So you’re a hot young eukaryote sporting all kinds of fancy internal membranous organelles, with a need to prove yourself better than the old guard prokaryotes — what do you do? Well, make scads of ATP, of course! And seemingly effortlessly at that, using only the dregs left over after glycolysis has taken its pass at a glucose molecule: NADH and pyruvate. Glycolysis in eukaryotes, as be ts its prokaryotic origins, happens in the cytoplasm. The TCA cycle (also called the citric acid cycle) happens inside the matrix of the mitochondria, a double-membraned organelle.
The pyruvate needs to make its way from the cytoplasm, through both outer and inner mitochondrial membranes, and into the mitochondrial matrix. How does this work? The outer membrane is porous, being riddled with large relatively nonspecific anion channels known as voltage-dependent anion channels (VDACs), and will readily admit pyruvate. In contrast, the inner mitochondrial membrane is highly impermeable, and entry of pyruvate is specifically regulated by a pyruvate transporter protein.
Figure 3. Acetyl-Coenzyme A is composed of four distinct molecular parts.
1. Once the pyruvate enters the mitochondrial matrix, the pyruvate dehydrogenase complex (consisting of three enzyme subunits E1, E2, and E3) converts it to acetyl-CoA (fig. 3) for entry into the tricarboxylic acid cycle (TCA). This reaction generates NADH and liberates CO2.
Pyruvate dehydrogenase complex (fig. 4) is actually an amalgamation of three enzymes. That is, there are three subunits to the complex: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). These three subunits are associated by noncovalent bonds. The pyruvate dehydrogenase subunit E1 acts first, using the cofactor thiamine pyrophosphate (TPP) to help remove a CO2 from the pyruvate to generate hydroxyethyl-TPP.
This is immediately used as a substrate by E2, resulting in regeneration of TPP and reactivation of pyruvate dehydrogenase, and also making the intermediate acetyl-dihydrolipoamide. Coenzyme A, which is also a substrate for E2, has a sulfhydryl group that attacks the acetyl group of acetyl-dihydrolipoamide. The acetyl group is immediately transferred to Coenzyme A to form the Acetyl-CoA that enters the TCA cycle.
The final step is for the dihydrolipoamide to be oxidized back to lipoamide by E3. It is this oxidation step that generates the NADH from NAD+.
Figure 4. Pyruvate dehydrogenase is a complex of three enzymatic subunits, E1, E2, and E3, which work in sequence as depicted here.
Now consider the breakdown of glucose. Recall that the complete breakdown of that six-carbon sugar should yield six single-carbon molecules of carbon dioxide. In glycolysis, the glucose is broken down into two molecules of three-carbon pyruvate. As the pyruvate is converted to acetyl-CoA, one CO2 is generated per molecule of pyruvate. That leaves just four carbons (in two 2-carbon molecules of acetyl-CoA) out of the original glucose 6. The TCA cycle will liberate each of those carbons as CO2 as well. Knowing the reactions in which the remaining carbons are released is a good way to study the first half of the TCA cycle.
As an integral part of coenzyme A, vitamin B5, or pantothenic acid, is needed for the TCA cycle, and therefore, for normal efficient generation of ATP. However, unlike some other vitamins, B5 deficiency is rare, and usually associated with deficiency in other vitamins or general malnourishment. On the other hand, deficiency in another B vitamin involved in pyruvate dehydrogenase activity (fig. 4), thiamine (B1), can lead to disease symptoms known as beriberi.
Arsenic, or more specifically arsenic-containing compounds such as arsenite and arsenate, are poisonous to cells by interfering with this reaction. The arsenic compound can interact with dihydrolipoamide, resulting in cyclization by bonding of both sulfhydryl sulfurs to the arsenic atom. This prevents E2 from working, and acetyl-CoA cannot be generated for ATP production via TCA cycle and oxidative phosphorylation. It should be noted that these arsenic compounds also affect other sulfhydryl-containing com- pounds, and within the context of the TCA cycle, it can also inactivate α-ketoglutarate dehydrogenase, which is similar to the pyruvate dehydrogenase.
Beriberi symptoms are classified in two groups: wet beriberi affects the cardiovascular system with symptoms such as enlarged heart, lung congestion, shortness of breath, swelling of lower legs, congestive heart failure; dry beriberi (also known as Wernicke-Korsakoff syndrome) affects the nervous system. Symptoms include polyneuritis in both central and peripheral nervous system, leading to pain, tingling, loss of sensation in extremities, loss of muscle function or paralysis of the lower legs, vomiting, nystagmus, and eventually mental confusion, speech difficulties, coma and death.
Genetic deficiencies in the pyruvate dehydrogenase complex lead to similar, but more immediately severe problems. The most common mutation is an X-linked dominant mutation in the a subunit of E1. PDC loss-of-function mutations as well as mutations in pyruvate carboxylase and mutations in cytochrome oxidase, are considered causes of Leigh’s disease, which is often neonatally fatal, though exceptions have survived a little over a decade. Severe lactic acidosis and the inability to generate sufficient energy, especially in neurons (which would normally be able to metabolize fat - see section of fatty acid catabolism - but cannot in these patients) and muscle cells, is the underlying cause of the symptoms.
2. Acetyl-CoA enters the tricarboxylic acid cycle as a substrate of citrate synthase, which adds it to oxaloacetate to make citrate. This is the reason that this cycle is also called the citric acid cycle. Citrate, having three carboxyl groups, is a tricarboxylic acid, leading to the name that this text will use. The other common name for this is the Krebs cycle, as it was first proposed by Hans Krebs in 1937.
Citrate synthase is a dimeric enzyme that in its native form has a binding cleft for oxaloacetate. Binding of oxaloacetate causes a conformational shift closing the oxaloacetate binding site, locks it in and simultaneously reveals the acetyl-CoA binding site. The current model for this reaction involves three steps: Acetyl-CoA is converted to an enol intermediate, which attacks the oxaloacetate to form citronyl-CoA (S-citryl-CoA), which is then hydrolyzed to citrate and Coenzyme A.
3. In the next step, aconitase rearranges citrate to make isocitrate.
Aconitase pushes citrate into a cis-aconitate intermediate, which is then converted to isocitrate. Interestingly, while aconitase contains an Fe-S cluster, it does not appear to participate in redox reactions as is usually the case for such groups. Instead, its purpose is to hold the cis-aconitate in its place within the enzyme as it [the cis-aconitate] undergoes a bizarre molecular flip on its way to isocitrate.
Sodium fluoroacetate, also known as compound 1080, is a common pesticide that is used primarily against rodent and other mammalian pests, and can act in humans if ingested. Once introduced to the organism, it can be converted to fluoroacetyl-CoA and then to fluorocitrate, which then acts as a competitive inhibitor of aconitase. As such, the poisoning most severely and quickly affects tissues with high energy needs. No effective antidotes are recognized for lethal doses of fluoroacetate poisoning.
4. Isocitrate is a substrate for isocitrate dehydrogenase, which transfers a high energy electron from the isocitrate onto NAD+ to make NADH and α-ketoglutarate. This reaction also liberates one CO2. For those keeping track at home, that leaves two more carbons from the six in glucose.
The NAD+-dependent isocitrate dehydrogenase is actually found in two isoforms in mammals: an NAD+-utilizing isoform in the mitochondrial matrix, and an isoform that uses NADP+ that is found in the cytosol as well as the mitochondria. The reaction starts with NADH-generating oxidation of isocitrate to oxoalosuccinate, which is then decarboxylated with the help of a Mn2+ or Mg2+ cofactor to release the carbon dioxide and form α-ketoglutarate.
5. Alpha-ketoglutarate is also oxidized (by α-ketoglutarate dehydrogenase) generating NADH and succinyl-CoA. Like acetyl-CoA, this CoA-associated compound contains a high energy thioester bond. This reaction liberates the final CO2 from the glucose.
α-ketoglutarate dehydrogenase is very similar to pyruvate dehydrogenase complex structurally and mechanistically. There are three enzymes: the α-ketoglutarate dehydrogenase, a dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. Also similar to pyruvate dehydrogenase complex, the end product is a molecule containing a high energy thioester bond.
6. The CoA is regenerated by succinyl-CoA synthetase, which also forms succinate and GTP or ATP. This GTP is energetically an ATP equivalent, and made in animal cells. Bacterial and plant homologues of this enzyme use ADP and make ATP. Formation of this ATP/GTP is possible because the high-energy thioester bond of succinyl-CoA is broken.
Succinyl-CoA synthetase first brings together the succinyl-CoA and inorganic phosphate (in solution within the mitochondrial matrix as well as the cytosol) to produce succinyl phosphate and liberate the CoA. Then the phosphate is transferred from the succinyl phosphate, to the enzyme itself temporarily, which then drops the succinate. And finally, the phosphate is transferred to GDP/ADP.
7. Next, the succinate is oxidized. The enzyme that does this, succinate dehydrogenase, is a little different from the other dehydrogenases because this one happens to be embedded in the inner mitochondrial membrane, and instead of transferring the electron to NAD+, the electron is transferred to FAD, making FADH2, and fumarate. The energy in FADH2 can also be used to power ATP production similar to the energy in NADH.
Even though the usual intro-class simplification is that FADH2 is roughly an equivalent to NADH, the situation is actually more complicated. Unlike NAD+, and for that matter, unlike most occurences of FAD, the FAD is covalently bound to the succinate dehydrogenase. Therefore, it is not a soluble metabolite, nor is it available to be reoxidized quite like NADH. It is, of course, reoxidized. But, this occurs within the context of the electron transport chain (where it is known as Complex II), with the help of Coenzyme Q.
8. Fumarase catalyzes the addition of water to the fumarate to generate malate.
The carbon double bond of fumarate is attacked by a hydroxyl (OH-) to form a carbanion transition, which then takes on a proton (H+) from the enzyme to form the malate. The fumarase enzyme is protonated at the same time that it binds fumarate, and is deprotonated at the end to form the malate.
9. Malate is oxidized by malate dehydrogenase in the final reaction of the TCA cycle to generate more NADH, and oxaloacetate, the latter of which can then be added to acetyl-CoA to start the cycle all over again.
Malate dehydrogenase is similar in structure to the lactate dehydrogenase and the alcohol dehydrogenase mentioned in the fermentation section. Energetically, the standard free energy change of this reaction is very highly positive (29.7 kJ/mol) but the oxaloacetate is quickly converted to citrate, so that more formation of oxaloactetate is favored over malate formation.
Now that the complete cycle has been described, it should be noted that the regulation of this cycle is primarily through acetyl-CoA and oxaloacetate availability, as well as NADH concentration. As respiration rate increases, NADH levels drop as they are oxidized to make ATP (see next section). This drop in [NADH] then causes an increase in oxaloactetate, which is then used by citrate synthase. [Acetyl-CoA] is regulated by its synthesis by pyruvate dehydrogenase. On the reverse side of regulation, both NADH and succinyl-CoA are strong inhibitors of α-ketoglutarate dehydrogenase. Thus as NADH is used up, the enzyme is disinhibited and increases its production of more NADH.
Having taken the 2 pyruvates created during glycolysis through the TCA cycle to complete oxidation into CO2, what is our intrepid eukaryotic hero left with? Two ATP-equivalents (GTPs) six NADH, and two FADH2. This hardly seems to be a treasure trove of usable energy worth boasting about. Fortunately, the mitochondrion is not finished. Next, the high energy electrons will take a ride on the electron transport chain, and via the magic of oxidative phosphorylation, produce ATP by the bucket.