The Basics of Energy Production: The Citric Acid Cycle

Step 1: Citrate synthesis

The first reaction of the citric acid cycle is a condensation step catalyzed by an enzyme called citric acid synthase. In this step, the acetyl group from acetyl-CoA (C₂₃H₃₈N₇O₁₇P₃S) is attached to a four-carbon oxaloacetate molecule, forming a six-carbon molecule called citrate. 

CoA is bound to a sulfhydryl group (-SH) and diffuses away to combine with another acetyl group. This step is exergonic (reaction that releases free energy), making the process irreversible. The rate of which this reaction occurs is determined by the amount of ATP available. The less there is, the faster the reaction is – and vice versa.

Interestingly, oxaloacetate is also the final product of the citric acid cycle, making the whole process a closed loop. 

Step 2: Isomerization of citrate

In the second step of the citric acid cycle, citrate (C₆H₅O₇^-3) loses a water molecule and gains another. This reaction is catalyzed by aconitase, which essentially converts citrate into its isomer, isocitrate. 

Because this step consists of removing and attaching a water molecule, some scientists consider that the citric acid cycle consists of nine steps instead of eight. 

Step 3: Oxidative decarboxylation of isocitrate

The third step is catalyzed by isocitrate dehydrogenase, which oxidizes isocitrate (C₆H₈O₇) and removes a molecule of carbon dioxide, producing a five-carbon molecule α-ketoglutarate. The process also releases two electrons that reduce NAD⁺ to NADH and H⁺. This is also the first step of the cycle that produces a reduced coenzyme. 

This step is regulated by the positive feedback from ADP and negative feedback from NADH and ATP. Thus, a higher concentration of NADH and ATP inhibits this reaction and vice versa.

Step 4: Oxidative decarboxylation of α-ketoglutarate

In the fourth step of the citric acid cycle, α-ketoglutarate (C₅H₆O₅) loses a carbon dioxide molecule and coenzyme A takes its place, forming a four-carbon molecule called succinyl-CoA. This process is catalyzed by the enzyme α-ketoglutarate dehydrogenase.

Steps three and four are oxidation and decarboxylation steps. They release electrons that reduce NAD⁺ into NADH and release carboxyl groups that form CO₂ molecules.

The product of step three is α-ketoglutarate, whereas the products of step four is a succinyl group. CoA then binds the succinyl group to form succinyl-CoA. The enzyme that catalyzes this reaction is regulated by feedback inhibition of ATP, NADH, and succinyl CoA.

Step 5: Cleavage of succinyl coenzyme-A

The fifth step of the citric acid cycle is catalyzed by succinyl-CoA synthetase. Here, the CoA from succinyl-CoA (C₂₅H₄₀N₇O₁₉P₃S) is removed to produce a four-carbon molecule called succinate. The energy released is then used in substrate-level phosphorylation to form guanosine triphosphate (GTP) from guanosine diphosphate (GDP) and Pi. 

These enzymes, called isoenzymes, come in two different forms depending on what type of animal tissue they are found. The first one (ATP) is found in tissues with a large amounts of ATP, such as skeletal muscle tissue, heart, etc. The other form (GTP) is found in tissues with a high number of anabolic pathways, such as the liver.

The newly formed GTP is energetically similar to ATP, but its use is more limited. One example of this is protein synthesis, which mainly uses GTP.

Step 6: Oxidation of succinate

Step six is a dehydration process, where succinate (C₄H₆O₄) is converted by the enzyme succinate dehydrogenase, producing a four-carbon molecule called fumarate. This oxidation process also transfers two hydrogen atoms and their electrons from succinate to FAD, reducing it to FADH₂. 

Because the enzyme that catalyzes this reaction is embedded in the inner membrane of the mitochondrion, FADH₂ can immediately transfer its electrons into the electron transport chain. 

Step 7: Hydration of fumarate

In the seventh step of the citric acid cycle, water is added to fumarate (C₄H₄O₄), producing a four-carbon molecule called malate. This reaction is catalyzed by the enzyme fumarase.

Step 8: Oxidation of malate

In the eighth and last step of the citric acid cycle, malate (C₄H₄O₅^-2) is oxidized back into oxaloacetate with the help of an enzyme called malate dehydrogenase. This step also reduces NAD⁺ into another molecule of NADH as well as H⁺. 

Because the final pathway of the citric acid cycle produces the same four-carbon compound used in the first step (oxaloacetate), the entire process is considered a closed loop. This essentially allows the cycle to run indefinitely as long as there are sufficient reactants present. 

What happens to NADH and FADH₂?

The NADH and FADH₂ molecules formed during glycolysis and the Krebs cycle are known as electron carriers capable of transporting electrons into the inner membrane of the mitochondria. NADH especially has the capacity to produce vast amounts of ATP.

In the mitochondria, NADH and FADH₂ transfer their electrons into the electron transport chain. Through a series of reactions, these electrons provide the energy to pump protons from the mitochondrial matrix into the inner mitochondrial membrane. This gradual release of electrons creates a proton gradient that drives the synthesis of ATP.

This final phase of cellular respiration is known as oxidative phosphorylation and it produces a substantial amount of energy – 28 ATP.

Final thoughts

As you can see, the citric acid cycle is an incredibly complex process that may sometimes seem daunting to try and understand. Thus, it is no surprise that its discoverer Hans Krebs was awarded a Nobel prize in physiology or medicine in 1953. This is also why it is often referred to as the Krebs cycle. 

The citric acid cycle is an important phase of cellular respiration that sits between glycolysis and oxidative phosphorylation. It utilizes the end products from glycolysis and breaks them down further in a series of eight reactions. 

Once all eight reactions have taken place, a single pyruvate molecule is broken down into 2 CO₂, 3 NADH, 1 FADH₂, and 1 GTP. While this entire process produces only 2 ATP by itself, the remaining electron carriers also drive the electron transport chain and oxidative phosphorylation. This allows the oxidation of glucose to continue, producing vast amounts of more ATP. 

Did you learn anything new about the citric acid cycle? Let us know in the comments.