Bioc 811

Biochemistry 811

Lecture on the TCA Cycle

D. Nelson, last modified Dec. 2, 2004 7AM

Reading: Berg, Tymoczko and Stryer 5th edition, Chapter 17

Today and for the next two lectures, we will examine how living organisms have survived
a self inflicted catastrophe that began over 3.5 billion years ago. That event was the
evolution of chlorophyll leading to the development of photosystems I and II. These
electron transfer pathways generate glucose, ATP and NADPH from the energy of
sunlight. Why was this a catastrophe? Because the ultimate source of electrons for
photosystem II is water and removal of electrons from water produces molecular oxygen.

Oxygen is not poisonous by itself except for some enzymes like nitrogenase that have
reactive metal centers that can be ruined by molecular oxygen. The problem for early life
was oxygen by-products like hydrogen peroxide (H₂O₂),
hydroxyl radicle OH•, and superoxide anion O₂¯•.
These are very reactive species that can damage cell components, including lipids, proteins
and DNA. Without some protection oxygen can kill cells. The organisms that evolved
photosystem II that makes oxygen had to evolve some protective systems also. These
include small molecule free radical scavengers like vitamin C and vitamin E, and enzymes
like superoxide dismutase that converts superoxide to hydrogen peroxide, and catalase that
converts hydrogen peroxide to oxygen and water. It is no accident that we find a specific
form of superoxide dismutase in mitochondria, where oxygen is reduced, and catalase is
prominent in red blood cells where oxygen is transported. (Placing hydrogen peroxide on
a cut causes bubbles to form. This is caused by catalase producing oxygen. People who
are missing the catalase gene do not make bubbles with hydrogen peroxide.)

Once bacterial cells protected themselves from the harmful effects of oxygen, oxygen
offered a new opportunity for biochemistry to exploit. Transfer of electrons from one
organic compond to another organic compound, as in glycolysis, does not result in a very
large amount of free energy being released. That is why glycolysis only generates 2 ATP
per glucose molecule. However, electron transfer to oxygen does produce much more free
energy that can be trapped as ATP, almost 20 times more. The way aerobic cells do this
is via the TCA (tricarboxylic acid) cycle and electron transfer down a series of electron
carriers in a membrane embedded chain.

The TCA cycle is also called the citric acid cycle or the Krebs cycle for Hans Krebs who
won the Nobel Prize in 1953 for deciphering this pathway in 1937. Your book (p484) has a rejection
letter to Krebs from Nature, showing that journal editors cannot always see great discoveries
when they are really new. The TCA cycle strips electrons from pyruvate, eventually oxidzing
it completely to 3 CO₂ molecules. The electrons are recovered as the reduced coenzymes
NADH and FADH₂. These reduced coenzymes are the main source of captured energy in this
cycle. Only one GTP is made directly during the TCA cycle. This is called substrate level
phosphorylation. The rest of the ATP is made later from reoxiding the coenzymes NADH and
FADH₂ in a process called oxidative phosphorylation.

It is critical to note that the TCA cycle captures energy in the form of NADH. This also
happened in glycolysis. There is only so much NAD+ in cells and it has to recycle. If all
the NAD+ is converted to NADH then the glycolysis pathway would come to a stop
because glyceraldehyde 3 phosphate dehydrogenase requires it. Under anaerobic
conditions pyruvate is reduced to lactate so NADH can be reoxidized to NAD+. It is a
waste of the energy stored in the NADH, but it has to happen or the pathway will be shut
down. Under aerobic conditions the NADH can be reoxidized to NAD+ by giving the
electrons to the electron transfer pathway. This frees pyruvate to be used in another way.
Instead of being reduced to lactate, it is oxidized all the way to CO₂. This is
done in the TCA cycle.

The key intermediate that feeds into the TCA cycle is acetyl CoA. Acetyl CoA can come
from three sources: pyruvate from glycolysis, amino acid degradation or fatty acid beta-
oxidation. We will only talk about puruvate as the source for this discussion. Before we
proceed, we need to discuss compartmentation of the various reactions and intermediates in
glycolysis and the TCA cycle. The TCA cycle reactions take place in the matrix of the
mitochondria with succinate dehydrogenase being the only membrane bound enzyme in the
cycle. Glycolysis is in the cytosol, so we have the problem of moving across the two
membranes of the mitochondrion. Pyruvate is the substrate that has to be transported
across. Pyruvate crosses the outer membrane through porin. A bacterial porin has been
crystallized and its structure determined. It is shown on page 331. Porins are beta-barrel
structures with open channels about 10 Angstroms across that admit small molecules of 5-
10,000 Da or less. The mitochondrial inner membrane is tight, even to protons, as we will
see, so pyruvate crosses the inner membrane through a protein carrier called the pyruvate
transporter. This is one of 46 related mitochondrial carrier genes in humans that code for
mitochondrial and peroxisomal transport proteins. These carriers are very important for
establishing communication between the cytosol and the mitochondrial matrix. We will run
into them again as we discuss mitochondrial metabolism.

After pyruvate enters the mitochondria it is converted to acetyl CoA by the pyruvate
dehydrogenase complex (pp 467-472). The PDH complex ia a very large enzyme
assembly composed of three main enzymes

1) Pyruvate decarboxylase E1 (24 copies) phosphorylated, contains thiamine pyrophosphate

2) Dihydrolipoyl transacetylase E2 (24 copies) has a lipoic acid group, inhibited by arsenite

3) Dihydrolipoyl dehydrogenase E3 (12 copies) contains NAD+

in addition, for regulation, there are about 5 copies of pyruvate dehydrogenase kinase and
fewer copies of pyruvate dehydrogenase phosphatase. This is for regulation by
phosphorylation and dephosphorylation(pp. 480-481).

The MW of the complex is 9 x 10⁶. A bacterial ribosome is 2.3 x
10⁶. A eukaryotic ribosome is 4 x 10⁶, so PDH is twice as
massive as a eukaryotic ribosome.

The initial reaction in the PDH complex is decarboxylation of pyruvate by the pyruvate
dehydrogenase portion of the PDH complex, also called E1. This is a difficult
reaction because once the carboxyl group is released as CO₂, the carbonyl
carbon (C2 of pyruvate) will exist as a carbanion. Even as a temporary condition this is a
highly unfavorable situation. The reaction is identical to yeast ethanolic fermentation where
pyruvate is decarboxylated to make acetaldehyde, which is then reduced to ethanol. The
yeast enzyme pyruvate decarboxylase and the PDH complex are both doing the same
chemistry, decarboxylation of an alpha keto acid. A cofactor used in nearly all reactions of
this type is thiamine pyrophosphate TPP. The thiazolium ring on TPP attacks the alpha
carbon of pyruvate to produce a structure that will delocalize the charge of the carbanion.
In this way the carboxyl group can leave.

The PDH E1 enzyme does not release acetaldehyde. The intermediate is passed along to the
other enzymes in the complex directly. The dihydrolipoyl transacetylase or E2, contains another
cofactor lipoic acid that has a cyclic disulfide bond on the end of a long tether. This
disulfide reacts with the carbanion intermediate and picks up the acetyl group from the
thiamine pyrophosphate as a thioester. The same long arm then moves the acetyl thioester
into position on E2 where it is exchanged for a new thioester with Coenzyme A. This forms the
acetyl CoA. During the process, the lipoic acid is released in the reduced form
(dihydrolipoic acid) with two free sulfhydryls. These are important, because the electrons
in these sulfhydryls are now removed by the third enzyme in the complex E3 or dihydrolipoyl
dehydrogenase. This forms NADH and reoxidizes the lipoic acid to the disulfide form.
Now the cycle can repeat and we have one molecule of acetyl CoA and one molecule of
NADH. Figures 17.7 and 17.8 on page 470 show the structure of the complex.

Acetyl CoA is the starting point for the TCA cycle. In the first reaction Acetyl CoA condenses
with oxaloacetate (4 carbons) to produce citrate (6 carbons). The enzyme is citrate synthase.
The cycle is named for this TriCarboxylic Acid. The driving force for this reaction is the
hydrolysis of the CoA from the product. This makes it an irreversible step.

Your book describes on pp. 472-473 how a large conformational change in the enzyme after oxaloacetate
binds causes the formation of the acteyl CoA binding site. Once citroyl CoA is formed another change
brings residues into place that will allow hydrolysis of the citroyl CoA. The sequential changes
in structure prevent the hydrolysis of acetyl CoA, that would be a wasteful reaction.

There is some reasoning behind having a cycle with eight steps to accomplish the
oxidation of acetate to two CO₂s. Why not just split it directly and cleave off one
CO₂ leaving behind a one carbon piece? In biochemistry new reactions evolve from old
enzymes and new difficult chemistry does not appear from nowhere. It is easier to take an
exisiting enzyme and adapt it to a new substrate rather than make a whole new enzyme with a new
mechanism. One common way to break a carbon-carbon bond in biochemistry is to cleave the bond
between the alpha and beta carbons next to a carbonyl group. Since acetate is two carbons and
it has no beta carbon, it is added to oxaloacetate. The product, citrate has a central OH, but
no carbonyl group that is two carbons away from a carboxyl. You might suggest oxidizing the
central OH to make a carbonyl, but that would make pentavalent carbon (5 bonds) and that is not
permitted. The solution is to move the central OH to an adjacent carbon and then oxidize it to
a carbonyl. Then the central carboxyl can be released as CO₂.

Aconitase is the enzyme that shifts the hydroxyl. It does this by removing water and then
adding it back to the newly formed C=C bond in the reverse orientation. You should be aware
that aconitase has an iron sulfur cluster in the active site. We will talk about these centers
next time when we discuss electron transport.

Isocitrate dehydrogenase does the oxidation of the shifted hydroxyl to make the carbonyl and
remove the CO₂. Two electrons are taken from the isocitrate and added to NAD+ to
make NADH. These electrons are destined to move down the electron transport chain to oxygen.

At this point it is probably good to mention some general features of the the TCA cycle. The
cycle is catalytic because it regenerates itself. However, it cannot get started without a
little of one of the intermediates. If you have all the enzymes present with lots of acetyl CoA
and NAD+ nothing will happen. If you then add a very small amount of any intermediate, all the
pyruvate will be converted to CO₂ and NADH. This happens because the intermediate
that you added gets converted to oxaloacetate and then reacts with Acetyl CoA so the cycle can
start.

For each turn of the cycle, two carbons enter as acetyl CoA and two carbons leave as
CO₂, but the carbons that leave are not the two carbons that just came in. They are
from the oxaloacetate.

Isocitrate dehydrogenase produces alpha ketoglutarate, the only five carbon compound in the
cycle. AlphaKG is decarboxylated in a second dehydrogenase reaction. The alpha KG
dehydrogenase enzyme complex is similar to the pyruvate dehydrogenase complex and it uses the
same dihydrolipoyl dehydrogenase to regenerate the lipoic acid. Once again the product is a CoA
deriviative, succinyl-CoA. It is important to remember similarities in biochemistry. As we go
through these pathways, I will point out where similar enzymes occur and you should try to keep
this in mind. They make good exam questions. We will see another complex like the PDH and
alphaKGDH complexes again in amino acid metabolism.

The next step in the cycle is succinyl CoA synthetase (named for the reverse reaction, since it
is assayed backwards). This enzyme uses the energy from CoA hydrolysis to make GTP. The GTP
gets converted to ATP by nucleoside diphosphate kinase. This is a substrate level
phosphorylation, meaning that the energy to make the high energy phosphate bond comes from
reaction of the substrate. This is different from oxidative phosphorylation where the energy
comes from a proton gradient.

The succinyl CoA step gives succinate, the first four carbon free (not coupled to CoA)
intermediate. The last three reactions are concerned with interconversions of succinate to
oxaloacetate. These steps also remove four more electrons as NADH.

Succinate dehydrogenase oxidizes succinate to fumarate forming a C=C double bond. The electrons
are removed and transfered to FADH₂ that is covalently bound to the enzyme. The
electrons are not energetic enough to reduce NAD+, that is why FAD is used here instead. This
enzyme is unusual because it is membrane bound and it is a member of the electron transport
chain called complex II. The FADH₂ passes the electrons along to three successive
iron sulfur clusters and then on to ubiquinone (Coenzyme Q or CoQ).

Fumarate is converted to malate by addition of water across the double bond. The enzyme
fumarase does this addition. A single gene codes for two different forms, a cytosolic form and
a mitochondrial form. The mRNA seems to be the same, but there are two different in frame AUG
start sites, one that includes a signal peptide and one that does not. Recently, experiments in
yeast have shown that replacement of the second start MET has no effect on the cytosolic and
mitochondrial distribution of the enzyme. (Sass et al. J Biol Chem 2001 Dec 7;276(49):46111-7).
These authors suggest that after the signal peptide is cleaved, an incompletely folded fumarase
in the mitochondria is exported backward out of the mitochondrial import machinery to form
the cytosolic fumarase.

AL591898 Human DNA sequence from clone RP11-409K12 on chromosome 1, complete
Query: 1     MYRALRLLARSRPLVRAPAAALASAPGLGGAAVPSFWPPNAARM 44 human mitochondrial fumarase
             MYRALRLLARSRPLVRAPAAALASAPGLGGAAVPSFWPPNAARM 
Sbjct: 56207 MYRALRLLARSRPLVRAPAAALASAPGLGGAAVPSFWPPNAARMgt 56340 exon 1 signal peptide

Query: 45      ASQNSFRIEYDTFGELKVPNDKYYGAQTVRSTMNFKIGGVTERMP 89
               ASQNSFRIEYDTFGELKVPNDKYYGAQTVRSTMNFKIGGVTERMP
Sbjct: 58611 agASQNSFRIEYDTFGELKVPNDKYYGAQTVRSTMNFKIGGVTERMP 58747 exon 2

The last step is done by malate dehydrogenase. As the name suggests, this is another oxidation step where two more electrons are removed to make NADH and oxaloacetate. The energetic barrier is high in this reaction so there is a low concentration of oxaloacetate in the mitochondria. The reaction is pulled forward by removal of the oxaloacetate by the citrate synthase reaction. There is a model for the TCA cycle that proposes a three dimensional association between the enzymes called a metabolon. (Velot C, Mixon MB, Teige M, Srere PA. Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon. Biochemistry. 36, 14271-6 1997). The metabolon falls apart during isolation, so experimental evidence for it is not strong. There is evidence of channeling of substrate between malate DH and citrate synthase..That means that the product of MDH goes directly to citrate synthase without release and rebinding. A radioactive labeled malate goes to citrate and is not diluted by unlabeled oxaloacetate.
Origin of the TCA cycle
No pathway emerges complete in evolution. Each step must evolve separately, so how can a cycle evolve? Some clues come from genome sequences of bacteria. The first bacterial genome to be sequenced was Haemophilus influenza. (Science vol. 269, July 28, 1995) When pathways were examined in H. flu it was found that the TCA cycle was incomplete. The first three enzymes were missing. Citrate synthase, aconitase and isocitrate DH were gone. No acetyl CoA could react with oxaloacetate to make citrate. Is all lost? It would be if acetyl CoA was the only entry point in the cycle. In reality, the eight intermediates can be drained off for other purposes and more of some intermediates can be added back to replenish the ones that are drained off. In H. flu, alpha ketoglutarate is added to bypass the first three enzymes. H. flu needs high levels of glutamate in the media to grow. This was not understood until the genome was sequenced. Glutamate is deaminated to make alpha ketoglutarate and ammonium. The ammonium ion goes to another pathway and alpha KG is used as the starting compound for the TCA cycle. Now the cycle is broken and it is linear not cyclic. What has the cell lost by doing this? It lost one oxidation step by isocitrate DH, so one NADH is lost. However, glutamate dehydrogenase in the mitochondria that makes alpha ketoglutarate from glutamate makes one NADH so no reducing equivalents are lost. Only the cyclic nature of the pathway is lost. So we see that it is possible to run a shortened linear version of this cycle. Another clue about the origin of the TCA cycle comes from asking what happens in anaerobic conditions (bacteria or yeast)? The TCA cycle is organized to make NADH and FADH₂ to be used in an electron transfer chain ending in oxygen. Oxygen is reduced to water. What if there is no oxygen? Does the cycle shut down? No oxygen is required for any reaction in the cycle, so it could keep running making NADH and FADH₂, but what to do with these. They must be reoxidized or the cycle will stop. Some bacteria have what is called an alternative oxidase (alternative to cytochrome oxidase that uses oxygen). These alternative oxidases can use another electron acceptor for the electron transport chain, like nitrate or nitrite. This would allow the cycle to run and NADH to be reoxidized by giving the electrons to a different end compound. It is not desirable to shut down the cycle completely, since it is a source of intermediates for biosynthesis of many other compounds. Alpha KG is a precursor to 4-5 amino acids. Acetyl CoA is used in fatty acid biosynthesis and some additional pathways, so it is almost a requirement to keep the PDH complex and the first few steps of the cycle running so these two compounds can be made. If there is no alternative oxidase to reoxidize the NADH from the PDH complex and the isocitrate DH, then what can be done? One answer is to convert pyruvate to oxaloacetate by pyruvate carboxylase (requires one ATP) and then run the cycle backwards from oxaloacetate to succinate. A special anaerobic enzyme called fumarate reductase (NADH linked) operates in reverse to go from fumarate to succinate in E. coli and yeast. Remember that succinate DH is an FAD-linked enzyme so it cannot consume NADH in the reverse reaction. So under anaerobic conditions the TCA cycle is broken into two linear pathways. One makes two NADH, one from the PDH complex and one from isocitrate DH. The other pathway consumes two NADH at malate DH and fumarate reductase. There is no net production of NADH, everything is balanced. There is no production of ATP from this anaerobic version of the pathway, but the intermediates can be made. In fact succinyl CoA is also a required intermediate to make heme, so succinyl CoA synthetase enzyme would probably also be there running backwards from succinate (I told you before that succinyl CoA synthetase is usually assayed in reverse mode.) Since heme is not needed in large amounts the pathway would usually stop at succinate. Before photosystem II made oxygen available, these two pathways were present. They are seen today in anaerobic bacteria and yeast. Only one enzyme is needed to close the cycle, alpha ketoglutarate DH. Existence of an electron transfer system that would reoxidize the NADH would then favor the joining of the two pathways to make a cycle. The reverse linear pathway consumes 1 or 2 ATPs per pyruvate (one at pyruvate carboxylase and one at succinyl CoA synthetase running backwards). The cyclic pathway makes 28 ATP per two pyruvates instead. Thus, we have seen a possible origin of the TCA cycle by joining two linear pathways by adding one enzyme, alpha ketoglutarate DH. Remember that alphaKGDH is related to PDH and they shared a common ancestor. Some of the subunits are even identical.
Other inputs and exits from the TCA cycle
We need to discuss the fates of the intermediates and how the cycle is replenished when some intermediates are used for other purposes. If you eat a large carbohydrate meal, your body will make pyruvate from the glucose and send it to the TCA cycle as acetyl CoA. Your body will want to save it as fat and acetyl CoA is the precursor for fat biosynthesis. There is one problem, fatty acid biosynthesis occurs in the cytosol and the acetyl CoA is made by the PDH complex in the mitochondria. Acetyl CoA must be moved out to the cytosol, but there is no acetyl CoA transporter. There is a citrate carrier which can move citrate out, so acetyl CoA is coupled to oxaloacetate and moved out to the cytosol as citrate. Once outside the mitochondria, it is broken down by ATP citrate lyase to give acetyl CoA and oxaloacetate. (p. 622) which costs one ATP. Fatty acid biosynthesis is a major drain on the TCA cycle because it removes citrate. We already saw that succinyl CoA is used for heme synthesis and alpha ketoglutarate is a source of carbon skeletons for amino acid biosynthesis. Oxaloacetate is another source for amino acid biosynthesis since it is the alpha keto acid of aspartate. Figure 17.19 p. 482 shows most of the exits from the TCA cycle. How can the cycle be filled up if it is depleted? The most important reaction is pyruvate carboxylase (p. 482). This enzyme is activated by acetyl CoA, so when there is plentiful acetyl CoA some pyruvate is converted to oxaloacetate to supply substrate for citrate synthase. The breakdown of amino acids also leads to many intermediates of the TCA cycle. This might lead to too many intermediates. The cycle is catalytic and all components get converted to oxaloacetate. If there is too much oxaloacetate the enzyme phosphoenol pyruvate carboxykinase can convert it to phosphoenol pyruvate (PEP) see pages 450-453. This costs one GTP, but pyruvate kinase will make one ATP when converting PEP to pyruvate. The pyruvate can then enter the TCA cycle and be oxidized to CO₂.
Regulation
As we have seen, the TCA cycle can serve two main functions. It can supply electrons to the electron transport chain in the form of NADH and FADH₂ or it can supply metabolic precursors for biosynthesis. Right at the beginning of the pathway these two choices are apparent. Pyruvate can go either way. For generation of NADH it will be made into acetyl CoA. For replenishing intermediates depleted by biosynthesis it can be converted to oxaloacetate. The enzymes involved are both regulated by levels of acetyl CoA. If acetyl CoA levels are high then there is a need for more oxaloacetate. Pyruvate carboxylase is stimulated and the PDH complex is inhibited. This causes a shift of carbon entering the TCA cycle from acetyl CoA (oxidative mode) to oxaloacetate (biosynthetic mode). The PDH complex is one of the more heavily regulated enzyme systems. In addition to acetyl CoA inhibiting activity, ATP and NADH also inhibit. This makes sense since these are the eventual products of the cycle. There is also a PDH kinase and phosphatase. The kinase is activated by high ATP/ADP ratios and high NADH/NAD+ ratios and also by high acetyl CoA. Phosphorylation of the pyruvate decarboxylase enzyme of the complex inhibits the whole complex. When these ratios drop a phosphatase dephosphorylates the enzyme and reactivates acetyl CoA synthesis. (see pp. 480-481) Inside the cycle three more enzymes are regulated. The first one is citrate synthase. It is inhibited by high ATP/ADP ratios and high NADH/NAD+ ratios. The next regulatory point is isocitrate dehydrogenase. Its activity affects the citrate concentration in mitochondria and cytosol. Since citrate is inhibitory to glycolysis and stimulatory to fatty acid biosynthesis the citrate concentration is key in determining a cells metabolic direction. Isocitrate DH is inhibited by high ATP and NADH and it is stimulated by high NAD+, ADP and isocitrate. The alpha ketoglutarate DH is also at a key branchpoint in the cycle, since alpha KG can be converted to glutamate for use in amino acid biosynthesis and protein synthesis. The biosynthetic path would be favored by conditions of high energy, so if NADH is high the enzyme is inhibited and alpha KG is diverted to glutamate. The enzyme is also inhibited by its product succinyl CoA. If this intermediate is accumulating it is safe to assume that alpha KG can be used elsewhere.
The glyoxylate cycle
Yeast can grow on acetate or ethanol as a carbon source. These two carbon units must be used for all the required carbon compounds in the yeast, including sugars. We have already seen that the TCA cycle cannot be a source for carbohydrate because for every two carbon unit (acetyl CoA) going in, two CO₂ come out. There is no net gain. Yeast and other organisms such as some bacteria, fungi, algae and plants have another pathway to accomplish net synthesis of larger carbon compounds from two carbon units like acetyl CoA. It shares three of the TCA cycle enzymes and adds two more that bypass the decarboxylation steps in the cycle. The two new enzymes are isocitrate lyase and malate synthase. Isocitrate lyase cleaves isocitrate into two acids. Succinate and glyoxylate (glyoxylate is pyruvate minus a methyl group). Malate synthase adds acetyl CoA to glyoxylate to make malate. The cycle converts two Acetyl CoA to malate a four carbon acid. Notice that the acetyl CoA is now making a product that can be converted to sugar by gluconeogenesis. In bacteria these two cycles are in the same compartment, but in plants and some other eukaryotes, the glyoxylate cycle is in a unique organelle called the glyoxosome. Succinate is not processed further in the glyoxosome since the succinate DH and fumarase are not present. Succinate must be exported to the cytosol and retransported into the mitochondria to be futher converted to oxaloacetate. The citrate synthase and aconitase of glyoxosomes are distinct enzymes from the mitochondrial ones. In yeast the glyoxylate enzymes are in the cytosol, succinate enters the mitochondria on another carrier. Recently, isocitrate lyase has been shown to be a crucial enzyme in the pathogenesis of Candida albicans (source of yeast infections) and Mycobacterium tuberculosis. Mycobacterium hides out in cells by using the glyoxylate cycle as its main sources of energy from fatty acids broken down to acetyl CoA. Mycobacterium with this gene knocked out was easily eliminated from infected mice, but normal Mycobacterium is very hard to get rid of. Drugs are now being develpoed to inhibit the isocitrate lyase of Mycobacterium. Humans do not have this enzyme so it should not affect us.