8.6: Citric Acid Cycle and Related Pathways

Source: BiochemFFA_6_2.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Citric acid cycle

The primary catabolic pathway in the body is the citric acid cycle because it is here that oxidation to carbon dioxide occurs for breakdown products of the cell’s major building blocks - sugars, fatty acids, and amino acids. The pathway is cyclic (Figure 6.63) and thus, doesn’t really have a starting or ending point. All of the reactions occur in mitochondria, though one enzyme is embedded in the organelle’s inner membrane. As needs change, cells may use a subset of the reactions of the cycle to produce a desired molecule rather than to run the entire cycle (see HERE).

Figure 6.63 - Amino acid metabolism and the citric acid cycle. Amino acids boxed in yellow are made from the indicated intermediate. Amino acids in blue are made into the intermediate in catabolism. Image by Aleia Kim

Acetyl-CoA

The molecule “feeding” the citric acid cycle is acetyl-CoA and it can be obtained from pyruvate (from glycolysis), from fatty acid β-oxidation, from ketone bodies, and from amino acid metabolism. Molecules from other pathways feeding into the citric acid cycle for catabolism make the citric acid cycle ‘cataplerotic’. It is worth noting that acetyl-CoA has very different fates, depending on the cell’s energy status/needs (see HERE). The description below describes oxidation (catabolism) in citric acid cycle.

Anabolically, acetyl-CoA is also very important for providing building blocks for synthesis of fatty acids, ketone bodies, amino acids and cholesterol. Other citric acid cycle intermediates are also important in amino acid metabolism (Figure 6.63), heme synthesis, electron shuttling, and shuttling of acetyl-CoA across the mitochondrial inner membrane. The ability of the citric acid cycle to supply intermediates to pathways gives rise to the term ‘anaplerotic.’ It means ‘to fill up.’ Before discussing the citric acid cycle, it is important to first describe one important enzyme complex that is a major source of acetyl-CoA for the cycle.

Figure 6.64 - E1 Subunit of Pyruvate Dehydrogenase. Wikipedia

Pyruvate decarboxylation

The pyruvate dehydrogenase enzyme is a complex of multiple copies of three subunits that catalyze the decarboxylation of pyruvate to form acetyl-CoA. The reaction mechanism requires use of five coenzymes. Pyruvate dehydrogenase is an enormous complex in mammals with a size five times greater than ribosomes.

Subunits

The three subunits are designated by E1, E2, and E3. E2 is also referred to as dihydrolipoamide acetyltransferase and E3 is more precisely called dihydrolipoyl dehydrogenase. Confusion arises with the name for E1. Some call it pyruvate dehydrogenase and others give it the name pyruvate decarboxylase. We will use pyruvate decarboxylase solely to refer to E1 and pyruvate dehydrogenase only to refer to the complex of E1, E2, and E3.

The catalytic actions of pyruvate dehydrogenase can be broken down into three steps, each taking place on one of the subunits. The steps, sequentially occurring on E1, E2, and E3, are 1) decarboxylation of pyruvate; 2) oxidation of the decarboxylated product; and 3) transfer of electrons to ultimately form NADH (Figure 6.65).

Figure 6.65 - Mechanism of action of pyruvate decarboxylation and oxidation by pyruvate dehydrogenase.

Catalysis

The catalytic process begins after binding of the pyruvate substrate with activation of the thiamine pyrophosphate coenzyme through formation of an ylide intermediate. The nucleophilic carbanion of the ylide attacks the electrophilic ketone carbon on the pyruvate, releasing carbon dioxide and creating an enol that loses a proton on the carbon to become a 1,3 dipole that includes the positively charged nitrogen of the thiamine. The reaction (step A in Figure 6.65) is a non-oxidative decarboxylation. Oxidation of the two carbon hydroxyethyl unit occurs in the transfer to the lipoamide.

Reductive acetylation

Reductive acetylation occurs next (Step B) as the 2-carbon hydroxyethyl unit is transferred to lipoamide on E2. (Lipoamide is the name for a molecule of lipoic acid covalently attached to a lysine side chain in the E2 subunit). In prokaryotes in the absence of oxygen, the hydroxyethyl group is not passed to lipoamide, but instead is released as free acetaldehyde , which can accept electrons from NADH (catalyzed by alcohol dehydrogenase) and become ethanol in the process of fermentation. In the presence of oxygen in almost all aerobic organisms, the process continues with transfer of the hydroxyethyl unit to E2 and continuation of the cycle below.

Figure 6.66 - Oxidized and reduced structures of lipoamide (lipoic acid linked to lysine)

Oxidation step

Transfer of the hydroxyethyl group from E1 to the lipoamide coenzyme in E2 is an oxidation, with transfer of electrons from the hydroxyethyl group to lipoamide’s disulfide (reducing it) and formation on the lipoamide of an acetyl-thioester (oxidizing it).

The acetyl group is then transferred from lipoamide to coenzyme A in E2 (Step C in Figure 6.65), forming acetyl-CoA, which is released and leaving reduced sulfhydryls on the lipoamide. In order for the enzyme to return to its original state, the disulfide bond on lipoamide must be re-formed. This occurs with transfer of electrons from reduced lipoamide to an FAD covalently bound to E3 (Step D). This reduces FAD to FADH2.

Formation of NADH

In the last step in the process, electrons from FADH2 are transferred to external NAD+, forming NADH (Step E) and completing the overall cycle. Then enzyme can then begin another catalytic round by binding to a pyruvate.

Pyruvate dehydrogenase regulation

Pyruvate deyhdrogenase is regulated both allosterically and by covalent modification - phosphorylation / dephosphorylation. Regulation of pyruvate dehydrogenase, whether by allosteric or covalent mechanisms has the same strategy. Indicators of high energy shut down the enzyme, whereas indicators of low energy stimulate it. For allosteric regulation, the high energy indicators affecting the enzyme are ATP, acetyl-CoA, NADH, and fatty acids, which inhibit it. AMP, Coenzyme A, NAD+, and calcium, on the other hand, stimulate it (Figure 6.67).

Figure 6.67 - Regulation scheme for pyruvate dehydrogenase (PD). Image by Aleia Kim

Covalent modification

Covalent modification regulation of pyruvate dehydrogenase is a bit more complicated. It occurs as a result of phosphorylation by pyruvate dehydrogenase kinase (PDK - Figure 6.67) or dephosphorylation by pyruvate dehydrogenase phosphatase (PDP).

PDK puts phosphate on any one of three serine residues on the E1 subunit, which causes pyruvate kinase to not be able to perform its first step of catalysis - the decarboxylation of pyruvate. PDP can remove those phosphates. PDK is allosterically activated in the mitochondrial matrix when NADH and acetyl-CoA concentrations rise.

Product inhibition

Thus, the products of the pyruvate dehydrogenase reaction inhibit the production of more products by favoring its phosphorylation by PDK. Pyruvate, a substrate of pyruvate dehydrogenase, inhibits PDK, so increasing concentrations of substrate activate pyruvate dehydrogenase by reducing its phosphorylation by PDK. As concentrations of NADH and acetyl-CoA fall, PDP associates with pyruvate kinase and removes the phosphate on the serine on the E1 subunit.

Figure 6.68 - Pyruvate dehydrogenase complex with three phosphorylation sites in red marked by arrows.Wikipedia

Low concentrations of NADH and acetyl-CoA are necessary for PDP to remain on the enzyme. When those concentrations rise, PDP dissociates and PDK gains access to the serine for phosphorylation. Insulin and calcium can also activate the PDP. This is very important in muscle tissue, since calcium is a signal for muscular contraction, which requires energy. Insulin also also activates pyruvate kinase and the glycolysis pathway to use internalized glucose. It should be noted that the cAMP phosphorylation cascade from the β-adrenergic receptor has no effect on pyruvate kinase, though the insulin cascade does, in fact, affect PDP and pyruvate kinase.

Figure 6.69 - The citric acid cycle. Image by Aleia Kim

Citric acid cycle reactions

Focusing on the pathway itself (Figure 6.69), the usual point to start discussion is addition of acetyl-CoA to oxaloacetate (OAA) to form citrate.

Acetyl-CoA for the pathway can come from a variety of sources. The reaction joining it to OAA is catalyzed by citrate synthase and the ∆G°’ is fairly negative. This, in turn, helps to “pull” the malate dehydrogenase reaction preceding it in the cycle.

In the next reaction, citrate is isomerized to isocitrate by action of the enzyme called aconitase.

Isocitrate is a branch point in plants and bacteria for the glyoxylate cycle (see HERE). Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase produces the first NADH and yields α-ketoglutarate.

This five carbon intermediate is a branch point for synthesis of the amino acid glutamate. In addition, glutamate can also be made easily into this intermediate in the reverse reaction. Decarboxylation of α-ketoglutarate produces succinyl-CoA and is catalyzed by α-ketoglutarate dehydrogenase.

The enzyme α-ketoglutarate dehydrogenase is structurally very similar to pyruvate dehydrogenase and employs the same five coenzymes – NAD+, FAD, CoA-SH, thiamine pyrophosphate, and lipoamide.

Regeneration of oxaloacetate

The remainder of the citric acid cycle involves conversion of the four carbon succinyl-CoA into oxaloacetate. Succinyl-CoA is a branch point for the synthesis of heme (see HERE). Succinyl-CoA is converted to succinate in a reaction catalyzed by succinyl-CoA synthetase (named for the reverse reaction) and a GTP is produced, as well – the only substrate level phosphorylation in the cycle.

The energy for the synthesis of the GTP comes from hydrolysis of the high energy thioester bond between succinate and the CoA-SH. Evidence for the high energy of a thioester bond is also evident in the citrate synthase reaction, which is also very energetically favorable. Succinate is also produced by metabolism of odd-chain fatty acids (see HERE).

Succinate Oxidation

Oxidation of succinate occurs in the next step, catalyzed by succinate dehydrogenase. This interesting enzyme both catalyzes this reaction and participates in the electron transport system, funneling electrons from the FADH2 it gains in the reaction to coenzyme Q. The product of the reaction, fumarate, gains a water across its trans double bond in the next reaction, catalyzed by fumarase to form malate.

Fumarate is also a byproduct of nucleotide metabolism and of the urea cycle. Malate is important also for transporting electrons across membranes in the malate-aspartate shuttle (see HERE) and in ferrying carbon dioxide from mesophyll cells to bundle sheath cells in C4 plants (see HERE).

Figure 6.70 - Succinyl-CoA synthetase mechanism

Figure 6.71 - Succinate dehydrogenase embedded in the mitochondrial inner membrane (top). Wikipedia

Figure 6.72 - Succinate dehydrogenase reaction. Image by Aleia Kim

Rare oxidation

Conversion of malate to oxaloacetate by malate dehydrogenase is a rare biological oxidation that has a ∆G°’ with a positive value (29.7 kJ/mol).

The reaction is ‘pulled’ by the energetically favorable conversion of oxaloacetate to citrate in the citrate synthase reaction described above. Oxaloacetate intersects other important pathways, including amino acid metabolism (readily converted to aspartic acid), transamination (nitrogen movement) and gluconeogenesis.

It is worth noting that reversal of the citric acid cycle theoretically provides a mechanism for assimilating CO2. In fact, this reversal has been noted in both anaerobic and microaerobic bacteria, where it is called the Arnon-Buchanan cycle (Figure 6.73).

Figure 6.73 - Arnon-Buchanon cycle. Alternative enzymes shown on right in lavender. Fd = ferredoxin. Wikipedia

Regulation of the citric acid cycle

Allosteric regulation of the citric acid cycle is pretty straightforward. The molecules involved are all substrates/products of the pathway or molecules involved in energy transfer. Substrates/products that regulate or affect the pathway include acetyl-CoA and succinyl-CoA .

Inhibitors and activators

High energy molecular indicators, such as ATP and NADH will tend to inhibit the cycle and low energy indicators (NAD+, AMP, and ADP) will tend to activate the cycle. Pyruvate dehydrogenase, which catalyzes formation of acetyl-CoA for entry into the cycle is allosterically inhibited by its product (acetyl-CoA), as well as by NADH and ATP.

Regulated enzymes

Regulated enzymes in the cycle include citrate synthase (inhibited by NADH, ATP, and succinyl-CoA), isocitrate dehydrogenase (inhibited by ATP, activated by ADP and NAD+), and α-ketoglutarate dehydrogenase (inhibited by NADH and succinyl-CoA and activated by AMP).

Anaplerotic/cataplerotic pathway

The citric acid cycle is an important catabolic pathway oxidizing acetyl-CoA into CO2 and generating ATP, but it is also an important source of molecules needed by cells and a mechanism for extracting energy from amino acids in protein breakdown and other breakdown products. This ability of the citric acid cycle to supply molecules as needed and to absorb metabolic byproducts gives great flexibility to cells. When citric acid cycle intermediates are taken from the pathway to make other molecules, the term used to describe this is cataplerotic, whereas when molecules are added to the pathway, the process is described as anaplerotic.

Cataplerotic molecules

The citric acid cycle’s primary cataplerotic molecules include α-ketoglutarate, succinyl-CoA, and oxaloacetate. Transamination of α-ketoglutarate and oxaloacetate produces the amino acids glutamate and aspartic acid, respectively. Oxaloacetate is important for the production of glucose in gluconeogenesis.

Glutamate plays a very important role in the movement of nitrogen through cells via glutamine and other molecules and is also needed for purine synthesis. Aspartate is a precursor of other amino acids and for production of pyrimidine nucleotides. Succinyl-CoA is necessary for the synthesis of porphyrins, such as the heme groups in hemoglobin, myoglobin and cytochromes.

Citrate is an important source of acetyl-CoA for making fatty acids. When the citrate concentration is high (as when the citric acid cycle is moving slowly or is stopped), it gets shuttled across the mitochondrial membrane into the cytoplasm and broken down by the enzyme citrate lyase to oxaloacetate and acetyl-CoA. The latter is a precursor for fatty acid synthesis in the cytoplasm.

Anaplerotic molecules

Anaplerotic molecules replenishing citric acid cycle intermediates include acetyl-CoA (made in many pathways, including fatty acid oxidation, pyruvate decarboxylation, amino acid catabolism, and breakdown of ketone bodies), α-ketoglutarate (from amino acid metabolism), succinyl-CoA (from propionic acid metabolism), fumarate (from the urea cycle and purine metabolism), malate (carboxylation of PEP in plants), and oxaloacetate (many sources, including amino acid catabolism and pyruvate carboxylase action on pyruvate in gluconeogenesis.