The citric acid cycle serves as the mitochondrial hub for the final steps in carbon skeleton oxidative catabolism for carbohydrates, amino acids, and fatty acids. Each oxidative step, in turn, reduces a coenzyme such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2). These reduced coenzymes contribute directly to the electron transport chain and thus to the majority of ATP production in the human body.
Acetyl-CoA, a significant carbon input into the citric acid cycle, can be derived from glucose or fatty acids; however, a substantial portion of acetyl-CoA comes from glucose or more specifically, pyruvate. The pyruvate dehydrogenase complex (PDC) facilitates the enzymatic conversion of pyruvate to acetyl-CoA. This complex has three protein subunits, in total requiring five cofactors and each with its unique enzymatic activity. The requirement of cofactors and the individual roles of each subunit allows for the complex to be highly regulated--in fact, the pyruvate dehydrogenase complex is an essential regulator of glucose metabolism.
Three separate mechanisms regulate the pyruvate dehydrogenase complex: covalent modification (the primary form of regulation), allosteric regulation, and transcriptional regulation. Covalent modification occurs as phosphorylation on the PDC’s first subunit, pyruvate decarboxylase. Phosphorylation by PDC kinase results in a reduction of PDC activity and an excess of ADP or pyruvate (indicating a need for more acetyl-CoA in the citric acid cycle) downregulates the PDC. Note that PDC kinase isoforms are tissue-specific. Dephosphorylation by phosphatase thus renders PDC active; the presence of calcium ions upregulates phosphatase's activity. Allosteric regulation of PDC involves the direct mechanism of product inhibition or substrate activation. For example, if E2 releases an excess of Acetyl-CoA or E3 an excess of NADH, these products will directly inhibit the PDC. On the other hand, an excess of CoASH (precursor to acetyl-CoA) or NAD+, these substrates will serve as direct activators of the PDC. Finally, transcriptional regulation is dependent on the amount of enzyme produced in fasting and fed conditions; enzyme production is reduced in the fasting state and increased in response to insulin in the fed state.
After the PDC synthesizes acetyl-CoA, it enters the metabolic process known as the citric acid cycle (or the tricarboxylic acid cycle). This cycle has eight steps, seven of which are within the mitochondrial matrix and the outlier, succinate dehydrogenase is associated with the electron transport chain on the inner mitochondrial membrane. As stated above, this cycle results in the final oxidative steps of acetyl groups, resulting in the release of two molecules of carbon dioxide gas. The citric acid cycle further yields reduced coenzymes with each oxidative step; these coenzymes include NADH, GTP, and FADH2. The details of these redox reactions are in the Molecular subsection, as the discussion of these reactions should take place at the molecular level for best comprehension.
The Pyruvate Dehydrogenase Complex's Reactions:
Pyruvate decarboxylase which is made up of 20 or 30 protein chains, is the first enzyme (E1) complex in the PDC. Its role is to release a molecule of carbon dioxide from pyruvate and subsequently attach the leftover carbons to thiamine pyrophosphate which is our first cofactor. The second (and more extensive, at 60 protein chains) enzyme (E2) is dihydrolipoyl transacetylase. This enzyme facilitates two carbon transfers (of those carbons that were once part of pyruvate). The first transfer involves moving these carbons from thiamine pyrophosphate to lipoic acid which is the endogenous second cofactor; the second carbon transfer moves these same carbons to coenzyme A which is the third cofactor. Therefore, the final enzyme, dihydrolipoyl dehydrogenase (E3) does not participate in a carbon transfer; instead, it reverses lipoic acid back to its disulfide form so that it can join in E2’s next carbon transfer. E3 does this by remaining bound to a flavin adenine dinucleotide which oxidizes said lipoic acid; flavin adenine dinucleotide is the fourth cofactor. The final step of the PDC pathway requires the transfer of protons and electrons from now FADH2 to NAD+, releasing NADH and H+ from the complex. This final reaction produces FAD which can then participate in the oxidation of lipoic acid.
Steps of the Citric Acid Cycle:
Citrate synthase catalyzes the condensation reaction of acetyl-CoA and oxaloacetate (the cycle’s final product) to form citrate, initiating the citric acid cycle. Note that this reaction is virtually irreversible with a delta-G-prime of -7.7 Kcal/M (thus strongly favoring citrate formation). Substrate and product availability regulate citrate synthase while citrate inhibits the enzyme oxaloacetate’s binding to the enzyme increases its affinity for acetyl-CoA. One should note that citrate serves as an inhibitory substrate for phosphofructokinase-1 in glycolysis and an activating substrate for acetyl CoA carboxylase in fatty acid synthesis. This point highlights the interconnectivity of our metabolic cycles - in short, no pathway occurs in a vacuum.
Isomerization of citrate
Aconitase, an enzyme with an iron-sulfur center facilitates the hydroxyl group migration that makes isocitrate out of citrate.
Oxidative decarboxylation of isocitrate
This is the first step of the citric acid cycle that produces a reduced coenzyme. Here, isocitrate dehydrogenase oxidizes isocitrate, releasing a carbon dioxide molecule and reduces NAD+ to NADH and a proton. The nature of the reaction (releasing a gas) makes it irreversible. Isocitrate dehydrogenase is allosterically regulated: ADP and calcium ions activate it while ATP and NADH inhibit its activity.
Oxidative decarboxylation of alpha-ketoglutarate by the alpha-ketoglutarate dehydrogenase complex
The alpha-ketoglutarate dehydrogenase complex functions analogously to that of the PDC. E1 of this complex decarboxylates alpha-ketoglutarate and transfers the four remaining carbons to thiamine pyrophosphate which is its first cofactor. Then E2 transfers the succinyl group to CoASH with the help of FAD. Finally, E3 resynthesizes FAD along with NADH from NAD+ so that the dehydrogenase complex maintains the substrates and cofactors necessary for continued reactions. The cofactors required in this complex are thiamine pyrophosphate, lipoic acid, coenzyme A, FAD, and NAD+.
Cleavage of succinyl coenzyme A
Succinate thiokinase catalyzes the cleavage of succinyl CoA’s thioester bond. The division of this high energy bond is coupled with the phosphorylation of guanosine diphosphate (GDP) and therefore produces GTP in addition to succinate. This coupled reaction is an example of substrate-level phosphorylation, as seen in glycolysis.
Oxidation of succinate
Succinate dehydrogenase oxidizes succinate to fumarate, producing a reduced FADH2 (from FAD). Note that succinate dehydrogenase is the one step in this pathway that is associated with the inner mitochondrial membrane and is thus directly part of the electron transport chain, where it is Complex II.
Hydration of fumarate
Fumarase is the catalyst in the hydration of fumarate to malate. This reaction is reversible. In another attempt to highlight the interconnectedness of metabolic pathways, note that the urea cycle also produces fumarate.
Oxidation of Malate
Malate dehydrogenase catalyzes malate’s oxidation to oxaloacetate, reducing NAD+ to NADH producing the final NADH. The delta-G-prime is positive, which would otherwise indicate the reaction favoring malate; however, the citrate synthase reaction to which oxaloacetate is a substrate drives the reaction forward.
Citric acid intermediates may leave the cycle to biosynthesize other compounds. Citrate can be diverted to fatty acid synthesis; alpha-ketoglutarate to amino acid synthesis, neurotransmitter synthesis, and purine synthesis; succinyl-CoA to heme synthesis; malate to gluconeogenesis and oxaloacetate to amino acid synthesis.
Intermediates can also be inserted into the citric acid cycle to replace cataplerotic processes and ensure the cycle continues. For example, throughout the whole body, pyruvate can enter the cycle by way of pyruvate carboxylase, thus inserting additional oxaloacetate into the cycle. This increase in oxaloacetate pushes the cycle forward towards the already exergonic citrate synthase reaction. The liver is a particular case in that it can produce alpha-ketoglutarate by transamination or oxidative deamination of glutamate.
Pyruvate Dehydrogenase Complex Deficiency
A pyruvate dehydrogenase complex deficiency diagnosis most often results from a defective pyruvate decarboxylase subunit due to a mutated X-linked PDHAD gene. This deficiency typically results in congenital lactic acidosis because pyruvate is converted to acetyl-CoA at a decreased rate, meaning pyruvate will instead be converted to lactate by lactate dehydrogenase. Symptoms vary with this deficiency; these symptoms can include neonatal-onset, hypotonicity, lethargy, neurodegeneration, muscle spasticity, and early death. Leigh syndrome or subacute necrotizing encephalomyelopathy is primarily caused by gene mutations that encode proteins of the PDC resulting in progressive neurodegeneration.
Early, acute thiamine (vitamin B1) deficiency is referred to as dry beriberi while chronic deficiency is referred to as wet beriberi, resulting in cardiac symptoms such as dilated cardiomyopathy. This deficiency results in an impaired pyruvate dehydrogenase complex due to a shortage of TPP. Like with PDC deficiency pyruvate is shunted to lactate dehydrogenase and converted to lactate. This chronic shunting of pyruvate can result in a fatal metabolic acidosis.
Isocitrate dehydrogenase 2 Mutation
Isocitrate dehydrogenase 2(IDH2), an isoform of isocitrate dehydrogenase, mitigates oxidative damage. IDH2 is also frequently mutated in adult patients with acute myeloid leukemia. This mutation causes IDH2 to catalyze its reaction to a final product of 2-hydroxyglutarate instead of the correct alpha-ketoglutarate. Increased levels of this oncometabolite results in DNA and histone hypermethylation, therefore causing epigenetic changes which make way for neoplasia. Note that 2-hydroxyglutarate is often a cancer biomarker in pediatric patients with inborn errors of metabolism.