Citric Acid Cycle and Glyoxylate Cycle Chapter 14 Citric Acid Cycle and Glyoxylate Cycle Copyright © 2013 Pearson Canada Inc.

Chapter 14 Outline: Overview of Pyruvate Oxidation and the Citric Acid Cycle Pyruvate Oxidation: A Major Entry Route for Carbon into the Citric Acid Cycle Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Action of the Pyruvate Dehydrogenase Complex The Citric Acid Cycle Stoichiometry and Energetics of the Citric Acid Cycle Regulation of Pyruvate Dehydrogenase and the Citric Acid Cycle Anaplerotic Sequences: The Need to Replace Cycle Intermediates Glyoxylate Cycle: An Anabolic Variant of the Citric Acid Cycle Copyright © 2013 Pearson Canada Inc.

Overview of Pyruvate Oxidation and the Citric Acid Cycle The citric acid cycle is the central oxidative pathway in respiration, the process by which all metabolic fuels—carbohydrate, lipid, and protein—are catabolized in aerobic organisms and tissues. Most of the energy yield from substrate oxidation in the citric acid cycle comes from subsequent reoxidation of reduced electron carriers. Copyright © 2013 Pearson Canada Inc.

Overview of Pyruvate Oxidation and the Citric Acid Cycle The three stages of respiration: Stage 1 - carbon from metabolic fuels is incorporated into acetyl-CoA. Stage 2 - the citric acid cycle oxidizes acetyl-CoA to produce CO2, reduced electron carriers, and a small amount of ATP. Stage 3 - the reduced electron carriers are reoxidized, providing energy for the synthesis of additional ATP. Copyright © 2013 Pearson Canada Inc.

Overview of Pyruvate Oxidation and the Citric Acid Cycle Schematic of a mitochondrion. Computer model generated from electron tomograms of a mitochondrion. Dehydrogenases catalyze substrate oxidations. Oxidases catalyze the subset of oxidations in which O2 is the direct electron acceptor. Copyright © 2013 Pearson Canada Inc.

Overview of Pyruvate Oxidation and the Citric Acid Cycle The fate of carbon in the citric acid cycle: Note that these departing CO2 groups derive from the two oxaloacetate carboxyl groups that were incorporated as acetyl-CoA in earlier turns of the cycle. Copyright © 2013 Pearson Canada Inc.

Pyruvate Oxidation: A Major Entry Route for Carbon into the Citric Acid Cycle Pyruvate oxidation to acetyl-CoA is catalyzed by the pyruvate dehydrogenase complex (PDH complex). It is an oxidative decarboxylation, which is virtually irreversible involving three enzymes and five coenzymes. Copyright © 2013 Pearson Canada Inc.

Pyruvate Oxidation: A Major Entry Route for Carbon into the Citric Acid Cycle Structure of the pyruvate dehydrogenase complex: Electron micrograph of the purified pyruvate dehydrogenasecomplex from E. coli. E2 core subcomplex (60 E2 monomers). E2-E3 subcomplex (E2 core 12 E3 dimers). Full PDH complex (E2-E3 subcomplex 1 ~30 E1 tetramers). Cutaway reconstruction of PDH complex. Copyright © 2013 Pearson Canada Inc.

Pyruvate Oxidation: A Major Entry Route for Carbon into the Citric Acid Cycle Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle In pyruvate oxidation, the acceptor of the active aldehyde (hydroxyethyl group) generated by TPP is lipoic acid, which is the internal disulfide of 6,8-dithiooctanoic acid. The coenzyme is joined to via an amide bond linking the carboxyl group of lipoic acid to a lysine e-amino group. Thus, the reactive species is an amide, called lipoamide, or lipoyllysine. Each lipoyllysine side chain is ~14Å long, and is located within a flexible lipoyl domain of E2 (and E3BP), allowing it to function as a “swinging arm” that can interact with the active sites of both the E1 and E3 components of the PDH complex. Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Oxidized and reduced forms of lipoamide: The cyclic disulfide of lipoamide can undergo a reversible two-electron reduction to form the dithiol, dihydrolipoamide. In pyruvate dehydrogenase, this reduction is coupled to the transfer of the hydroxyethyl group moiety from TPP, giving an acetyl thioester of the reduced dihydrolipoamide. Thus, lipoamide is a carrier of both electrons and acyl groups. Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Flavin coenzymes participate in two-electron oxidoreduction reactions that can proceed in 2 one-electron steps. Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Coenzyme A (A for acyl) participates in activation of acyl groups in general, including the acetyl group derived from pyruvate. The coenzyme is derived metabolically from ATP, the vitamin pantothenic acid, and b-mercaptoethylamine. Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Thioesters such as acetyl-CoA are energy rich because thioesters are destabilized relative to ordinary oxygen esters. Copyright © 2013 Pearson Canada Inc.

Coenzymes Involved in Pyruvate Oxidation and the Citric Acid Cycle Comparison of free energies of hydrolysis of thioesters and oxygen esters: Lack of resonance stabilization in thioesters is the basis for the higher DG of hydrolysis of thioesters, relative to that of ordinary oxygen esters. The free energies of the hydrolysis products are similar for the two classes of compounds. Copyright © 2013 Pearson Canada Inc.

Action of the Pyruvate Dehydrogenase Complex Mechanisms of the pyruvate dehydrogenase complex: Copyright © 2013 Pearson Canada Inc.

Action of the Pyruvate Dehydrogenase Complex Lipoamide is tethered to one enzyme (E2) in the PDH complex, but it interacts with all three enzymes via a flexible swinging arm. Copyright © 2013 Pearson Canada Inc.

Action of the Pyruvate Dehydrogenase Complex Arsenic poisoning, both intentional and unintentional, has had a long history, dating back to at least the eighth century. Trivalent As(III) compounds such as arsenite and organic arsenicals react readily with thiols, and they are especially reactive with dithiols, such as dihydrolipoamide, forming bidentate adducts: Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 1: Introduction of Two Carbon Atoms as Acetyl-CoA The initial reaction, catalyzed by citrate synthase, is akin to an aldol condensation. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Mechanism of the citrate synthase reaction: Step 1: Asp 375 extracts a proton from the methyl group, and His 274 donates a proton to the carbonyl oxygen of acetyl-CoA, creating an enol. Step 2: His 274 deprotonates the acetyl-CoA enol, stabilizing the nucleophilic enolate that attacks the keto carbon of oxaloacetate. His 320 protonates the aldol product (S)- citroyl-CoA. Step 3: The citroyl-CoA intermediate spontaneously hydrolyzes to citrate by a nucleophilic acyl substitution reaction giving exclusively the S stereoisomer of citroyl-CoA. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Three-dimensional structure of citrate synthase: The two forms of pig heart citrate synthase homodimer shown here were determined by crystallographic methods and support the induced fit model of enzyme catalysis. In the absence of CoA-SH the enzyme crystallizes in an “open” form. Citrate binds at the base of large clefts in both catalytic domains of the homodimeric protein. Binding of CoA-SH causes the enzyme to adopt a “closed” conformation, with the clefts essentially filled. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 2: Isomerization of Citrate The tertiary alcohol of citrate presents yet another chemical problem: tertiary alcohols cannot be oxidized without breaking a carbon–carbon bond. To set up the next oxidation in the pathway, citrate is converted to isocitrate, a chiral secondary alcohol, which can be more readily oxidized. This isomerization reaction, catalyzed by aconitase, involves successive dehydration and hydration, through cis-aconitate as a dehydrated intermediate, which remains enzyme-bound. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle The enzyme contains nonheme iron and acid-labile sulfur in a cluster called a 4Fe–4S iron–sulfur center. The iron–sulfur cluster coordinates the hydroxyl group and one of the carboxyl groups on the citrate molecule. The cis-aconitate intermediate must flip 180° during the reaction, presumably by releasing from the enzyme, then rebinding with the iron–sulfur cluster, but in the opposite orientation. Thus, of the four possible diastereomers of isocitrate, only one, the 2R,3S diastereomer, is produced. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle The prochirality of citrate when bound to aconitase: Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Fluoroacetate is an example of a mechanism-based, or suicide, inhibitor of aconitase. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 3: Generation of CO2 by an NAD+-Linked Dehydrogenase The first of two oxidative decarboxylations in the cycle is catalyzed by isocitrate dehydrogenase. Isocitrate is oxidized to a ketone, oxalosuccinate, an unstable enzyme-bound intermediate that spontaneously b-decarboxylates to give the product, a-ketoglutarate. The strategy here is to oxidize isocitrate’s secondary alcohol to a keto group b to the carboxyl group to be removed. The b-keto group acts as an electron sink to stabilize the carbanionic transition state, facilitating decarboxylation. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Two carbon atoms enter the citric acid cycle as acetyl-CoA, and two are lost as CO2 in the oxidative decarboxylations of steps 3 and 4. Step 4: Generation of a Second CO2 by an Oxidative Decarboxylation This is a multistep reaction entirely analogous to the pyruvate dehydrogenase reaction. An a-keto acid substrate undergoes oxidative decarboxylation, with concomitant formation of an acyl-CoA thioester. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Decarboxylation of a-ketoglutarate: The first step catalyzed out by the a-ketoglutarate dehydrogenase complex is a decarboxylation catalyzed by a-ketoglutarate decarboxylase (E1 of the complex), producing a four-carbon TPP derivative. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 5: A Substrate-Level Phosphorylation Succinyl-CoA is an energy-rich thioester compound, and its potential energy is used to drive the formation of a nucleoside triphosphate This reaction, catalyzed by succinyl-CoA synthetase, is comparable to the two substrate-level phosphorylation reactions that we encountered in glycolysis, except that in animal cells the energy-rich nucleotide product is not always ATP but, in some tissues, GTP. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Covalent catalysis by the succinyl-CoA synthetase reaction: Three successive nucleophilic substitution reactions conserve the energy of the thioester of succinyl-CoA in the phosphoanhydride bond of ATP (or GTP). An active site histidine side chain is transiently phosphorylated (N-phosphohistidine) during the reaction. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle A charge–dipole interaction stabilizes the phosphohistidine intermediate in the succinyl-CoA synthetase reaction. In this schematic, based on the E. coli enzyme structure, the permanent dipoles of two -helices (the “power” helices) are oriented such that the d+ at their N-termini interact with the negative charges on the phosphate group of the active site N-phosphohistidine, stabilizing this transient reaction intermediate. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 6: A Flavin-Dependent Dehydrogenation Completion of the cycle involves conversion of the four-carbon succinate to the four-carbon oxaloacetate. The first of the three reactions, catalyzed by succinate dehydrogenase, is the FAD-dependent dehydrogenation of two saturated carbons to a double bond. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Succinate dehydrogenase is competitively inhibited by malonate, a structural analog of succinate.Malonate inhibition of pyruvate oxidation was one of the clues that led Krebs to propose the cyclic nature of this pathway. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle A single C-C bond is more difficult to oxidize than a C-O bond. Therefore, the redox coenzyme for succinate dehydrogenase is not but the more powerful oxidant FAD. The flavin is bound covalently to the enzyme protein through a specific histidine residue. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 7: Hydration of a Carbon–Carbon Double Bond The stereospecific trans hydration of the carbon–carbon double bond is catalyzed by fumarate hydratase, more commonly called fumarase. Copyright © 2013 Pearson Canada Inc.

The Citric Acid Cycle Step 8: A Dehydrogenation that Regenerates Oxaloacetate Finally, the cycle is completed with the NAD+–dependent dehydrogenation of malate to oxaloacetate, catalyzed by malate dehydrogenase. Copyright © 2013 Pearson Canada Inc.

Stoichiometry and Energetics of the Citric Acid Cycle One turn of the citric acid cycle generates one high-energy phosphate (ATP or GTP) through substrate-level phosphorylation, plus three NADH and one FADH2 for subsequent reoxidation in the electron transport chain. Copyright © 2013 Pearson Canada Inc.

Regulation of Pyruvate Dehydrogenase and the Citric Acid Cycle Major regulatory factors controlling pyruvate dehydrogenase and the citric acid cycle: Red brackets indicate concentration dependence. NADH can inhibit through allosteric interactions, but apparent NADH inhibition can also be a reflection of reduced NAD+ availability. Copyright © 2013 Pearson Canada Inc.

Regulation of Pyruvate Dehydrogenase and the Citric Acid Cycle Regulation of the mammalian pyruvate dehydrogenase complex by feedback inhibition and by covalent modification of E1. A kinase and a phosphatase inactivate and activate the first component (E1) of the pyruvate dehydrogenase complex by phosphorylating and dephosphorylating, respectively, three specific serine residues. The active form of the pyruvate dehydrogenase complex is feedback inhibited by acetyl-CoA and NADH. Copyright © 2013 Pearson Canada Inc.

Regulation of Pyruvate Dehydrogenase and the Citric Acid Cycle The citric acid cycle is controlled primarily by the relative intramitochondrial concentrations of NAD+ and NADH. Copyright © 2013 Pearson Canada Inc.

Anaplerotic Sequences: The Need to Replace Cycle Intermediates Citric acid cycle intermediates used in biosynthetic pathways must be replenished to maintain flux through the cycle. Anaplerotic pathways serve this purpose. Copyright © 2013 Pearson Canada Inc.

Anaplerotic Sequences: The Need to Replace Cycle Intermediates Copyright © 2013 Pearson Canada Inc.

Anaplerotic Sequences: The Need to Replace Cycle Intermediates Because phosphoenolpyruvate is such an energy-rich compound, this reaction, catalyzed by phosphoenolpyruvate carboxylase, requires neither an energy cofactor nor biotin. This reaction is important in the C4 pathway of photosynthetic CO2 fixation Copyright © 2013 Pearson Canada Inc.

Anaplerotic Sequences: The Need to Replace Cycle Intermediates In addition to pyruvate carboxylase and phosphoenolpyruvate carboxylase, a third anaplerotic process is provided by an enzyme commonly known as malic enzyme but more officially as malate dehydrogenase (decarboxylating:NADP+). The malic enzyme catalyzes the reductive carboxylation of pyruvate to give malate. Copyright © 2013 Pearson Canada Inc.

Anaplerotic Sequences: The Need to Replace Cycle Intermediates Mechanism of the biotin-dependent pyruvate carboxylase reaction: Phase I is catalyzed by the biotin carboxylation (BC) domain. Phase II is catalyzed by the carboxyltransferase (CT) domain. Copyright © 2013 Pearson Canada Inc.

Anaplerotic Sequences: The Need to Replace Cycle Intermediates Mechanism of the biotin-dependent pyruvate carboxylase reaction: Copyright © 2013 Pearson Canada Inc.

Glyoxylate Cycle: An Anabolic Variant of the Citric Acid Cycle The glyoxylate cycle allows plants and bacteria to carry out net conversion of fat to carbohydrate, bypassing CO2-generating reactions of the citric acid cycle. Copyright © 2013 Pearson Canada Inc.

Glyoxylate Cycle: An Anabolic Variant of the Citric Acid Cycle Reactions of the glyoxylate cycle: Two acetyl-CoA molecules enter the cycle, one at the citrate synthase step, and the second at the malate synthase step. The reactions catalyzed by isocitrate lyase and malate synthase bypass the three citric acid cycle steps between isocitrate and succinate so that the two carbons lost in the citric acid cycle are saved, resulting in the net synthesis of oxaloacetate. Copyright © 2013 Pearson Canada Inc.

Glyoxylate Cycle: An Anabolic Variant of the Citric Acid Cycle Intracellular relationships involving the glyoxylate cycle in plant cells: Fatty acids released in lipid bodies are oxidized in glyoxysomes to acetyl-CoA, which can also come directly from acetate. Acetyl-CoA is then converted to succinate in the glyoxylate cycle, and the succinate is transported to mitochondria. There it is converted in the citric acid cycle to oxaloacetate, which is readily converted to sugars by gluconeogenesis. Copyright © 2013 Pearson Canada Inc.