Lecture: 11‐07‐2016

CHAPTER 19 Harvesting Electrons from the Cycle Central metabolic pathways and their association with key metabolic enzymes

©2015 by European Molecular Biology Organization Keren Yizhak et al. Mol Syst Biol 2015;11:817 The citric acid cycle is the biochemical hub of the cell, oxidizing carbon fuels, usually in the form of acetyl CoA, and serving as a source of precursors for biosynthesis Chapter 19 Outline • The citric acid cycle Main Functions of TCA Cycle oxidizes the acetyl fragment of acetyl CoA to

CO2.

• In the process of oxidation, high‐energy electrons are captured in the form of NADH and

FADH2.

• A key function of the citric acid cycle is to harvest high‐energy electrons from carbon fuels. • In the first stage, two carbons are introduced into the cycle by condensation of an acetyl group with a four‐carbon compound, oxaloacetate.

• The six‐carbon compound formed (citrate) undergoes two

oxidative decarboxylations, generating two molecules of CO2.

• In the second stage, oxaloacetate is regenerated.

• Both stages generate high‐energy electrons that are used to power the synthesis of ATP in oxidative phosphorylation. An overview of the citric acid cycle

The citric acid cycle oxidizes two‐carbon units, producing two molecules of CO2, one molecule of ATP, and high‐energy electrons in the form of NADH and FADH2. Cellular respiration

The citric acid cycle constitutes the first stage in cellular respiration, the removal of high‐energy electrons from carbon fuels (left). These electrons reduce O2 to generate a proton gradient (red pathway), which is used to synthesize ATP (green pathway).

The reduction of O2 and the synthesis of ATP constitute oxidative phosphorylation. The citric acid cycle The first stage generates two molecules of CO2 by oxidative decarboxylations.

Citrate synthase

Citrate synthase catalyzes the condensation of acetyl CoA and oxaloacetate to form citrate. • Citrate synthase exhibits induced fit.

• Oxaloacetate binding by citrate synthase induces structural changes that lead to the formation of the acetyl CoA binding site.

• The formation of the reaction intermediate citryl CoA causes a structural change that completes active site formation.

• Citryl CoA is cleaved to form citrate and coenzyme A. Aconitase catalyzes the formation of isocitrate from citrate.

Aconitase

Aconitase is inhibited, a suicide inhibitor, which irreversibly inhibits aconitase after aconitase forms by fluoroacetate fluorocitrate.

Fluoroacetate is found in the genus Gastrolobium, a flowering plant native to Australia. Aconitase is inhibited by a metabolite of fluoroacetate

Fluoroacetate, a toxin, is activated to fluoroacetyl CoA, which reacts with citrate to form fluorocitrate, a suicide inhibitor (p. 138) of aconitase.

After having been irreversibly inhibited, aconitase shuts down the citric acid cycle and cellular respiration, accomplishing its role as a pesticide. Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate, forming α‐ketoglutarate and capturing high‐energy electrons as NADH.

Isocitrate Isocitrate dehydrogenase dehydrogenase α‐Ketoglutarate dehydrogenase complex catalyzes the synthesis of succinyl CoA from α‐ketoglutarate, generating NADH.

The enzyme and the reactions are structurally and mechanistically similar to the pyruvate dehydrogenase complex.

α‐Ketoglutarate dehydrogenase complex Succinyl CoA synthetase catalyzes the cleavage of a thioester linkage and concomitantly forms ATP.

Succinyl CoA synthetase Cleavage of the thioester of succinyl CoA powers the formation of ATP.

The formation of ATP by succinyl coenzyme A synthetase is an example of a substrate‐ level phosphorylation because succinyl phosphate, a high phosphoryl‐transfer potential compound, donates a phosphate to ADP. Succinate dehydrogenase, fumarase, and malate dehydrogenase catalyze successive reactions to regenerate oxaloacetate.

FADH2 and NADH are generated.

Oxaloacetate can condense with another acetyl CoA to initiate another cycle.

Succinate Malate Fumarase dehydrogenase dehydrogenase

Methylene group The net reaction of the citric acid cycle is:

The electrons from NADH will generate 2.5 ATP when used to reduce oxygen in the electron‐transport chain.

The electrons from FADH2 will power the synthesis of 1.5 ATP with the reduction of oxygen in the electron‐transport chain.

Methylene group • The key control points in the citric acid cycle are the reactions catalyzed by isocitrate dehydrogenase and α‐ketoglutarate dehydrogenase.

• Recall that pyruvate dehydrogenase controls entry of glucose‐ derived acetyl CoA into the cycle. Control of the citric acid cycle

The citric acid cycle is regulated primarily by the concentrations of ATP and NADH.

The key control points are the enzymes isocitrate dehydrogenase and α‐ ketoglutarate dehydrogenase. Many of the components of the citric acid cycle are precursors for biosynthesis of key biomolecules. • Because the citric acid cycle provides precursors for biosynthesis, reactions to replenish the cycle components are required if the energy status of the cells changes.

• These replenishing reactions are called anapleurotic reactions.

• A prominent anapleurotic reaction is catalyzed by pyruvate carboxylase. Recall that this reaction is also used in gluconeogenesis and is dependent on the presence of acetyl CoA. Pyruvate carboxylase replenishes the citric acid cycle.

• The rate of the citric acid cycle increases during exercise, requiring the replenishment of oxaloacetate and acetyl CoA.

• Oxaloacetate is replenished by its formation from pyruvate. Acetyl CoA can be produced from the metabolism of both pyruvate and fatty acids.

• Defects in succinate dehydrogenase, fumarase, isocitrate dehydrogenase or pyruvate dehydrogenase kinase can contribute to the development of cancer.

• Mutant isocitrate dehydrogenase generates

• 2‐hydroxyglutarate which alters gene expression, leading to unrestrained growth.

• Other defects contribute to use of aerobic glycolysis by cancer cells. • The glyoxylate cycle is similar to the citric acid cycle but bypasses the two decarboxylation steps, allowing the synthesis of carbohydrates from fats.

• Succinate can be converted into oxaloacetate and then into glucose.

• The glyoxylate cycle is prominent in oil‐rich seeds such as sunflower seeds. The glyoxylate pathway

• The glyoxylate cycle allows plants and some microorganisms to grow on acetate because the cycle bypasses the decarboxylation steps of the citric acid cycle.

• The reactions of this cycle are the same as those of the citric acid cycle except for the ones catalyzed by isocitrate lyase and malate synthase, which are boxed in blue.

In plants, the glyoxylate takes place in organalles called glyoxysomes Sunflowers can convert acetyl CoA into glucose

[Javier Soriano/AFP/Getty Images.]

Tour de France cyclists pass a field of sunflowers. The glyoxylate cycle is especially prominent in sunflowers.