The Krebs cycle

Let us now trace the fate of pyruvate that is produced at the end of glycolysis under aerobic conditions. Remember our goal here is to understand how the catabolism of glucose and other carbohydrates is used to generate the ATP that is essential to cells and tissues.

Table 3.3. Summary of ATP production in glycolysis.

Fig. 3.6. The Cori cycle. In active muscle (e.g., with anaerobic conditions) the conversion of pyruvate into acetyl-CoA and processing via the TCA cycle is blocked (red X). This leads to lactate or lactic acid production. The lactate diffuses out of the muscle tissue into blood where the liver and kidney can convert lactate into glucose-6-phosphate for storage as glycogen or conversion to glucose-1-phosphate then glucose. Muscle or other tissues in the body can then use this regenerated glucose.

Once this foundation is established, we will then be able to understand how other nutrients can also be used for energy production. The next major biochemi­cal pathway for this processing is called the citric acid or Krebs cycle. These reactions occur inside the mito­chondria, so before pyruvate can be modified, it has to pass across the mitochondrial membrane.

This occurs via the action of a specific membrane transporter. The pyruvate is then quickly oxidatively decarboxylated (removal of CO₂) to produce acetyl­coenzyme A (acetyl-CoA). This reaction is catalyzed by the action of the multienzyme complex pyruvate dehydrogenase. The product, acetyl-CoA plays an especially central role in energy metabolism. This overall reaction involves two coenzymes:

Pyruvate + NAD⁺ + CoA

→ Acetyl-CoA + NADH + CO₂.

The oxidized-reduced NAD⁺ and NADH are familiar from the action of glyceraldehyde 3-phosphate dehy­drogenase or lactate dehydrogenase and reactions of glycolysis. Now a description of CoA is in order. CoA is a complex molecule derived from pantothenic acid (common in meats and grains), thioethanolamine, and ATP. The essential feature is that CoA acts as a carrier of acyl groups. Specifically, the thiol group of the thio­ethanolamine residue of the molecule functions in this manner in a variety of reactions involved in fatty acid oxidation and fatty acid synthesis and acetylation reactions. The molecule is also important in oxidative decarboxylation reactions as with pyruvate. Acommon convention is to abbreviate the structure of the reduced form of the molecule as CoA∙SH, which designates the reactive SH group of the molecule. The acetyl group that is now part of acetyl-CoA is derived from the catabolism of pyruvate (two carbons remaining after decarboxylation of pyruvate). As we will see, acetyl- CoA is at the confluence of a variety of major meta­bolic pathways. Almost all of the many specific carbohydrates and fats that are Catabolized for energy production are utilized to generate acetyl-CoA. In addition, a number of the nonessential amino acids from degraded proteins also are cannibalized to gen­erate acetyl-CoA. As a special case in ruminants, one of the major products from fermentation of dietary carbohydrates is acetate, which is readily converted to acetyl-CoA for subsequent processing through the mitochondria.

Back to our story: at this point, the two carbons of the acetyl group of acetyl-CoA and the 4-carbon mol­ecule Oxaloacetate condense to create the 6-carbon compound citrate. This is the first step of the Krebs cycle as outlined in Figure 3.7. There are two critical physiological points to the Krebs cycle reactions. The first is that some ATP is produced directly via substrate-level phosphorylation of ADP similar to that which occurs in glycolysis.

As was the case with glycolysis, the coupled oxidation-reduction reactions are a critical part of the processing. The combination NAD⁺ and NADH ap­pears again along with FAD and FADH. The enzymes that catalyze these oxidation reactions by removal of hydrogen atoms are dehydrogenases, for example, lactate dehydrogenase whose action is illustrated in Figure 3.9. For these reactions to take place, the enzymes require the assistance of coenzymes that act to hold or carry these hydrogen atoms. It can be a source of confusion, but transfer of the hydrogen atom, with its lack of a neutron but paired electron and proton, is effectively viewed as an electron transfer. Thus, oxidation-reduction reactions defined by either elec­tron acceptance or electron donation are often linked with movement of the hydrogen atom. This explains the abbreviations related to FAD versus FADH₂ or

Fig. 3.7. Outline of the Krebs cycle reactions. Since each glucose molecule yields two pyruvate molecules (in the presence of oxygen), this allows two turns of the cycle. With each turn, two carbons are removed from the citric acid (six carbons) by decarboxylation reactions; this leads to the production of the 4-carbon intermediate oxaloacetic acid.

NAD⁺ versus NADH + H⁺ in the Krebs cycle reactions outlined in Figure 3.7. Figure 3.8 gives an example of this type of reaction.

At this point you are probably wondering just how much ATP is generated from glucose catabolism and when this actually occurs. As we have seen, each turn of the Krebs cycle only generates two molecules of ATP via substrate-level phosphorylation. The key depends on a cluster of interrelated membrane-bound enzymes that make up the electron transport chain. The activity of these enzymes also accounts for essen­tially all of our need for oxygen. As the electron chain enzymes function, the hydrogen atoms (electrons) that are removed as various intermediates of glycolysis and Krebs cycle are oxidized and are progressively

Fig. 3.8. Reduction of FAD. Coupled reduction of FAD in conjunction with the conversion of succinic acid to fumaric acid as occurs in the Krebs cycle is shown. Arrows indicate the fate of hydrogen atoms (electrons).

passed along until they are combined with oxygen. Oxygen is the final electron acceptor in the chain so that water is formed.

Interestingly, the position along the electron trans­port chain at which FADH₂ or NADH + H⁺ donate their electrons differs. Because of this, the amount of energy that is produced is greater for NADH com­pared with FADH₂. Specifically, each pair of hydrogen atoms from NADH + H⁺ supplies energy for the cre­ation of three ATP, but the two hydrogen atoms from FADH₂ yield only two ATP Most of the proteins of the electron transport chain are closely linked clusters within the inner mitochondrial membrane along with more mobile proteins (coenzyme Q and cytochrome C) that act as carriers between complexes. Figure 3.9 illustrates the release of energy associated with oxida­tion of NADH or FADH₂ in the electron transport chain, and Table 3.4 summarizes ATP production from completed catabolism of glucose.

Under ideal conditions, the complete oxidation of one glucose molecule to CO₂ and water yields 36-38 ATP Alternative figures come from uncertainty about the energy yield of reduced NAD⁺ that is produced

Fig.

Table 3.4. Summary of ATP production from the Krebs cycle and electron transport chain reactions.

from glycolysis. For these molecules to be utilized, they must be passed across the mitochondrial mem­brane by active transport. An estimate of this "expense" is that the net ATP gain from reduced NAD⁺ derived from the cytoplasm is only two ATP per molecule instead of the usual three ATP for those created inside the organelle. Since two of these molecules are pro­duced in the cytoplasm during glycolysis, the total yield is reduced to 36 ATP per molecule of glucose. Regardless, when oxygen is available, energy capture from the biological oxidation of glucose is highly effi­cient. If a mole of glucose is completely combusted, as in a calorimeter, it yields 686 kcal. Energy obtained in the creation of high-energy ATP bonds equals 262 kcal for an efficiency of 38% [262/686 ? 100]. This is mark­edly more efficient than most machines. Energy not captured in the formation of ATP is liberated as heat.