The Citrate Cycle

How does the citrate cycle use redox reactions to convert energy?

• The citrate cycle is considered the “hub of metabolism” because (1) it is central to aerobic metabolism by generating the bulk of the NADH and FADH₂ used to generate ATP by oxidative phosphorylation; (2) it oxidizes metabolic fuels from a variety of sources (carbohydrates, fatty acids, proteins); and (3) it provides metabolites for biosynthetic pathways.

• The citrate cycle can be thought of as a metabolic engine in which the fuel is acetyl-CoA, the exhaust is CO₂, and the work performed is the transfer of electrons by use of a series of linked redox reactions.

• The citrate cycle is also called the Krebs cycle in honor of Hans Krebs, who discovered it; alternatively, it is also called the citric acid cycle or the tricarboxylic acid (TCA) cycle. The descriptive term citrate cycle reflects that all three carboxyl groups on citrate are deprotonated at physiologic pH, making it a conjugate base inside cells. Moreover, the first enzyme in the citrate cycle is citrate synthase, which reflects the physiologic ionic species.

• Oxidation reactions are a loss of electrons, whereas reduction reactions are a gain of electrons. The energy available from redox reactions is due to differences in the electron affinity of reactants and is based on molecular structure.

• Coupled redox reactions consist of two half-reactions containing a conjugate redox pair. Compounds that accept electrons are called oxidants (oxidizing agents) and are reduced in the reaction, whereas compounds that donate electrons are called reductants (reducing agents) and are oxidized in the reaction.

• The reduction of NAD⁺ to NADH involves the transfer of a hydride ion (:H⁻), which contains 2 e⁻ and 1 H⁺, and the release of a proton (H⁺) into solution. FAD is reduced by sequential addition of one hydrogen (1 e⁻ and 1 H⁺) at a time.

• The biochemical standard reduction potential (E°′), measured in volts (V), represents the electron affinity of a given conjugate redox pair. Oxidants with a higher affinity for electrons than that of H⁺ have positive E°′ values (E°′ > 0); oxidants with a lower affinity for electrons than that of H⁺ have negative E°′ values (E°′ < 0).

• The amount of energy available from a coupled redox reaction is directly related to the difference between two reduction potentials and is defined by the term ΔE°′. The ΔE°′ of a coupled redox reaction is determined by subtracting the E°′ of the reductant (e⁻ donor) from the E°′ of the oxidant (e⁻ acceptor).

• The ΔE°′ for a coupled redox reaction is proportional to the biochemical standard Gibbs energy change, ΔG°′, as described by the equation ΔG°′ = −nFΔE°′.

• To calculate the actual reduction potentials for conjugate redox pairs, the concentrations of the oxidant (e⁻ acceptor) and reductant (e⁻ donor) need to be accounted for by using the Nernst equation.

How does the pyruvate dehydrogenase complex link glycolysis to the citrate cycle?

• Pyruvate is oxidatively decarboxylated in the mitochondrial matrix by the multi-subunit pyruvate dehydrogenase complex, which uses a six-step reaction mechanism that requires three distinct enzymes and five different coenzymes.

• The five coenzymes used by the pyruvate dehydrogenase complex are NAD⁺, FAD, CoA, TPP, and α-lipoic acid (lipoamide).

• Severe niacin deficiency causes the disease pellagra, which leads to insufficient levels of NAD⁺/NADH for metabolic reactions. Corn-rich diets can lead to pellagra if the cornmeal is not prepared properly to release the protein-bound niacin.

• Thiamine, also called vitamin B₁, is the precursor to TPP, an important coenzyme in the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase reactions. Thiamine deficiency causes the human disease beriberi.

• The eukaryotic pyruvate dehydrogenase complex contains three enzymes: E1, pyruvate dehydrogenase; E2, dihydrolipoyl acetyltransferase; and E3, dihydrolipoyl dehydrogenase.

• The pyruvate dehydrogenase reaction can be broken down into six distinct catalytic steps: steps 1, 2, and 3 lead to the formation of acetyl-CoA, whereas steps 4, 5, and 6 regenerate the oxidized form of lipoamide and reduce NAD⁺ to NADH + H⁺.

• Pyruvate dehydrogenase activity is regulated by allosteric control in response to energy charge, NADH to NAD⁺ ratios, and CoA to acetyl-CoA ratios. It is also regulated by serine phosphorylation, which is mediated by kinase and phosphatase enzymes that are themselves regulated by energy charge metabolites.

What are the eight enzymatic reactions of the citrate cycle?

• The citrate cycle consists of eight enzymatic reactions that can be divided into two sets of four reactions each. The first four reactions include a condensation reaction, an isomerization reaction, and two consecutive oxidative decarboxylation reactions, each of which releases a CO₂ and generates NADH. The second four reactions include a substrate-level phosphorylation reaction that generates GTP, a redox reaction producing FADH₂, a hydration reaction, and a redox reaction that generates NADH and produces oxaloacetate to restart the cycle.

• Citrate synthase catalyzes the first reaction in the pathway, which is the condensation of oxaloacetate and acetyl-CoA to form citrate.

• Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate by transferring two electrons to NAD⁺ to form NADH, releasing CO₂ in the process.

• Mitochondrial aconitase catalyzes an isomerization reaction in the citrate cycle that converts citrate to isocitrate by using an iron–sulfur cluster (4 Fe–4 S) in a two-step reaction. Cytosolic aconitase converts citrate to isocitrate in the cytosol by using a similar iron–sulfur cluster, but it also has regulatory functions that control the synthesis of iron-metabolizing proteins. The human mitochondrial and cytosolic aconitase proteins are encoded by separate genes.

• The Australian gidgee tree contains the natural product fluoroacetate, which is converted to fluoroacetyl-CoA by the enzyme acetyl-CoA synthase. Citrate synthase converts fluoroacetyl-CoA into fluorocitrate, a potent inhibitor of aconitase and the active ingredient in the animal poison Compound 1080.

• α-Ketoglutarate dehydrogenase is functionally similar to pyruvate dehydrogenase and requires the same five coenzymes. It catalyzes an oxidative decarboxylation reaction that produces CO₂, NADH, and succinyl-CoA.

• GTP (or ATP) is generated by a substrate-level phosphorylation reaction catalyzed by the enzyme succinyl-CoA synthetase. In organisms and tissues in which GTP is the product of the succinyl-CoA synthetase reaction, the GTP is readily converted to ATP by the enzyme nucleoside diphosphate kinase.

How is the citrate cycle regulated to meet the energy needs of the cell?

• The three main control points for regulation of the citrate cycle are the reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase.

• Regulation of citrate cycle reactions is accomplished by several mechanisms, including substrate availability, product inhibition, and feedback inhibition.

• Citrate synthase is inhibited by citrate, succinyl-CoA, NADH, and ATP; inhibition by ATP is reversed by ADP. Isocitrate dehydrogenase is activated by ADP and Ca²⁺ and inhibited by NADH and ATP. α-Ketoglutarate dehydrogenase is activated by Ca²⁺ and AMP and is inhibited by NADH, succinyl-CoA, and ATP.

• Citrate, a flavoring for food and a preservative for medicines, is produced commercially by using a proprietary biotechnology process that is based on Aspergillus niger fermentation of sucrose to generate citrate, which is exported to the culture medium and collected.

How are citrate cycle metabolites used in other pathways and how are they replenished?

• The citrate cycle provides biosynthetic precursors for several metabolic pathways; it is considered to be an amphibolic pathway because it functions in both catabolism and anabolism.

• Excess citrate in mitochondria is exported to the cytosol, where it is cleaved by the enzyme citrate lyase to release acetyl-CoA and oxaloacetate. Cytosolic acetyl-CoA is used for fatty acid and cholesterol biosynthesis, whereas oxaloacetate is used to generate phosphoenolpyruvate for gluconeogenesis.

• Oxaloacetate and α-ketoglutarate are metabolic precursors to aspartate and glutamate, respectively, and succinyl-CoA is a precursor in heme biosynthesis. Malate can be used as a source of carbon in the gluconeogenic pathway.

• The enzyme pyruvate carboxylase balances the input of oxaloacetate and acetyl-CoA into the citrate cycle by converting pyruvate into oxaloacetate using an ATP-dependent carboxylation reaction that is stimulated by acetyl-CoA.

• Anaplerotic (meaning “to fill up”) reactions replenish citrate cycle intermediates such as oxaloacetate to maintain flux through the pathway. Pyruvate carboxylase converts pyruvate to oxaloacetate, whereas phosphoenolpyruvate carboxylase generates oxaloacetate from phosphoenolpyruvate.

• Malic enzyme, another anaplerotic enzyme, generates malate from pyruvate in a reversible reaction requiring NADH.