9 Pyruvate dehydrogenase and the TCA cycle

  • You should know how nutrients enter the TCA cycle.
  • Be aware of the central role of acetyl-CoA.
  • Understand the cyclic nature of the TCA cycle and how CO2 is generated from Acetyl-CoA.
  • Appreciate storage of electrons in NADH and FADH2 during the TCA cycle.
  • Understand how the TCA cycle is regulated.
  • Understand the amphibolic role of the TCA cycle
  • Role of acetyl-CoA in metabolism
  • Oxidation and generation of CO2 in the TCA cycle
  • Role of vitamins in enzymatic catalysis
  • Amphibolic nature of the TCA cycle
  • Enzyme regulation by protein phosphorylation
Thus far you have seen how carbohydrates are digested and absorbed (Chapter 5) and how glucose is partially broken down during glycolysis (chapter 7) to form pyruvate. From chapter 2 you know that glucose is eventually fully oxidised CO2 and H2O. Thus, we need to understand how pyruvate is broken down into its final products. Metabolism of glucose occurs in several steps. In the cytosol glucose is converted into 2 molecules of pyruvate.  Some other nutrients can be converted into pyruvate, as well, for instance the amino acid alanine or glycerol, derived from fat. In the next step pyruvate is converted into a two-carbon compound called acetyl-CoA. In chapter 3 you saw that Coenzyme A (CoA) is a molecule used to chemically activate fatty acids. Thus, acetyl-CoA is a chemically reactive form of acetic acid. Acetyl-CoA is arguably the most central metabolite in our cells. All nutrients can be broken down to form acetyl-CoA (Fig. 1), directly or indirectly. Thus, much of our nutrient intake ends up as a chemically reactive two carbon compound. The conversion of pyruvate into acetyl-CoA is catalysed by the  pyruvate dehydrogenase complex, which we will look at in detail below.
Fig. 1 Sources of Acetyl-CoA

The final pathway that generates CO2 from acetyl-CoA is the Tricarboxylic acid cycle or TCA cycle. Before we look at individual reactions let us get an overview of the pyruvate dehydrogenase reaction and the TCA cycle (Fig. 2).

Fig. 2 Overview of the TCA cycle reactions

We can already learn most of the important facts about the TCA cycle (including the pyruvate dehydrogenase reaction) from the overview of Fig. 2.

  • The TCA cycle (including pyruvate dehydrogenase) takes place inside mitochondria (where the electrons can be passed to the respiratory chain).
  • It generates 3 molecules of CO2, corresponding to the 3 carbons of pyruvate.
  • It oxidises pyruvate via acetyl-CoA, removing a total of 10 electrons. Eight (8) electrons are stored as NADH, 2 electrons are stored as FADH2.
  • It generates a molecule of GTP (equivalent to ATP) through substrate-level phosphorylation.
  • It is a cyclic process regenerating the acceptor oxaloacetate to take on another acetyl-CoA.
  • Coenzyme A is also recycled in two reactions of the TCA cycle.
  • The TCA cycle is subject to metabolic coupling, without regeneration of NAD+ and FAD it will stop.

Pyruvate enters mitochondria through the mitochondrial pyruvate carrier. The preparation step for the TCA cycle is the pyruvate dehydrogenase reaction generating acetyl-CoA, CO2 and NADH. (Fig. 3).  

Fig. 3 The enzymatic reactions of the pyruvate dehydrogenase complex

Figure 3 illustrates that the pyruvate dehydrogenase complex actually comprises 3 enzymes, of which multiple copies are in each complex. Without going into all the details of the reaction, we can extract the following information about this enzyme. Initially pyruvate is decarboxylated by an attack of a carbanion belonging to Thiamine pyrophosphate (TPP). The remaining hydroxyethyl-moiety forms a covalent intermediate with TPP (orange area).  The hydroxyethyl-moiety is then transferred to lipoamide (purple area), which is part of the second enzyme. The hydroxyethyl-group is oxidised during the transfer to an acetyl-covalent intermediate forming a thioester with one of the two sulphurs of dihydrolipoamide. The acetyl-group is then picked up by coenzyme A to form acetyl-CoA, another thioester. Please note that because lipoamide became dihydrolipoamide (-S-S- to -SH HS-) in the reaction, it picked up two electrons. These two electrons plus the protons are transferred onto a protein-bound FAD forming FADH2  (green area). This FADH2 then passes the electrons finally onto NAD+, which forms NADH + H+. It is noteworthy how elegantly each individual enzyme is coming back to its initial configuration in this complicated process. Particularly unusual is dihydrolipoyltransacetylase, which is depicted as a rotating arm in Fig. 3. How could this look at the molecular level? Watch the video below.

Another reason to look into the pyruvate dehydrogenase reaction in more detail is its link to nutrition. Vitamins form an important part of our nutrition, but only trace amounts are required. Why is that the case? Most water-soluble vitamins form co-factors of enzymes. As co-factors, vitamins provide chemical reactivity that amino acid side-chains cannot provide (Table 1, only vitamins listed that are enzyme cofactors). Some vitamins remain attached to the enzymes, while others move between enzymes. 

Vitamin Cofactor Chemical reactivity Enzymes
B1
Thiamine-pyrophosphate
ylid, Aldehyde transfer
Decarboxylases
B2
FAD/FADH2
Redox reactions
Dehydrogenases
B3
NAD(P)/NAD(P)H
Redox reactions
Dehydrogenases
B5
Pantothenate
Thioesters
Fatty acid activation
B6
Pyridoxal phosphate
Aldehyde
Transaminase
B7
Biotin
CO2 transfer
Carboxylases
B9
Folate
C1-transfer
Formyltransferase
B12
Cobalamine
Methyl-transfer
Methyltransferase

Pyruvate dehydrogenase is a good example to illustrate the role of vitamins because it uses several of them (Fig. 3 and 4), namely Niacin, Riboflavin, Thiamine and Pantothenic acid. Because enzymes operate catalytically, the amount of vitamins only needs to match the amount of enzyme. Some cofactors remain attached to the enzyme (e.g. FAD in pyruvate dehydrogenase), while NAD/NADH moves between different enzymes to deliver electrons. All four vitamins are not only used by pyruvate dehydrogenase, but are used by many other enzymes in our body, as well. Vitamins provide chemical capabilities to enzymes, which cannot be provided by amino acid side chains, such as electron storage (Niacin, Riboflavin), carbanion attack (Thiamine), and transferable thioester formation (Pantothenic acid).

Fig. 4 Vitamins required for a functional pyruvate dehydrogenase complex.

Thioester formation is another example where vitamins provide extra chemical reactivity. Although cysteine residues in enzymes can form thioesters as well (for example in the fatty acid synthase complex), coenzyme A allows the transfer of a thioester from one enzyme to another. The ability to form carbanions is entirely dependent on vitamins and cannot be achieved by amino acid side-chains.

Vitamins and personalized medicine

Personalized medicine is an approach in which genome sequencing is used to tailor specific treatments.

This example is taken from New Engl J Med 384;22 nejm.org June 3, 2021:

A 5-week-old, previously healthy male infant was admitted after 2 hours of inconsolable, atypical crying and irritability. Ten years earlier, his parents, who were first cousins, had had a child with a similar neurologic presentation that rapidly progressed to epileptic encephalopathy; the child died at 11 months of age without an etiologic diagnosis, despite extensive evaluation. Genome sequencing revealed a mutation in the thiamine transporter SLC19A3 which provides cells with the vitamin. Thiamine and biotin administration was started 37.5 hours after admission, and phenobarbital administration was started 2 hours later (to suppress seizures). One 15-second seizure was recorded thereafter. Six hours later, the patient was alert, calm, and bottle feeding.  After a further 24 hours passed without seizures, the patient was discharged and has been thriving ever since. The lack of Thiamin would have caused death if untreated

Your body has now converted pyruvate to acetyl-CoA inside the mitochondria and released the first  two CO2 derived from the original glucose molecule (1 glucose = 2 pyruvate). Now we want to have a closer look at the TCA cycle itself (Fig. 5).

Fig. 5 Reactions of the TCA cycle. The atoms derived from acetyl-CoA are shown in blue. Because of the symmetry of the succinate molecule, the carbons of acetyl-CoA can show up in any part of the following metabolites (magenta).

The citrate synthase reaction makes use of the activated thioester Acetyl-CoA, to condensate oxaloacetate with acetate. The transferred atoms are labelled in blue. Citrate is rearranged to isocitrate, which is oxidised (2 electrons to NAD+) and decarboxylated to become alpha-ketoglutarate. These are  CO2 no. 3 and 4 derived from the initial glucose molecule. Please note that the released CO2 is not the same as the carbon atoms going into the cycle (blue). This does not matter as we only need to keep the overall balance. The carbons from acetyl-CoA will be converted to CO2 eventually when the cycle turns over again. The alpha-ketoglutarate is oxidised (2 electrons to NAD+) and decarboxylated to form succinyl-CoA. These are CO2 no. 5 and 6 derived from the original glucose molecule. If you look closer at the reaction it is the same as the pyruvate dehydrogenase reaction and in fact both enzyme complexes look very similar and are evolutionary closely related. Your body has now oxidised all 6 carbon atoms of glucose to CO2. 

Despite what you might guess, when monitoring your breathing, your body doesn't care whether you're inhaling enough oxygen. It cares only whether you're expelling enough carbon dioxide - that's the gas that sets off the panic button when you're suffocating.
Sam Kean
Author

Please note, we have no yet generated any water to account for the full oxidation of glucose into 6H2O and 6CO2. We will come back to this in a moment. Subsequently succinyl-CoA is hydrolysed, and in the process a molecule of GTP is generated. This is an example of substrate-level phosphorylation. Succinate dehydrogenase removes another 2 electrons, this time in the form of FADH2. The reason for the use of FAD instead of NAD is the position of the electrons in the molecule. NAD removes two electrons from a single carbon atom, while FAD removes one electron and proton each from two adjacent carbon atoms in a two-step reaction (Fig. 6).

Fig. 6 The use of FADH in oxidation reactions introduces a double bond.

Succinate dehydrogenase is a part of the respiratory chain (complex 2), we will look at this in more detail in chapter 10. In the next step water is added to the double bond generating malate. Malate is then oxidised (2 electrons to NAD+) to form oxaloacetate. Oxaloacetate can now initiate a new cycle by condensating with acetyl-CoA. Let us do some accounting of electrons. To form 6 molecules of water from 3 molecules of oxygen (O2), we need 12 electrons and 12 protons. The glyceraldehyde-3-phosphate dehydrogenase reaction (GAPDH) of the glycolysis pathway (chapter 7) accounts for 2 electrons (x 2 glyceraldehyde-3-phosphate per glucose). The pyruvate dehydrogenase reaction (2 x 2 electrons), citrate dehydrogenase (2 x 2), alpha-ketoglutarate dehydrogenase (2 x 2), succinate dehydrogenase (2 x 2) and malate dehydrogenase (2 x 2). In summary a total of 24 electrons, which can generate 12 molecules of water.

Where did the water go? The oxidation of glucose as outlined in the preceding paragraph yields 12 molecules of water, but we need only six to balance our equation C6H12O6 + 6O2 –> 6CO2 + 6H2O. We need to account for an additional 6 molecules of water. The three water molecules are incorporated in the TCA cycle (inspect the TCA cycle reactions). We need to multiply x 2, because 1 Glucose generates 2 pyruvate. The full oxidation of glucose in your body hence has a different equation:

C6H12O6 + 6O2 + 6H2O –> 6CO2 + 12H2O 

This allows for the removal of an additional 12 electrons from water, which flow to the respiratory chain, where they combine with protons and oxygen to reform water. This metabolic trick maximises the energy yield of the reaction: Full oxidation of glucose yields -2874 kJ/mol, oxidation of one molecule of NADH yields -220kJ/mol, FADH yields -158kJ/mol. Thus 10 x -220 +2 x -158 = -2516 kJ/mol, plus the energy incorporate in ATP (2) and GTP (2) = -120 kJ/mol makes a total of -2636 kJ/mol stored as energy-rich molecules for other purposes.

So far we have got to know the TCA cycle as an energy generating catabolic pathway, but it is more than that. The TCA cycle provides a lot of intermediates for biosynthetic processes. This is called the amphibolic nature of the TCA cycle (Fig. 7).

Fig. 7 The TCA cycle as a hub for biosynthetic reactions (blue). Anaplerotic metabolites are shown in orange.

Intermediates of the TCA cycle are used to synthesise amino acids, heme for hemoglobin and other proteins, and glucose (gluconeogenesis). The reactions are shown in blue. This poses a problem for a cycle. If you remove intermediates, the cycle loses capacity until it disappears. Please note that acetyl-CoA cannot refill the TCA cycle with intermediates, because the entry of two carbon atoms is balanced by the release of 2 CO2 (see Fig 2).   As a result, we need reactions to refill the cycle when intermediates are removed. These reactions are called anaplerotic reactions (orange). There needs to be a careful balance between using intermediates and refilling, because energy generation might otherwise be compromised. One of the uses of GTP generated by the TCA cycle is to serve as an energy sensor providing information about the state of the TCA cycle.

Can you use intermediates of the TCA cycle for energy generation? If you add citrate to the TCA cycle it becomes citrate again after 1 cycle, the molecule is not used up. You need a special enzyme to do this, namely malic enzyme which converts malate to pyruvate. Pyruvate can then be converted to acetyl-CoA to generate CO2 from intermediates of the TCA cycle.

Now that you understand the metabolic purposes of the TCA cycle, we want to look at its regulation. First, we will have a look at pyruvate dehydrogenase. You saw above that the enzyme is a complex arrangement of multiple units of three individual enzymes. We learned about allosteric enzymes during the glycolysis lecture. (please read section on allosteric regulation if you do not feel familiar with the concept) We saw that phosphofructokinase was tightly regulated by the energy state of the cell. Pyruvate dehydrogenase is also an allosteric enzyme and it is negatively regulated by NADH and Acetyl-CoA, which are both products of the enzyme (Fig. 8). In addition pyruvate dehydrogenase is regulated via covalent modification, in this case protein phosphorylation (Fig. 9). Allosteric regulation and protein phosphorylation are the two most common mechanisms of enzyme regulation.

The principle of protein phosphorylation is the same as we observed for allosteric regulation. Instead of binding an allosteric activator or inhibitor, the protein becomes phosphorylated (Fig. 9). Phosphorylation occurs on serine, threonine or tyrosine residues, which have hydroxyl-groups available for the phosphoryl-transfer. Enzymes can be switched “on” or “off” by phosphorylation. There are many examples where a phosphorylated protein is active, but it can also switch it to inactive, as in the case of pyruvate dehydrogenase. There are two enzymes required for this type of regulation a protein kinase, which adds a phosphate group, and a protein phosphatase, which removes the phosphate group. Often protein kinases are tightly regulated themselves, while protein phosphatases act more constitutively. This would mean that the enzyme is normally dephosphorylated (the  phosphatase is more active than the kinase), until a physiological trigger activates the protein kinase. In the case of pyruvate dehydrogenase the corresponding kinase itself is regulated by allosteric activators (NADH, acetyl-CoA) and inhibitors (pyruvate, NAD, CoA). Magnesium ions and calcium ions activate the phosphatase. The latter is very important for muscle cells and neurons, where calcium ions are used to activate cell function (contraction, nerve impulse).

Fig. 8 Regulation of Pyruvate dehydrogenase

Often allosteric behaviour is found to act on the same enzymes as protein phosphorylation. This makes sense because both cause conformational changes that affect the catalytic efficiency of the enzyme (Fig. 9).

Fig. 9 Regulation through protein phosphorylation is similar to allosteric regulation.

The TCA cycle is subject to tight metabolic control. Like glycolysis it can speed up 100-fold during exercise. In the small volume of the mitochondrial matrix NADH has to be recycled immediately, otherwise NAD will be depleted and slow down the TCA cycle. The coupling is so tight in the mitochondria that the speed of the TCA cycle is directly regulated by the use of ATP and in turn by the rate of the respiratory chain (metabolic coupling). In addition, some of the enzymes in the TCA cycle are regulated by allosteric modulators (Fig. 10). Isocitrate dehydrogenase, the main regulatory point of the TCA cycle, is negatively regulated by its reaction product NADH and activated by ADP (see end of chapter question). Several enzymes of the TCA cycle are activated by calcium ions, which are associated with muscle contraction and nerve cell activity. NADH, the main product of the TCA cycle is an allosteric inhibitor of several enzymes.

Fig. 10 Allosteric regulation of the TCA cycle. Inhibitors are shown in red, activators are shown in green.

It is instructive to have a look at the pool sizes of TCA cycle intermediates (Fig. 11). The free energy of the combined reactions of the TCA cycle is +15 kJ/mol. The TCA cycle is “held back” by the small amounts of oxaloacetate, because the malate dehydrogenase reaction strongly favours formation of malate. As a result, the TCA cycle is controlled tightly be the removal of NADH, allowing it to move in clockwise direction in its usual depiction. The equilibrium of the malate dehydrogenase reaction is also relevant for gluconeogenesis, where carbons flow from pyruvate to malate. Isocitrate dehydrogenase is the second most important regulator of the TCA cycle, also drawing on a small pool. Anaplerotic reactions refill the critical pools of the TCA cycle.

Fig. 11 Relative pool sizes of TCA cycle intermediates.
  • The TCA cycle metabolises Acetyl-CoA to carbon dioxide and reducing equivalents (NADH, FADH).
  • The main sources of Acetyl-CoA are fatty acids and glucose.
  • It is a catalytical process. Oxaloacetate is regenerated. When intermediates are withdrawn, they need to be replaced. This is carried out by anaplerotic reactions.
  • The TCA cycle itself produces very little energy (1ATP equivalent).
  • Pyruvate dehydrogenase and isocitrate dehydrogenase are highly regulated enzymes that determine the flow through the TCA cycle.
  • The TCA cycle has an important role in biosynthetic pathways.
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