glycolysis occurs in the cytoplasm while the
citric acid cycle occurs in the matrix space of mitochondria.
Thunberg Discovers SUCCINIC DEHYDROGENASE
The development of our understanding of the sequence of events in the TCA cycle originates with the
discovery of the enzyme succinate dehydrogenase (SDH). The discovery of this enzyme was made by Thunberg using
a special piece of apparatus that still bears his name-the Thunberg Tube.
Thunberg was studying the ability of minced pigeon flight skeletal muscle to oxidize organic compounds.
To measure these reactions he would place the minced tissue plus the blue dye, methylene blue (MB), in the main
body of his apparatus and the compound to be tested in the bulb of the side-arm. The apparatus was assembled and
the air removed by applying a vacuum to the side-arm. Then the apparatus was inverted and the reagents mixed; if the
organic compound, AH2, could be oxidized by the tissue then
AH2
MB (Blue)
A
MBH2 (Colorless)
The reduction of methylene blue eliminates its blue color and thus the efficacy of the tissue to oxidize the
particular compound can be gauged in a semi-quantitative way by simply measuring the time taken to abolish the
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blue color (it was necessary to remove the oxygen because MBH2 reacts rapidly with oxygen to regenerate MB).
Thunberg showed that many organic compounds could be oxidized by this tissue; of these compounds he found that
succinic acid was by far the most effective. It was oxidized to fumaric acid.
COOH
COOH
CH2
CH
CH2
CH
COOH
COOH
The relevant enzyme he called succinate dehydrogenase (SDH). Thunberg also found that this activity of SDH is
inhibited by malonate, an analog of succinate lacking 1 CH 2; inhibition by malonate was competitive. This
sensitivity to malonate became an important diagnostic test for the participation of SDH in a metabolic process.
The Sparker Effect Of Dicarboxylic Acids
The next important contribution was made by Szent-Gyorgi who was studying the endogenous (intrinsic)
respiration of pigeon flight muscle. As you might expect, this very active tissue is highly enriched in energy
producing enzymes. Furthermore a freshly prepared mince contains a sufficient quantity of endogenous substrates
(probably from the metabolism of glycogen which functions as a source of pyruvate) that it can support the
consumption of oxygen without the necessity for adding any additional substrate. In following the time dependence
of oxygen consumption by this preparation Szent-Gyorgi found that, while the initial rate of oxygen uptake was
very significant, this ability decreased with time and eventually stopped.
O2 Consumed
510 uliters
+ 1 umole fumarate
Endogenous Rate
Time
If at this point succinate or fumarate was added to the incubation mixture the original, brisk, rate of O 2
consumption was restored. This additional uptake did not simply represent the direct oxidation of the succinate for
the amount of additional O2 consumed was very much more than that required for the complete combustion of the
fumarate added. For example, in one experiment, 1 umole of fumarate was added. Complete oxidation of this
requires 3 umoles of O2:
1 fumarate + 3 O2 ====> 4 CO2 + 2 H 2O.
3 umoles of O2 = 3 x 22.4 uliters = 67.2 uliters.
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The observed consumption of O2 was 510 uliters, some 8-times that expected from simple oxidation. It
thus appeared that the succinate and fumarate were being used in a semi-catalytic manner and this phenomenon was
called the sparking effect of dicarboxylic acids.
The phenomenon actually raises two questions; (i) What is the nature of the sparking process; and (ii) Why
does the sparking action decay with time?
The sparking effect of succinate was eliminated by prior addition of malonate (implying SDH was involved)
and it was thus inferred that the sequence was:
? ⇒ succinate ⇒ fumarate ⇒ ?
Subsequently ADDITIONAL dicarboxylate SPARKERS were discovered. The most notable were:
Malate
Oxalacetate (OAA)
COOH
COOH
HOCH
C O
CH2
CH2
COOH
COOH
However 3-C acids such as pyruvate did not restore activity. Malate can be produced from fumarate by
hydration of the double bond while oxalacetate can be produced by the oxidation of malate; the enzyme MDH (malate
dehydrogenase) was already known at the time. So the sequence
COOH
COOH
COOH
COOH
CH2
CH
CH2
CH
CH2
CH2
COOH
COOH
COOH
COOH
succinate
HOCH
fumarate
C O
malate
oxalacetate
became likely. Indeed the postulated participation of OAA provided an explanation for the limited duration of the
sparking phenomenon. Like all β-keto acids OAA is unstable in solution and spontaneously decarboxylates to
pyruvate and CO2. Pyruvate itself has no sparking action and depletion of the pool of 4-C dicarboxylic acids would
lead to a decrease in the endogenous respiration.
More Sparkers.
Subsequently Krebs found that a 6-C acid (citrate) and a 5-C acid (alpha-ketoglutarate, α-Kg) were active in
Szent-Gyorgi's system
COOH
COOH
CH2
CH2
CH2
HO C COOH
CH2
C O
COOH
COOH
citrate
alpha-ketoglutarate
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He also found that the stimulating effect of these two acids was eliminated by malonate, thus implicating
SDH in the process. Krebs rationalized
citrate ⇒ α-Kg
α-Kg ⇒ Succinate
COOH
COOH
CH2
by citrate dehydrogenase, an enzyme already known
by spontaneous decarboxylation.
COOH
COOH
CH2
CH2
CH2
CH
CH
CH2
C O
CH2
COOH
COOH
COOH
COOH
HO C COOH
But, as you can see, in citrate the O atom is on C3 (as OH) while in α-ketoglutarate it is on C4 (as =O), so citrate
cannot be converted to α-Kg directly. The logical possibility is that citrate is first converted to isocitrate in which
the -OH is moved from C 3 to C 4. Shortly thereafter Martius and Knoop found an enzyme, aconitase, that catalyses
this interconversion. So:
citrate ⇒ isocitrate ⇒ αΚg ⇒ succinate ⇒ fumarate ⇒ malate ⇒ OAA
The key, Nobel Prize winning, experiment was performed in 1939 by Krebs and Johnston who incubated
OAA plus pyruvate together with muscle mince in the absence of oxygen. Because there was no O2 present no
oxidative metabolism could occur. They discovered that citrate accumulated in the reaction mixture and thus
proposed the conceptually important step in which OAA and pyruvate condensed with one-another to produce citrate
(via an intermediate 7C compound) and closed the circle on what had, to that time, been a linear scheme.
citrate ⇒ isocitrate ⇒ αΚg ⇒ succinate ⇒ fumarate ⇒ malate ⇒ OAA
⇑
⇓
⇑⇐⇐⇐⇐⇐⇐⇐⇐⇐⇐ [ C7] ⇐⇐⇐⇐⇐⇐⇐⇐⇐⇐⇐⇐ + pyruvate
In this cyclic scheme pyruvate is oxidized to 3 CO2 and 6 H while OAA functions catalytically as a carrier
of pyruvate and its degradation products. As the OAA decomposes to pyruvate the cycle runs down and mechanisms
must be found to replenish the pool of dicarboxylic acids; these were the sparkers.
Our current picture of the Citric Acid Cycle is not fundamentally different from that deduced by Krebs and
his contemporaries; however in one important respect his original depiction of the cycle was in error. We now know
that it is not pyruvate that enters the TCA but acetyl CoA and that the product of the crucial condensation reaction is
not a 7-C compound, as Krebs had supposed, but is citrate itself.
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THE ENZYMES OF THE C ITRIC ACID C YCLE
Condensing enzyme -The Committing Reaction of the Citric Acid Cycle.
The initiating reaction of the Citric Acid Cycle is catalyzed by an enzyme familiarly known as the
condensing enzyme but formally called Citrate Synthase or Synthetase. This is an example of 'Tail Activation', a
Type II reaction of CoA in which the α-carbon of the acetyl moiety of CoA condenses with the carbonyl group of
oxalacetate. The reaction is initiated by the binding of OAA which induces a large conformational change creating a
site for acetyl CoA.
B:
BH+
H-CH 2 -CO-SCoA
:CH 2 CO-S-CoA
COOH
C O
CH2
COOH
CO-SCoA
S-citryl CoA
CH 2
HO C COOH
CH2
COOH
COOH
CH 2
+ CoA
HO C COOH
CH2
COOH
A base labilizes the proton on the methyl carbon of acetyl-CoA and the carbanion produced performs
nucleophilic addition on the electron deficient carbonyl carbon of the oxalacetate. The hydrolytic cleavage of the
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thioester bond is effected by the same enzyme. This is an extremely exergonic reaction (∆Go' = -9 kcal/mole) and
thus, for all practical purposes, the reaction is irreversible, driven by the hydrolysis of the acyl thio-ester.
Aconitase.
The conversion of citrate to α-ketoglutarate involves both chain shortening via decarboxylation and
oxidation of the hydroxyl function to a carbonyl. As ketoglutarate is an α-keto acid the carbonyl is next to the COOH group, whereas in citrate the -OH is displaced from this position by a CH2 group. The next step is
consequently the swapping of a -H with an -OH so that the -OH is moved into position ready for oxidation. The
enzyme that catalyses this interconversion is called aconitase.
A+
CH2
C
A+
CH2
AOH
+HB
C
H :B
H
COOH
citrate (89%)
CH2
H
C COOH
HO C COOH
H
COOH
COOH
COOH
C COOH
HO- C-H
COOH
COOH
iso-citrate (7%)
cis-aconitate (4%)
The reaction is initiated by removal of the proR proton of the proR acetate. The reaction proceeds by
labilization of the hydroxyl function due to withdrawal of the proton by the base followed by capture of the
hydroxide by a Lewis acid. Thus the citrate is dehydrated, the double bond is introduced and cis-aconitate is formed;
this intermediate is planar (sp2). It appears that, for isocitrate to be formed, the cis-aconitate dissociates from the
dehydrating species, reorients itself within the active center cavity, and binds back to the dehydrating center in the
alternative orientation. In the dehydrating step the citrate is bound by the central COOH (+ OH). In the rehydrating
step it is bound by the bottom COOH. This seems very likely because isotope studies show that the proton
removed by the base is retained in the isocitrate (although the hydroxyl group is in equilibrium with the solvent).
The product is the 2R3S, one of four possible isomers.
The intermediate participation of cis-aconitate is not a hypothesis. When aconitase is incubated with either
of the three compounds the equilibrium mixture has the composition 89% citrate, 4% cis-aconitate and 7% isocitrate.
However the cis-aconitate only comes off about once for every ten cycles of catalysis.
An interesting aspect of the activity of aconitase is that the isolated enzyme requires ferrous iron for activity
and it has recently been established that aconitase contains an iron-sulfur cluster. We will discuss these clusters in
detail later; suffice it to say that native, functional, aconitase contains a typical tetranuclear iron-sulfur cluster:
Fe
Fe
S
S
S
S
Fe
Fe +2
Fe
S
Fe
S
S
Fe
Fe
S
It is a property of these clusters that they are unstable and in the case of aconitase one of the iron atoms is lost
during enzyme purification, so that in the laboratory the isolated enzyme contains a cluster in which one corner of
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:B
the cube is vacant. When Fe plus a thiol is added to the enzyme, the cluster is spontaneously reconstituted and
activity is regained. Furthermore these clusters can be studied by a variety of techniques suited to the characterization
of iron (epr, Mossbauer). These techniques show that citrate binds to the Fe that has been restored (Fea). The
oxidized cluster has a formal charge of 2+ , and because of its chemical nature is well suited to disperse electron
density. Thus the cluster is presumed to be the Lewis acid responsible for initiating the dehydration.
Citrate is prochiral and aconitase distinguishes between the upper (Pro-S) and lower (Pro-R) acetate
fragments. This is rationalized using Ogston's 3 point scheme.
Isocitrate Dehydrogenase-The First Chain Shortening Reaction.
In the next reaction isocitrate is both oxidized and decarboxylated to produce α-ketoglutarate by the action of
a single enzyme. The dehydrogenase requires Mn+2 for activity, presumably to bind the substrate and act as a
superacid.
Because a β-hydroxyl moiety, unlike a β-keto moiety, cannot function as an electron sink (no double bond
to absorb the transiently appearing lone pair as the C-C bond is broken), there is no obvious element to facilitate
decarboxylation. Consequently a reasonable path would be first oxidation of the -C-OH to -C=O followed by
decarboxylation.
The decarboxylation is essentially irreversible and serves to pull the reaction to the right. It is hypothesized
that the Mn chelates to the two adjacent C=O groups promoting electron flow from the lone pair on the oxygen of
the center carboxylate and breaking of the HC-COOH bond.
H
COOH
COOH
CH2
CH2
C COOH
COOH
C COOH
H
HO- C-H
H
C=O
NAD+
COOH
NADH
CH2
Mn+2
O-
CH2
H
C
H
C
COOH
COOH
alpha-Kg
O
C=O
COOH
COOH
CH2
CH2
+H+
C
C O-
H
C
H
C=O
Mn+2
Mn+2
COO-
H
C=O
CO2
[oxalosuccinate]
COOH
C
COO-
COO-
The initial oxidation step is linked to reduction of NAD+ (in the mitochondrion where the citric acid cycle
proceeds but also NADP+ in the cytosol where a form of this enzyme exists for biosynthetic purposes. This latter
enzyme is much more active). The mitochondrial enzyme is subject to allosteric control by a number of
metabolites, notably AMP and ADP which are activators, and ATP and NADH which are inhibitors (also citrate,
isocitrate, Mg and pH).
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activity
+ADP
-ADP
[isocitrate]
In the presence of ADP the Km for isocitrate decrease by a factor of 10 so that much less isocitrate is
required to achieve Vmax. The two enzymes phosphofructokinase and isocitrate dehydrogenase are called pacemaker
enzymes and they fill analogous roles in their respective pathways viz.: energy demand turns them on while energy
sufficiency turns them off (Both are activated by ADP).
The
-Ketoglutarate Dehydrogenase complex-the second chain shortening step.
This reaction is very similar to the Pyruvate Dehydrogenase complex. We make the substitutions
Pyruvate Dehydrogenase Complex Ketoglutarate Dehydrogenase
Substrate:
α-Ketoglutarate
Pyruvate
First intermediatehydroxyethyl-TPP
succinic semialdehyde-TPP
Product
acetyl CoA
succinyl CoA
Regulated?
Regulated by
phosphorylation
Not regulated.........
The subsequent metabolism of the dihydrolipoamide is identical in the two cases. So in general terms if we
write the reaction as the processing of R-CO-COOH, with R = CH3- for PDC and R = COOH-CH2-CH2- for KDC,
then the reactions are identical. (A reminder: the first enzyme in the complex is called the keto-acid dehydrogenase
(or sometime decarboxylase) and the assembly of the three enzymes is called the keto-acid dehydrogenase complex.)
The Fate of Succinyl CoA
The succinyl CoA that is produced can be metabolized in three alternative ways:
[1]. The first and least important enzyme is SUCCINYL CoA HYDROLASE which simply hydrolyzes the
thioester bond and dissipates the high-energy bond.
[2]. The second usage of succinyl CoA is by the enzyme SUCCINYL CoA ACETOACETATE CoA
TRANSFERASE. This catalyses the exchange reaction
succinyl CoA + acetoacetate <===> succinate + acetoacetyl CoA
a reaction that proceeds with the intermediate formation of a CoA enzyme intermediate. This transferase reaction is
important in all tissues except liver. It is the only way to activate acetoacetate, an important compound in lipid
metabolism. Liver can't metabolize acetoacetate.
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[3]. The third way is particularly important in the liver where it is used to produce a mole of ATP. It
requires the sequential action of two enzymes:
1) Succinyl CoA + GDP + Pi ⇔ Succinate + GTP + CoA
succinate thiokinase/succinyl CoA synthase
2)
GTP + ADP ⇔ GDP + ATP
nucleoside diphosphokinase
This is the second example of substrate level phosphorylation.
The thiokinase reaction proceeds by 3 steps:
a) E + Succinyl CoA + Pi ⇔ E-succinyl-phosphate + CoASH
(This presumably occurs by the sequential binding of succinyl CoA and then Pi)
b) E-succinyl-P
⇔ E-P + succinate {3-phospho-histidine}
c) E-P + GDP
⇔ E + GTP
Exchange Reaction
1)
2)
3)
4)
*GDP
*Pi
*succinate
*CoA
Additions
⇔ *GTP
⇔ *GTP
⇔ *succinyl CoA
⇔ *succinyl CoA
None
CoA, succinate (or succinyl CoA)
Pi
Pi
Exchange Rxn 1 establishes step c
Exchange Rxn 2 establishes steps a,b and c.
Exchange Rxns 3 and 4 establish steps a and b.
Succinate Dehydrogenase.
At this point all of the reactions that result in reduction in carbon chain length are complete, 2 CO2 have
been eliminated 2 NADH and 1 ATP have been made and we are back with a 4-carbon acid. However the acid is
succinate, whereas to start a new cycle we need oxalacetate. This requires the oxidation of a methylene function to a
carbonyl, a 4-electron oxidation. This basic reaction type occurs in a number of metabolic pathways. In each case it
is accomplished by the same sequence of 3 reactions, though not by the same enzymes. You will discover that this
is a very important general sequence in metabolic reactions consisting of an oxidation followed by a hydration
followed by a second oxidation with the oxidations catalyzed by a flavin enzyme and a NAD(P) enzyme respectively.
In the TCA the first step is the oxidation of succinate to fumarate catalyzed by succinate dehydrogenase.
This enzyme contains flavin and iron-sulfur clusters. However as the enzyme is most conveniently considered along
with the enzymes of the electron transport system we will postpone discussion of its detailed properties and restrict
our present attention to the organic substrate. Overall the reaction is
`
E.FAD + succinate ⇔ E.FAD-succinate ⇔ E.FADH2-fumarate
E.FADH2-fumarate + A ⇔ E.FAD-fumarate + AH2
E.FAD-fumarate ⇔ E.FAD + fumarate
i.e. the fumarate doesn't dissociate from the enzyme until the electron equivalents are passed on to the next
component. The identity of the physiological electron acceptor, A, will be discussed in the lectures on the
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mitochondrial electron transfer chain. In the laboratory a variety of inorganic or organic electron acceptors can be
employed. e.g. potassium ferricyanide or dichlorophenol-indophenol.
The stereochemistry of the reaction.
Mechanistically the reaction is dehydrogenation α,β to a carbonyl (in this instance a C00H on both the α
and β carbons). The removal of the two H is trans (a+d or b+c)!
COOH
Ha
Hc
Hb
Hd
COOH
This was established as follows: If oxidation is CIS then a+b or c+d will be the pair removed, if it is TRANS then
a+d or b+c will be removed. Note that either possibility results in the COOH's being trans in fumarate. This issue
was resolved by the synthesis of both trans-dideutero and cis-dideutero succinate. In the former case cis oxidation
leads to the production of 100% monoD fumarate while trans oxidation gives 50% diD and 50% diH-fumarate.
Conversely cis-oxidation of the latter compound gives zero monoD while trans oxidation gives 100% monoD. So
the enzyme was incubated with the different substrates, the fumarate formed was isolated and its deuterium content
established by mass spectrometry. With the trans dideuterosuccinate the product contained 0 monoD; with cisdideuterosuccinate the product contained 100% monoD. Therefore the 2H are eliminated with TRANS
stereochemistry (not cis as stated in V&V).
Each methylene of succinate is prochiral. However the oxidation to fumarate removes 1H from each
methylene and the enzyme is not required to differentiate between the two H on a single C atom, i.e. it uses both of
these prochiral centers-the product is symmetric. Consequently the reaction has no asymmetry and the succinate is
treated as a completely symmetric molecule-both ends are equivalent (c.f. citrate).
Fumarase
The fumarate produced as a result of succinic dehydrogenase activity is now hydrated by the enzyme
fumarase. There is the trans addition of the elements of water!
C
HOOC
COOH
COOH
H
c
HOCH
CH
H
H2O
2
COOH
L-malate (OH on left)
Is the addition of the water cis, (H and OH attack from same side), or is it trans, (H and OH attack from opposite
sides). This can be established by nmr by running the reaction in D2O> The original protons on the 2 central
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carbons are either relatively near (cis) or far apart (far away); the former yields a larger proton-proton splitting in the
nmr spectrum. Product found to be trans!
Malate Dehydrogenase.
The final reaction is the cycle, and the bootstrap for the whole process, is the oxidation of malate to OAA
by malate dehydrogenase.
COOH
COOH
HOCH
C O
+ NAD+
CH2
CH2
COOH
COOH
(S)
+ NADH + H+
(re)
This reaction is highly endergonic, ∆Go' = 7 kcal, hence the equilibrium lies very much in the favor of
malate synthesis, and it is only because citrate synthetase catalyses such an exergonic reaction that the cycle
functions. Note however that this is not as stupid as it might seem. The juxtaposition of the two strongly
opposing reactions (malate dehydrogenase and citrate synthase) means that the common intermediate, OAA, will
only be present in extremely small concentrations. As we have already learnt OAA is unstable and this instability is
a major source of depletion of intermediates from the cycle. The device just noted has the effect of minimizing this
depletion.
Malate dehydrogenase comes in two forms, one is soluble and is found in the cytoplasm, while the other is
found in the mitochondrial matrix. The significance of these two forms will become apparent in the lectures on
gluconeogenesis later in the semester.
The Overall Energetics
Pyruvate ⇒ Acetyl-CoA
Isocitrate ⇒ α-KG
α-KG
⇒ Succinate
Succinate ⇒ fumarate
malate ⇒ OAA
1 NADH( = 3 ATP)
1 NADH
1 NADH + 1 GTP
1 FADH2 (= 2 ATP)
1 NADH
Net
15 ATP
Pyruvate + 5/2 O2 ⇒ 3 CO2 + 2H 2O
∆Go' = -283 kcal/mole
100 x (15 ATP x -7.6)/-283 = 40%
If we had started with acetate we would have expended energy in making acetyl CoA and we would not have
obtained the 3 ATP from the oxidation of pyruvate. So the net yield would have been 11 ATP. (Free energy of
combustion of acetate is -214 kcal/mole, so efficiency is still close to 40%).
Starting from glucose we get 2 ATP from substrate level phosphorylation, 2 NADH (= 6 ATP) from
phosphoglyceraldehyde dehydrogenase plus the 30 ATP from the oxidation of 2 moles of pyruvate making a total of
38 ATP. The complete combustion of glucose yields -686 kcal so the efficiency is 42%.
The above book-keeping makes the assumption that NADH linked substrates are worth 3 ATP and
succinate yields 2 ATP. THESE ARE THE VALUES YOU WILL FIND IN ALL TEXT-BOOKS. However the
many investigators in the field believe that the values are 2.5 and 1.5 respectively. This will be covered in the
lectures on BioEnergetics.
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When considering the "ATP-value" of cytoplasmic NADH we must account for the fact that this molecule
cannot enter the mitochondrion directly. There are two pathways available:
1) Via Malate. This consumes a NADH in the cytoplasm but restores it in the matrix. So the net penalty
is zero
2) Via reduction of DHAP to glycerophosphate. This is a NAD-linked enzyme in the cytoplasm but the
oxidation of glycerophosphate back to DHAP in the matrix proceeds by a flavoprotein and only 2 (1.5) ATP is
recovered. The net loss is 1 ATP per NAD and thus the overall yield is 36 ATP. Because the flavoprotein is
essentially irreversible and the malic dehydrogenase reaction is biased heavily in favor of malate the second pathway
seems more likely.
Isotope Tracing.
The fate of the carbon atom of acetyl CoA after entering the TCA is a little subtle. We have seen that
ultimately 2 CO2 are produced for each acetyl CoA that is utilized but we will now see that there is not a simple
relationship between the C atoms that are condensed with oxalacetate and those emitted as CO2. To do this we
will follow the trace the path of C around the cycle using labeled acetyl CoA as a device to keep track of the two C
atoms.
In the first reaction the acetyl CoA is condensed with oxalacetate to give citrate in which C atoms 1 and 2
are derived from the acetyl CoA.
COOH
CH2
HO
C COOH
CH2
COOH
Citrate is a symmetric molecule so that, in principle, aconitase can dehydrate either at C 3-C5 or at C2-C3.
In fact, as we have seen, citrate is dehydrated stereospecifically at C3-C5 (the Ogston Triangle). The isocitrate
subsequently made is consequently labeled:
COOH
CH2
H
C COOH
HO- C-H
COOH
This is then processed by isocitrate dehydrogenase and α-KG dehydrogenase to yield succinyl CoA
COOH
COOH
COOH
CH
2
CH
2
CH
HOCH
2
CH
2
CH
CH
COOH
COOH
CH
CO-SCoA
COOH
COOH
2
At this point all of the CO2 has been liberated while all of the radioactivity is still present in the 4-C acid.
The asymmetry in labeling is maintained through here. However at this point succinate is liberated and succinate is
another prochiral molecule. In this case however the dehydrogenation and subsequent steps do not discriminate
between the two ends of the molecule so that the isotope effectively becomes distributed over all of the molecule
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with an "intensity" half of that present in the original acetyl CoA. i.e. all 4 C-atoms of succinate are labeled with a
specific activity 50% of the original acetyl CoA. (While any individual molecule has only two carbons radio-labeled,
in a large sample of molecules the addition of the OH from water to fumarate occurs 50% of the time at the radiolabeled end and the other 50% of the time at the non radio-labeled end. As a result it appears that each carbon is
labeled to 50% of the original level.)
Notice that the 2 CO2 evolved came from the original oxalacetate while the C-atoms of acetyl CoA are
retained. As an exercise you should show that the CO 2 liberated during the second rotation of the cycle has a
specific activity 50% of the acetyl CoA, and if you have the patience, that the specific activity of the CO2 evolved
during the third cycle is 75%. Try to do this without consulting your textbook. There is nice diagram of this
process on p. 336 of the first edition of Zubay (regrettably omitted in subsequent editions).
A NAPLEROPTIC R EACTIONS
The TCA cycle, being catalytic, should be completely self-sustaining. In fact, there are two drains on the
components of the cycle which would , if uncompensated, eventually lead to the cessation of the process. These two
drains are:
1) The instability of oxalacetate which spontaneously decarboxylates into pyruvate and CO2.
2) The demands of biosynthetic pathways many of which use the intermediates of the TCA as a launching pad.
Because the TCA is involved in both anabolism and catabolism it is said to be AMPHIBOLIC. These anabolic
pathways put a drain on the mitochondrial concentrations of:
a) Citrate. The mitochondrial membrane is impermeable to acetyl CoA. Consequently when this
metabolite is needed in the cytoplasm it is transported there in the form of citrate. There is a specific carrier system
for transporting citrate across the membrane. In the cytoplasm acetyl CoA is regenerated:
Citrate + CoA + ATP ⇔ Acetyl CoA + OAA + ADP + Pi
Citrate Cleavage Enzyme.
b) α-Ketoglutarate, a starting point in the biosynthesis of glutamate, proline, hydroxyproline,
γ -aminobutyrate (neurotransmitter), arginine and ornithine.
c) Succinyl CoA, a starting point in the biosynthesis of porphyrins.
d) Malate, which is used to export reducing equivalents to the cytosol e.g. for gluconeogenesis.
e) OAA, which is consumed in the biosynthesis of aspartate and pyrimidines.
If no attempt were made to replenish these intermediates then the cycle would eventually be drained and stop.
Obviously mechanisms must exist to furnish these intermediates. These mechanisms are called the anapleroptic
(filling up) sequences. There are two basic paths:
(1) CO 2 fixation. This is characteristic of mammals.
(2) The glyoxylate pathway; Found in bacteria and plants.
CO 2 Fixation.
There are 4 enzyme reactions with the capability to fix CO2. They produce either malate, oxalacetate or succinyl
CoA.. Because decarboxylation reactions are very exergonic reversal of these reactions needs an energy source.
(1) MALIC ENZYME. This enzyme is present in muscle and catalyses the reductive carboxylation
Pyruvate + CO2 + H + + NADPH ⇔ malate + NADP + ∆Go' = 0.4 kcal/mole
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Note that CO 2 in aqueous solution is really bicarbonate: observe analogy to isocitrate dehydrogenase. in
reverse
(2) PEP CARBOXYKINASE (usually works in opposite direction, i.e. gluconeogenesis)
PEP + GDP(IDP) + CO2 ⇔ OAA + GTP(ITP)
∆Go' = 4.0 kcal/mole
(3) The third is PYRUVATE CARBOXYLASE (requires Mn). This enzyme is present in liver and kidney
and catalyses the reaction
Pyruvate + CO2 + ATP ⇔ OAA + ADP + Pi ∆Go' = -0.5 kcal/mole
Physiologically this is probably the most important. The enzyme is also important in gluconeogenesis and has an
absolute requirement for acetyl CoA which functions as a positive allosteric effector. It uses biotin as a prosthetic
group.
Biotin
There are 6 well characterized carboxylase enzymes that have a requirement for ATP and bicarbonate as
substrates, and which utilize biotin as obligate prosthetic group. We will encounter several of them, notably the
pyruvate, acetyl-CoA and propionyl-CoA carboxylases, and possibly the transcarboxylase. Biotin has the structure:
O
HN
NH
imidazolone
tetrahydrothiophene
S
CH2
CH2
CH2
CH2
COOH
valerate
It consists of an imidazolone ring cis-fused to a tetrahydrothiophene ring; the latter has a valerate sidechain.
In all of the enzymes examined biotin is covalently bound to the respective apoenzyme by means of an amide bond
between the COOH of this sidechain and an active center lysine (analogous to the attachment of lipoic acid even to
the long flexible sidechain; the product is called biotinyl lysine or biocytin).
The function of the biotin is to mediate the carboxylation reaction usually by coupling it to ATP cleavage.
This is accomplished in a two-stage process. In the presence of carbon dioxide (actually present as bicarbonate in
aqueous solution) and ATP, N1 of the biotin is converted to carboxybiotin. It is believed that the actual substrate is
bicarbonate but that this is converted to CO2 by the enzyme in a reaction using ATP (with the hypothesized
carboxyphosphate as an intermediate. The C of CO2 is very electrophilic and adds to the N of biotin.
O
O
+ CO2 , ATP
C
O
N
N
+ ADP + Pi
..
R
+ RCOOH
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S
CH2
This compound then functions as a carbon dioxide donor (∆Go' for hydrolysis = -5 kcal/mole). This can be
envisioned as an attack of a nucleophile on the electron deficient carbon of the carboxy function with elimination of
the biotin and production of the carboxylated nucleophile. In the case of pyruvate carboxylase the reaction begins
with attack of a base onto the pyruvate methyl producing a carbanion which then attacks the carboxy-biotin. With
the acyl CoA's tail activation of the alpha-carbon is the initial step. The products of the reaction with acetyl-CoA
and propionyl-CoA are malonyl CoA and methylmalonyl CoA respectively.
Attacking Nucleophile
Product
:CH 2COCOOH (from pyruvate)
oxalacetate
acetyl CoA
(tail activation)
malonyl CoA
propionyl CoA
methylmalonyl Co A
Biotin enzymes are strongly inhibited by the protein, avidin. This protein is a significant component of egg-white.
It has an incredible affinity for free biotin (ca. 10-15M) principally due to an off-rate of about 2 years, and still has
a high affinity for the prosthetic group when bound to a protein.
(4) PROPIONYL CoA CARBOXYLASE (leads to formation of succinyl CoA).
(a) Propionate arises from the β-oxidation of fatty acids containing an odd number of carbon atoms. The
reaction begins with the synthesis of propionyl CoA from propionate by acetyl CoA synthetase (which we met in
the CoA lecture: this enzyme functions equally well with acetate and propionate).
CH3CH2COOH + CoASH + ATP ⇔ CH 3CH2CO-S-CoA + ADP + Pi
(b) Carboxylation of propionyl-CoA by the biotin requiring carboxylase
CH3CH2CO-S-CoA + CO2 + ATP ⇔ COOH-CH-CO-S-CoA
CH 3 methylmalonyl CoA
This reaction forms the D-(S)-isomer of methylmalonyl CoA..
(c) Epimerisation by methylmalonyl racemase to give the L(R)-isomer (interchanges H and COOH).
H
CH 2
C
(c)
H
CO
COOH
SCoA
(d)
d) Mutation of methylmalonyl CoA to give succinyl CoA. The enzyme is methylmalonyl CoA mutase.
This last enzyme uses adenosyl cobalamin, a derivative of Vitamin B12.
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Coenzyme B12
Porphyrins (which will be considered in detail in Ch. X) are macrocycles made by linking together 4
pyrroles into a square array using methine bridges between the α carbons as the linking functions. In adenosyl
cobalamin the prosthetic group contains a modified porphyrin ring with the south and west pyrrole rings fused
directly without the intervening methine bridge. One of these "pyrroles" (west) is saturated and thus the macrocyclic
ring is called a CORRIN. The central cobalt atom is coordinated equatorially to the pyrrole nitrogens. A fifth
nitrogen is present at the lower axial coordination site; this is provided by a benzimidazole function. The remaining
site at the upper axial position is very unusual. It is occupied by a carbon atom provided by the 5' methylene of the
ribose of adenosine. This reaction can be envisaged as arising by attack of the nucleophile Co(II) on the 5'-C of the
ribose of ATP eliminating PPP (triphosphate), possibly driven by subsequent hydrolysis of the triphosphate. A
second form of B12, methyl B12, is an important reagent in methylation reactions. In vitamin B12 the adenosyl
group is replaced by cyanide.
The rearrangement reactions catalyzed by B12 can be represented:
where X may be a carbon atom with substituents, an oxygen of an alcohol, or an amino group.
The reactions occur by the following sequence:
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(1) Homolytic cleavage of the Co-C bond to produce the methylene radical and reduce the cobalt from 3+ to
2+.
(2) Abstraction of a H atom from the substrate by the methylene radical to yield the methyl group and
producing a carbon radical of the substrate. This may bind to the Co.
(3 & 4) A mysterious rearrangement whereby the R group on the second atom from the Co and the radical
site on the first atom interchange. The details of this reaction are still unknown.
(5 & 6) Reverse of Steps 2 and 1.
An environmentally important reaction of B12 is the alkylation of mercury and arsenic. The deep mud of
lakes contain bacteria (called methanogens) which synthesize methane. In certain species part of this reaction is the
transfer of the methyl carbanion from methyl B12 to an electrophile, normally the proton. Some lakes contain
mercuric salts (Hg2+ ) as the result of dumping of mercury, or arsenate–a contaminant of domestic phosphate-based
detergents. These agents are themselves relatively innocuous because they have low solubility and there is no easy
way for them to get into living systems. The bugs use these compounds as methyl acceptors and make
methylmercury and dimethyl arsine(CH3Hg+, (CH 3)2AsH); these compounds can be assimilated and are very toxic.
The Glyoxylate Cycle
Bacteria can often be grown on acetate as the sole source of organic carbon. As these bacteria possess the
tricarboxylic acid cycle it follows automatically that they have mechanisms for synthesizing the TCA intermediates.
This they achieve by means of the glyoxylate cycle, a metabolic sequence that converts acetate to longer chain
carbon cycle intermediates. It is derived from the tricarboxylic acid cycle.
In summary:
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Citrate
Acetyl CoA
Oxalacetate
Isocitrate
Malate
Glyoxylate
NADPH
NADP+
+ succinate
Acetyl CoA
Thus the net reaction is the conversion of 2 acetyl CoA + NADP+ to yield succinate + NADPH + 2 CoA. This
cycle contains two novel enzymes.
1) Isocitrate Lyase (ISOCITRITASE, equiv. to ALDOLASE).
H
COOH
COOH
CH2
CH2
CH2
C COOH
COOH
+
H
H-C-OH
COOH
C O
COOH
An aldol cleavage of isocitrate yielding succinate and glyoxylate.
2). Malate Synthase (equiv. to Citrate synthase-condensing enzyme)
COOH
H
C O
CH2
+ CH3SCoA
HOCH
COOH
COOH
Tail activation
glyoxylate
acetyl CoA
malate
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+ CoA
Routes for the Formation of Acetyl CoA
1) That provided by pyruvate dehydrogenase complex has already been considered.
2) β-Oxidation of fatty acids, a future topic.
3) Acetate activating Enzyme or ACETATE THIOKINASE or acetyl CoA synthetase. This was covered in
the CoA section.
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