How are electrons extracted from the citric acid cycle for use in the electron transport chain

Abstract

The citric acid cycle utilizes mitochondrial enzymes. The first step is fusion of the acetyl group of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase. CoA-SH and heat are released and citrate is produced. Citrate is isomerized by dehydration and rehydration to isocitrate. The enzyme aconitase catalyzes these two steps using cis-aconitate as the intermediate. The next two steps are catalyzed by isocitrate dehydrogenase. Dehydrogenation of isocitrate forms oxalosuccinate which decarboxylates to alpha-ketoglutarate. Alpha-ketoglutarate is further oxidatively decarboxylated by alpha-ketoglutarate dehydrogenase—a multienzyme complex. Succinyl-CoA is formed in this unidirectional reaction.

Succinate thiokinase converts succinyl-CoA to succinate, while first generating ATP/GTP by substrate-level phosphorylation. Succinate is acted upon by succinate dehydrogenase, requiring FAD and Fe-S proteins to form fumarate. Fumarase adds water to a double bond of fumarate yielding malate. Malate regenerates oxaloacetate by action of NAD+-dependent malate dehydrogenase, completing the cycle.

Citric acid cycle

The CAC marks the center of interconnected energy providing pathways and cycles. In the mitochondrial matrix, enzymes of the CAC (also known as Krebs cycle or tricarboxylic acid cycle -TCA) produce the reducing equivalents NADH and FADH2. They deliver electrons to complexes of mitochondrial electron-transport chain, which builds up a proton gradient that drives ATP production. This key process links glycolysis, fatty acid oxidation, and also amino acid oxidation. In contrast to glycolysis and fatty acid oxidation, which can be described as linear pathways, cycles such as the CAC are energetically more efficient. ATP output of a full cycle is shown in Table 4.3 [31,32]. The centrality of the Krebs cycle for metabolism is remarkable. It not only connects all catabolic substrate oxidation pathways to the respiratory chain, but also represents the source of biosynthetic products for many anabolic processes [33]. Thus, although the cycle does not lose intermediates (i.e., moieties) by its own reactions, it still needs to be replenished continuously (anaplerosis, see later in the chapter).

Table 4.3. Catalytic Efficiency of the Citric Acid Cycle

The CAC stepwise catabolizes one molecule of acetyl-CoA, the high-energetic end product of glycolysis and β-oxidation into two molecules of carbon dioxide and the reducing equivalents NADH and FADH2. A schematic overview of the full CAC is shown in Fig. 4.6. Initially, acetyl-CoA reacts with the cycle intermediate oxaloacetate. This reaction is driven by citrate synthase, which is the key enzyme of the cycle and may also be seen as a marker of mitochondrial activity. The product citrate is dehydrated to the unstable intermediate cis-aconitate, which is subsequently hydrated to isocitrate. Both reactions are catalyzed by aconitase. In the third step of the CAC, NAD+-dependent isocitrate dehydrogenase transforms isocitrate to oxalosuccinate with the result of the first reducing equivalent NADH. Decarboxylation of oxalosuccinate leads to formation of α-ketoglutarate and CO2. In the next step, α-ketoglutarate dehydrogenase catalyzes the oxidation and carboxylation of α-ketoglutarate to succinate by formation of succinyl-CoA as an unstable intermediate. This reaction is comparable to the pyruvate dehydrogenase reaction and leads to formation of one molecule of high-energetic GTP. Efficiency of α-ketoglutarate dehydrogenase is crucial and controls the substrate flux through the complete CAC [34]. Further oxidation of succinate by FAD+-dependent succinate dehydrogenase results in the reducing equivalent FADH2 and fumarate, which is then hydrogenated to malate by fumarase. In the “last” step of the CAC, the NAD+-dependent malate dehydrogenase converts malate to oxaloacetate and another NADH. Consequently, oxaloacetate may react with another molecule of acetyl-CoA and start a new round of the cycle.

The CAC as the pivotal element of energy metabolism is strictly regulated. The cycle is triggered by ADP, inorganic phosphate, and calcium while it is inhibited by NADH and ATP. Additionally, most CAC enzymes are regulated by their educts and products in a kinetic manner. For example, succinate dehydrogenase is inhibited by oxaloacetate but activated by succinate. Although the amount of the intermediates of the CAC is strictly regulated, they also represent the basic building blocks for many synthesis processes. The pool of CAC intermediates is therefore continuously “in change” and needs to be replenished if synthesis processes have prevalence – a process that is called anaplerosis.

Entry via Succinyl-CoA

The citric acid cycle intermediate succinyl-CoA plays an important role in fatty acid and amino acid metabolism because it is the entry point of odd-chain fatty acids, propionate, and the branched chain amino acids valine and isoleucine into the citric acid cycle. Substrate entry as α-ketoglutarate or succinyl-CoA, in contrast to other anaplerotic pathways, is associated with the generation of energy-rich phosphates via a substrate-level phosphorylation. The reaction catalyzed by succinyl-CoA synthetase (E.C. 6.2.1.4) normally favors the conversion of succinyl-CoA to succinate and leads to substrate level phosphorylation of GDP to GTP. This energy-providing pathway becomes important under anaerobic conditions when ATP generation by oxidative phosphorylation is inhibited, such as in the setting of ischemia.

Citric Acid Cycle

The CAC interfaces with several other pathways (Fig. 7-7). It serves not only as a destination for the oxidation of carbon skeletons from amino acids but also as a source of precursors for biosynthesis pathways.

If the citrate concentration increases beyond that needed for energy generation by the CAC, then it is transported to the cytoplasm, where it is converted to acetyl-CoA and OAA by citrate lyase (see Chapter 10).

Carbon skeletons from deamination of amino acids enter at acetyl-CoA, α-ketoglutarate, succinyl-CoA, fumarate, or OAA. Carbons entering the CAC at succinyl-CoA, fumarate, or OAA can contribute their carbon skeletons to gluconeogenesis; they are termed glucogenic (see Chapter 12).

α-Ketoglutarate and OAA can leave the cycle, also through transamination, to be used for the synthesis of the carbon skeletons of the nonessential amino acids.

Succinyl-CoA can leave the cycle to serve as a precursor in the synthesis of porphyrins (see Chapter 12). It can also contribute to the use of ketone bodies in peripheral tissues by donating its CoA group to acetoacetate. Succinyl-CoA is formed from propionyl-CoA, a product of odd-chain fatty acid oxidation and the catabolism of several amino acids.

Acetyl-CoA carbons are always oxidized to CO2 and energy and never contribute carbon skeletons to gluconeogenesis. Therefore, fatty acid carbons cannot be used for glucose synthesis even though the fatty acids can be used to energize this pathway.

5.4 Block Effect on Enzyme Inhibition: Aconitase Inhibition in the Citric Acid Cycle

The citric acid cycle (also called as Krebs cycle or tricarboxylic acid cycle) takes place in the mitochondria and is an integral part for the generation of adenosine triphosphate (ATP). In the citric acid cycle, 36 mol of ATP are formed from a single glucose molecule, and reduced nicotinamide adenosine diphosphate and other organics are also formed, which serve as intermediates for the biosynthesis of amino acids (e.g., glutamate is synthesized from α-ketoglutaric acid, an intermediate in the citric acid cycle).

Fluoroacetate is the starting compound for the biosynthesis of the fluorinated compounds in certain soil bacteria. However, fluoroacetyl-CoA (18), derived from the fluoroacetate, also competes with acetyl-CoA in its citrate-synthase-catalyzed reaction with oxaloacetate to form (2R, 3R)-2-fluorocitrate (20), which undergoes aconitase-catalyzed dehydration to give the 2(R)-2-fluoro-cis-aconitic acid (21) (in analogy to the normal citric acid cycle, in which cis-aconitic acid is formed from acetyl-CoA). Whereas the hydration of cis-aconitic acid gives isocitric acid, the next intermediate in the normal citric acid cycle, 21 undergoes SN2′ type of hydroxylation–defluorination to give (R)-hydroxy-trans-aconitic acid (22), which no longer can participate in the citric acid cycle and, moreover, it acts as an irreversible inhibitor of the aconitase enzyme, resulting in shutting down of the citric acid cycle, and thereby exerting cytotoxicity in humans and other mammals.41 In order to establish the mechanism of the aconitase inhibition by the secondary metabolite 22, a single-crystal X-ray structure determination was undertaken. The single-crystal structure of this enzyme–inhibitor complex revealed the tight binding of 22 to the aconitase at the active site.41 His101, His147, and His167 have strong hydrogen bonding interactions with the substrate carboxylate moieties, and the resulting protonated His101 also forms hydrogen bond to the inhibitor hydroxyl group, which, in turn, forms a hydrogen bond to Asp165 (Figure 21). Thus the enzyme inhibitory action of the toxic metabolite 22 is not through its covalent binding to the amino acid residues at the enzyme active site, but rather through its tight fitting in, and thereby blocking, the active site.41

Importantly, the 2(S)-2-fluoro-cis-aconitic acid, (enantiomer of 21) would not be able to undergo the similar SN2′ type of hydroxylation–defluorination and therefore would not be able to inhibit the aconitase enzyme. The citrate-synthase-catalyzed reaction of oxaloacetate with Z-enolate-CoA (Z-19), followed by aconitase catalyzed dehydration would give the 2(S)-2-fluoro-cis-aconitic acid, whereas E-enolate-CoA (E-19) gives predominantly 21. The citrate synthase converts the fluoroacetyl-CoA (18), stereoselectively (98:2 diastereoselectivity) to E-19, resulting in the cytotoxicity associated with the fluoroacetate (Figure 22). Using ab initio quantum mechanics/molecular mechanics modeling Mulholland and coworkers42 have rationalized the high stereoselectivity of the citrate synthase (i.e., the stereoselective formation of the E-19): the computed enzyme–substrate complex for the E-19 is about 2 kcal/mol more stable than that for Z-19, which is also in accord with the estimated Δ(ΔGǂ) value based on the experimentally observed ∼98:2 diastereomeric ratio of the 2(R)- and 2(S)-2-fluorocitrates.

Aconitase

The citric acid cycle enzyme aconitase belongs to the family of (^’”-sensitive, [4Fe-4S]-containing dehydratases. Both the mitochondrial and cytosolic forms of aconitase are inactivated by O2•−, so that activity can be used to monitor O2•− production. The drawback of this assay is that it measures intracellular O2•− production from all sources, not only from NAD(P)H oxidases, and it is not easily adaptable to the measurement of NADPH-dependent O2•− production, except with the use of antisense or knockout strategies (see above).

Cell extracts are prepared according to Gardner et al.35 by sonication in hypotonic Tris buffer [50 mM Tris-HCl (pH 7.4), 0.6 mM MnCl2, 20 μM (±)fluoro-citrate]. The lysate is then immediately stored at − 80 °. Fluorocitrate and MnCl2 are included to limit the inactivation of aconitase by O2•− during extract preparation and storage, and lysates can be stored for up to 2 weeks. Cysteine (1 mM) can be substituted for fluorocitrate.36

Aconitase activity is measured spectrophotometrically by monitoring the increase in absorbance at 240 nm due to the formation of cis-aconitate from isocitrate. For this assay, lysates are incubated in 50 mM Tris-HCl (pH 7.4), 20 mM isocitrate, and 0.5 mM MnCl2. After addition of the extract, A240 is determined for 1–2 min (ε240 = 3.6mM− 1 cm− 1).

Aconitase activity can also be assessed by monitoring the formation of NADPH at 340 nm. Here, citrate is used as the substrate for aconitase, and its product, iso-citrate, is converted to α-ketoglutarate by the NADP+-dependent enzyme isocitrate dehydrogenase. Lysates are clarified by centrifugation for 20 sec at 14,000g, and 10- to 100-μg aliquots of the supernatant are incubated in a reaction mixture containing 50 mM Tris-HCl (pH 7.4), 5 mM sodium citrate, 0.6 mM MnCl2, 0.2 mM NADP+, and isocitrate dehydrogenase (1 U/ml). Absorbance at 340 nm (ε340 = 6.22 mM− 1 cm− 1) is monitored at 25 °, and rates are measured in the latter half of a 60-min assay. One milliunit of aconitase activity is defined as the amount catalyzing the formation of 1 nmol of isocitrate per minute. To obtain a quantitative measure of O2•−, one must also determine the rate constant of aconitase reactivation according to Hausladen and Fridovich36 and then [O2•−] = [aconitaseinactive]kreactivation/[aconitaseactive]kinactivation, although many investigators simply report the relative ability of two samples to inactivate aconitase to obtain a comparative measure of O2•− levels.

7.27.5.1.4 Inhibitors of the glycolytic pathway

A functional citric acid cycle is absent in plasmodium parasites, and their main energy source is considered to be the ATPs generated during anaerobic fermentation of glucose. To meet the high energy requirement of the parasites, the infected erythrocytes consume 30–100 times more glucose than uninfected ones, and glycolysis is essential for parasite survival.315 Almost all enzymes involved in this process have been identified from P. falciparum, and the crystal structures of three of them, triosephosphate isomerase (PfTIM),316 lactate dehydrogenase (PfLDH),317 and fructose-1,6-bisphosphate aldolase (PfALDO),318 are currently available. Since a number of key differences exist between these enzymes and their human counterparts, many of them have gained attention as possible targets for therapeutic intervention.

During the second stage in glycolysis, fructose-1,6-bisphosphate aldolase catalyzes the conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Comparison of the crystal structures of PfALDO and human aldolase A has indicated a few key features that could be useful in drug design. One of these is the presence of a ‘pocket’ formed by residues in the loop region 290–300 and those from two nearby loops, which is hydrophobic and more constricted in PfALDO than that in human aldolase A.318,319 Even though reports on small molecule inhibitors of PfALDO are limited, homologous peptides of human band-3 protein and those derived from P. falciparum alpha-tubulin have been shown to inhibit PfALDO.320 In another approach, antisense oligonucleotide technique was used to target PfALDO messenger RNA (mRNA), which subsequently led to the growth inhibition of erythrocytic P. falciparum.321

Triosephosphate isomerase is involved in the isomerization of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate, which is essential to maintain the efficiency of glycolysis. Screening a database of compounds against the crystal structure of PfTIM led to the identification of several anionic dyes with good docking scores and activities, and one of the compounds investigated, congo red (62), showed an IC50 value of 30.4 μM against the enzyme.322 In another interesting study by Singh et al., peptide fragments corresponding to loops 1 and 3 of PfTIM were used to disrupt the dimerization of TIM which is essential for its enzymatic activity.323 One of these peptides, which contained residues 68–79 of loop 3, was able to inhibit the enzyme with submillimolar IC50 values. Even though a number of studies pertaining to the conformational details of PfTIM are available,324 reports on rational drug design based on this enzyme are currently very limited. Future exploration in this direction could give new lead compounds useful in drug development against malaria.

Lactate dehydrogenase (LDH) is the last enzyme in the glycolytic pathway and is responsible for the conversion of pyruvate to lactate with concomitant formation of NAD+ from NADH. Since a constant supply of NADH is essential for glycolysis, LDH also assists in the regeneration of NADH from NAD+. Even though the inhibition of this enzyme could kill the parasite, it is important to develop inhibitors which can differentiate it from human LDH isoforms. Important structural and kinetic differences that are useful in studies with this enzyme are (1) a change in the positioning of NADH in PfLDH compared to that in the human enzyme, as indicated by a displacement of the nicotinamide ring by about 1.2 Å due to sequence changes in the cofactor binding region, (2) changes in the sequence and secondary structure of a loop adjacent to the active site, (3) larger size of the active site cleft in PfLDH compared to its human counterpart, (4) unlike human LDH, the malarial enzyme is not inhibited by high concentrations of pyruvate, and (5) malarial LDH is very active with a synthetic coenzyme, 3-acetylpyridine adenine dinucleotide (APAD), which makes it useful as a probe to test parasitemia in human blood.319,325,326

Over the years, a number of investigations have been conducted to identify new drug candidates that can inhibit PfLDH. One of the important compounds identified was gossypol (63), a polyphenolic binaphthyl disesquiterpene isolated from cotton seeds. It is competitive for NADH and showed LDH inhibition with a submicromolar IC50 value (0.7 μM). Although this compound retained similar activity when tested in vitro against malaria parasites (IC50=7 μM), its cytotoxicity, arising from the aldehyde groups, and poor selectivity against human LDHs were limiting factors in drug development efforts.327 A synthetic analog based on the structure of gossypol, 7-p-trifluoromethylbenzyl-8-deoxyhemigossylic acid (64), was later developed, which showed Ki values of 13, 81, and 0.2 μM, respectively, against human muscle, heart, and PfLDHs.328 More recently, high-throughput screening of a library of compounds led to the discovery of a number of azole-based small molecules having good selectivities and activities against PfLDH. Thus, compound 65 showed an IC50 value of 0.65 μM against PfLDH (relative value against human LDH=72.05 μM) and exhibited promising in vitro (IC50=18.6 μM against K1 strain of P. falciparum) and in vivo (41% reduction in parasitemia after a 4-day test) activities against malaria parasites.329 Crystallographic studies showed that these analogs bind in a similar fashion to that of pyruvate in the active site of the enzyme, and suggest the possibility of developing new antimalarial agents based on such scaffolds. More studies are needed in this area to identify better drug candidates having activities ideal for therapeutic use, with good selectivities over human enzymes.

Carbohydrate Metabolism

Glycolysis and the citric acid cycle are the major energy-producing catabolic pathways and they are conserved in all kingdoms of life. Rickettsia and Orientia alike are non-glycolytic and utilize citric acid cycle intermediates, mainly derived from pyruvate, glutamine and glutamate, and aspartic acid and asparagine to drive oxidative phosphorylation and ATP synthesis [159,160]. Orientia lack a complete glycolysis pathway in which glucose is oxidized to pyruvate, though it does have three glycolytic enzymes (Gap, Pgk and TpiA). These enzymes are absent in R. conorii and R. prowazekii – consistent with the failure to detect glycolytic activity from bacterial extracts – but they possess a glycerol-phosphate transporter (glpT) gene, which allows expression of the transporter for internalization of substrates from the host cell [157]. Thus orientae do not synthesize pyruvate by the glycolytic pathway (Fig. 112.6), but it is possible that pyruvate is acquired from the host, or synthesized from malate by malate dehydrogenase (MaeA), and subsequently converted to pyruvate by pyruvate phosphate dikinase (PPDK), both of which are present in all of the Rickettsiales members. As three enzymes involved in the initial steps of the citrate acid cycle are lacking in Orientia, they also produce no acetyl-CoA [13,142]. Among the members of Rickettsiales, Orientia are the only members that lack a functional pyruvate dehydrogenase complex and rely on the host cell as a source of acetyl-CoA, which is an essential coenzyme in diverse biosynthetic pathways [158]. These enzymes serve to generate glycerol-phosphate, the starting material for glycerophospholipid synthesis and for generation of energy from glycerol-3-phosphate by a reverse reaction [158].

Citric Acid Cycle Energy Balance

Oxidation of acetate in the citric acid cycle has high phosphoryl transfer potential and yields significant chemical energy. The oxidative reactions of the cycle donate the reduced hydrogen atoms from coenzymes to the respiratory chain. There, the flow of electrons is coupled to the pumping of protons from the mitochondrial matrix to the intermembrane space creating an electrochemical H+ gradient. Return of H+ through the ATP synthase channel provides the energy for ATP synthesis from ADP and Pi. Each pair of hydrogen ions transferred from NAD generates 2.5–3 molecules of ATP (in order to simplify calculations, factor 3 is used). The hydrogen atoms transferred from flavoproteins (FAD) produce 1.5–2 ATP (to simplify calculations, factor 2 is used). The total energy balance per mole of metabolized acetate of the citric acid cycle is summarized in Table 14.4.

Table 14.4. Energy Yield of Acetate Oxidation in the Citric Acid Cycle

4 Methods for the Detection of Oxidative Stress

Several methods have already been described above which are suitable techniques for the accurate measurement of ROS formation or elimination inside the mitochondrion. ROS formation and elimination are always present in parallel and the selective measurement of one of the two processes does not provide information about the status of the other. In order to establish whether there is an oxidative stress condition, stress-sensitive mitochondrial functions also need to be monitored. The measurement of mitochondrial aconitase activity is a suitable candidate for this purpose.