Biochem 12.1 PDF - The Citric Acid Cycle

# The Citric Acid Cycle ## Introduction Lesson 11.1 examines glucose catabolism, including glycolysis, in which glucose is converted to two pyruvate molecules. Glycolysis requires NAD+, and under anaerobic conditions (ie, when oxygen is absent or scarce) NAD+ is regenerated by fermentation. Under...

# The Citric Acid Cycle ## Introduction Lesson 11.1 examines glucose catabolism, including glycolysis, in which glucose is converted to two pyruvate molecules. Glycolysis requires NAD+, and under anaerobic conditions (ie, when oxygen is absent or scarce) NAD+ is regenerated by fermentation. Under aerobic conditions, however, NAD+ is regenerated through aerobic respiration (Figure 12.1). After glycolysis, aerobic respiration continues with the citric acid cycle, which produces NADH and FADH2. These molecules then enter the electron transport chain, where they ultimately pass their electrons to molecular oxygen (O2). These reactions provide the energy needed for ATP synthesis. ## Stages of Aerobic Respiration 1. Glycolysis 2. Pyruvate processing 3. Citric acid cycle 4. Electron transport and chemiosmosis ## Overview of Glucose Catabolism **Figure 12.1** Overview of glucose catabolism through aerobic respiration. This lesson focuses on the citric acid cycle, also known as the Krebs cycle or the tricarboxylic acid (TCA) cycle. Specifically, it focuses on the citric acid cycle using the pyruvate generated by glycolysis as a starting material. Other molecules such as fatty acids and amino acids may also enter the citric acid cycle, and these processes will be covered in Chapter 13. ## Pyruvate Entry into the Citric Acid Cycle The end product of glycolysis is pyruvate. Once formed, pyruvate typically has one of two fates. Under anaerobic conditions, pyruvate is fermented to form either ethanol or lactate, depending on the organism (see Concept 11.1.04). Under aerobic conditions (ie, abundant oxygen), many organisms instead send pyruvate into the citric acid cycle. In eukaryotes, the citric acid cycle occurs in the mitochondria, whereas glycolysis occurs in the cytosol. Consequently, pyruvate must be transported from the cytosol into the mitochondria prior to use in the citric acid cycle. Mitochondria consist of two membranes that surround the mitochondrial matrix. The outer membrane is porous and permeable to most ions and small molecules. The inner membrane is much more selective but contains transport proteins that carry pyruvate into the matrix, as shown in Figure 12.2. **Figure 12.2** Pyruvate transport into the mitochondrial matrix. The mitochondrial matrix contains multiple proteins involved in catabolism, including the pyruvate dehydrogenase complex (PDC) shown in Figure 12.3. This complex consists of three individual enzymes that collectively catalyze the decarboxylation of pyruvate to form acetyl coenzyme A (acetyl-CoA) and CO2. These enzymes are 1. Pyruvate dehydrogenase - Often called the E1 component of the complex, this enzyme uses the molecule thiamine pyrophosphate (TPP) as a coenzyme. TPP nucleophilically attacks the electrophilic carbon at position two (C2) of pyruvate, converting the carboxyl group at position one into CO2. 2. Dihydrolipoyl transacetylase - Often called E2, this enzyme uses a sulfur-containing molecule called lipoic acid (or lipoate) as a cofactor. Lipoic acid attacks the electrophilic carbon and forms a bond with it, forming acyl-lipoate and releasing TPP as a leaving group. Coenzyme A, often represented as CoA-SH, then removes the acyl group (specifically, a 2-carbon acetyl group) from lipoate to form acetyl-CoA. 3. Dihydrolipoyl dehydrogenase - Called E3, this enzyme uses the coenzyme FAD to oxidize lipoic acid so that it can repeat the E2 reaction. The FAD is converted to FADH2, which then transfers its electrons to NAD+, forming NADH and regenerating FAD. **Figure 12.3** Overview of mechanism of the pyruvate dehydrogenase complex. The net reaction catalyzed by the PDC, then is: $Pyruvate + NAD+ + CoA-SH \rightarrow Acetyl-CoA +NADH + CO_2$ The resulting acetyl-CoA enters the citric acid cycle, the NADH enters the electron transport chain (see Lesson 12.2), and the CO2 is eventually exhaled. Pyruvate dehydrogenase is inactivated by phosphorylation. The phosphorylation reaction is carried out by pyruvate dehydrogenase kinase (PDK), which is allosterically activated by the products of the PDC: acetyl-CoA and NADH. PDK activation results in the inactivation of the PDC, and therefore this pathway works as a form of feedback inhibition. In contrast, PDK is downregulated by the reactants of the PDC: pyruvate, coenzyme A, and NAD+. In this way, buildup of PDC reactants results in the release of inhibition—and therefore an indirect activation-of the PDC. Interactions between PDK, PDC, and several metabolites are shown in Figure 12.4. **Figure 12.4** Pyruvate dehydrogenase kinase (PDK) action on the PDC, and allosteric regulation of PDK. The phosphate is removed by pyruvate dehydrogenase phosphatase, which reactivates the PDC. Pyruvate dehydrogenase phosphatase is activated by insulin and by calcium ions. This regulation can help stimulate aerobic respiration during periods of glucose excess and vigorous muscular contraction, respectively. # Citric Acid Cycle Reactions Once pyruvate is converted acetyl-CoA, it can enter the **citric acid cycle**. The citric acid cycle is an eight-step metabolic pathway that begins when a two-carbon group (the acetyl component of acetyl-CoA) combines with a four-carbon molecule (oxaloacetate) to form the six-carbon molecule citrate. Over the course of the cycle, two carbon atoms are removed, one at a time, each in the form of CO2. This converts the six-carbon molecule first into a five-carbon molecule, and then into a four-carbon molecule, which eventually regenerates oxaloacetate. The regenerated oxaloacetate can then react with a new acetyl-CoA molecule to repeat the cycle. The first half of the citric acid cycle (ie, three of the first four steps) is predominantly irreversible. Under physiological conditions, the reactions are highly exergonic, and two of them release CO2, which quickly exists the mitochondria. The three irreversible reactions in the first half of the cycle are the primary points of regulation. The final four reactions, in contrast, are reversible. Figure 12.5 shows an overview of the citric acid cycle. **Figure 12.5** Overview of the citric acid cycle. ## Steps of the Citric Acid Cycle ### Step 1: Acetyl-CoA and Oxaloacetate Combine to Become Citrate The first step of the citric acid cycle is the condensation of the two-carbon acetyl group of acetyl-CoA and the four-carbon molecule oxaloacetate. A water molecule is also consumed in this reaction, while free coenzyme A (CoA-SH) is produced. The product is the six-carbon molecule citrate (the conjugate base of citric acid) from which the cycle takes its name. Citric acid contains three carboxylic acid groups, and accordingly the cycle is also called the tricarboxylic acid (TCA) cycle. The formation of citrate is catalyzed by the enzyme citrate synthase, a transferase that transfers an acetyl group from acetyl-CoA to oxaloacetate. The reaction is shown in Figure 12.6. **Figure 12.6** Step 1 of the citric acid cycle, catalyzed by citrate synthase. Importantly, this reaction is irreversible, meaning acetyl-CoA cannot be regenerated from citrate by this enzyme. Consequently, this step is carefully regulated. Citrate synthase activity is downregulated by citrate, NADH, succinyl-CoA, and ATP, each of which are products of citrate synthase or of downstream enzymes. In other words, these molecules cause feedback inhibition. In contrast, ADP allosterically upregulates citrate synthase activity. ### Step 2: Citrate Becomes Isocitrate Citrate is a symmetric molecule with a hydroxyl group on its central carbon. To facilitate formation of a-ketoglutarate in Step 3, the hydroxyl group must move to one of the methylene (-CH2-) groups of citrate. In other words, citrate must isomerize to form isocitrate. This reaction is the only reversible reaction in the first half of the cycle and is facilitated by the enzyme aconitase. Aconitase first removes the hydroxyl group along with a hydrogen atom from a methylene group, forming a water molecule. This dehydration results in the formation of a double bond, yielding the molecule cis-aconitate. This classifies aconitase as a lyase. After the dehydration reaction, water is then added back to cis-aconitate in a different configuration (ie, it is rehydrated) to form isocitrate. Consequently, the net reaction catalyzed by aconitase is an isomerization (the hydroxyl group and hydrogen atom switch places). However, because the enzyme accomplishes isomerization by catalyzing two separate lyase reactions, it is not classified as an isomerase despite its name. Figure 12.7 shows the conversion of citrate to isocitrate. **Figure 12.7** Conversion of citrate to isocitrate through a cis-aconitate intermediate. ### Step 3: Isocitrate Is Decarboxylated to Form a-Ketoglutarate From an energy standpoint, the primary purpose of the citric acid cycle is to produce the reduced cofactors NADH and FADH2, which later enter the electron transport chain. The third step of the citric acid cycle, in which isocitrate becomes a-ketoglutarate, is the first of three to produce NADH. This step is facilitated by the enzyme isocitrate dehydrogenase. Conversion of isocitrate (a six-carbon molecule) to a-ketoglutarate (a five-carbon molecule) occurs through oxidative decarboxylation. One of the carboxyl groups of isocitrate is oxidized to CO2 and released. In addition, the hydroxyl group of isocitrate is oxidized to a ketone. Consequently, isocitrate loses two electrons and CO2 in the reaction. CO2 cannot readily be added back to a-ketoglutarate, so this reaction is irreversible. When an oxidation reaction occurs, a reduction reaction must occur simultaneously, because the electrons lost by one molecule must be gained by another. In this case, the two electrons lost by isocitrate are transferred to NAD+ as a hydride ion, forming NADH. Because the reaction is a redox reaction, isocitrate dehydrogenase is an oxidoreductase. Figure 12.8 shows the oxidative decarboxylation of isocitrate. **Figure 12.8** Conversion of isocitrate to a-ketoglutarate. Isocitrate dehydrogenase is closely regulated. Like citrate synthase, isocitrate dehydrogenase is allosterically inhibited by ATP and upregulated by ADP. Interestingly, this enzyme is also upregulated by calcium ions (Ca2+). Calcium is released from the sarcoplasmic reticulum as part of the process of muscle contractions, which consume ATP. Therefore, Ca2+ signals that more ATP is needed. ### Step 4: a-Ketoglutarate Is Decarboxylated and Becomes Succinyl-CoA This step, facilitated by the a-ketoglutarate dehydrogenase complex, is another irreversible oxidative decarboxylation in which the carboxyl group linked to the ketone carbon of a-ketoglutarate is oxidized to CO2 (Figure 12.9). The ketone carbon is further oxidized by reacting with coenzyme A to form a thioester called succinyl-CoA. The succinyl component contains the four carbons remaining after the loss of CO2. The two electrons lost from a-ketoglutarate are transferred to NAD+ as a hydride, producing the second of three NADH molecules. The a-ketoglutarate dehydrogenase complex is another oxidoreductase. **Figure 12.9** Oxidative decarboxylation of a-ketoglutarate to form Succinyl-CoA. a-Ketoglutarate dehydrogenase, like citrate synthase, is allosterically downregulated by succinyl-CoA and NADH (its products). Like isocitrate dehydrogenase, this enzyme is allosterically upregulated by Ca2+. Note that at this point in the pathway, three CO2 molecules have been produced in the mitochondria. The first was produced in the step before the cycle began, when pyruvate became acetyl-CoA. The second CO2 molecule was produced when isocitrate became a-ketoglutarate (Step 3), and the third was produced when a-ketoglutarate became succinyl-CoA (Step 4). Pyruvate is a three-carbon molecule. Therefore, at this point in the process, the carbon atoms that entered as pyruvate have been offset by carbons that exit as CO2. Importantly, the two carbons contributed by acetyl-CoA are still present in succinyl-CoA at this point, while two other carbons were removed as CO2, with the net effect being that three carbons went into the mitochondria as pyruvate, and three came out as CO2. Each glucose molecule that entered glycolysis produced two pyruvate molecules (six carbons), so once both pyruvate molecules enter the mitochondria, all six carbons from glucose are effectively converted to CO2 by these steps. Figure 12.10 highlights the carbons contributed by acetyl-CoA and the carbons that become CO2. **Figure 12.10** The two carbons that acetyl-CoA contributes to citrate are still present in succinyl-CoA, while two other carbons (contributed by oxaloacetate) become CO2. ### Step 5: Succinyl-CoA Is Converted to Succinate The thioester bond in succinyl-CoA is a high-energy bond. In humans, the enzyme succinyl-CoA synthetase catalyzes formation of GTP by condensing GDP and inorganic phosphate. In the same reaction, succinyl-CoA is broken into the four-carbon molecule succinate and coenzyme A (Figure 12.11). Interestingly, this reaction is reversible because the high-energy phosphoanhydride bond in GTP is energetically similar to that of the thioester in succinyl-CoA. **Figure 12.11** Reversible conversion of succinyl-CoA to succinate. Some organisms transfer the phosphate group to ADP, forming ATP instead of GTP. Note that GTP and ATP are energetically equivalent (see Lesson 10.1). In addition, in organisms that produce GTP in this step, the phosphate group may subsequently be transferred to ADP to produce ATP. The enzyme in this reaction is named succinyl-CoA synthetase, but in the context of the citric acid cycle, succinyl-CoA is broken down; therefore, this enzyme is named for the reverse reaction: formation of succinyl-CoA using the energy provided by GTP hydrolysis. Based on this reaction, succinyl-CoA synthetase is a ligase, linking two molecules (succinate and coenzyme A) while separately hydrolyzing a nucleoside triphosphate. ### Step 6: Succinate Becomes Fumarate Succinate contains two methylene (-CH2-) groups linked to each other through a sigma bond. Oxidation of this bond removes one hydrogen atom (including its electrons) from each carbon to form a C=C double bond. The resulting molecule is called fumarate. This reversible reaction is facilitated by the oxidoreductase enzyme succinate dehydrogenase, which effectively transfers the two hydrogen atoms to FAD, forming FADH2. This is the only step in the citric acid cycle that produces FADH2. Figure 12.12 shows oxidation of succinate to fumarate. **Figure 12.12** Oxidation of succinate to fumarate. Importantly, succinate dehydrogenase is part of a larger enzyme complex found in the inner mitochondrial membrane: Complex II of the electron transport chain (see Lesson 12.2). The succinate dehydrogenase component of this complex does not use free FAD. Instead, FAD is bound to the enzyme as a prosthetic group. As soon as FADH2 is produced, it quickly transfers its electrons through the rest of the complex, regenerating FAD. ### Step 7: Fumarate Becomes L-Malate In this reversible step, the carbon-carbon double bond formed in the previous step is broken by addition of water. This reaction produces L-malate and is catalyzed by the enzyme fumarase. Although water is used to break a bond in this reaction, in this case it is a pi bond. Therefore, as noted in Lesson 4.2, fumarase is not classified as a hydrolase, but is instead classified as a lyase. Although the product of this reaction is commonly referred to simply as malate, it is important to note that malate is a chiral molecule. Specifically, the hydroxyl carbon is a chiral center, and the reaction catalyzed by fumarase is stereospecific; it always produces the L-form of malate. Figure 12.13 shows the reaction catalyzed by fumarase. **Figure 12.13** Hydration of fumarate to form L-malate. ### Step 8: L-Malate Is Oxidized to Oxaloacetate In the final step of the citric acid cycle, the hydroxyl group of L-malate is oxidized to a ketone to yield oxaloacetate. In this reversible process, which is catalyzed by an oxidoreductase called malate dehydrogenase, two electrons are transferred to NAD+ to form the final NADH molecule of the cycle (Figure 12.14). The resulting oxaloacetate molecule can then react with another acetyl-CoA molecule to begin a new cycle. **Figure 12.14** Oxidation of malate to oxaloacetate. ## Citric Acid Cycle Products The net reaction for the citric acid cycle is: $Acetyl-CoA + 3 NAD+ + FAD + 2 H_2O + GDP + Pi \rightarrow 2 CO2 + 3 NADH + FADH_2 + GTP + Coenzyme A + 3 H+$ Note that because oxaloacetate is consumed and then regenerated by the cycle, it is not included as a net reactant or product. Similarly, none of the intermediates are included because they are consumed immediately after they are produced. In addition to the reactions of the cycle itself, entry of pyruvate into the cycle follows the reaction: $Pyruvate + NAD+ + Coenzyme A \rightarrow Acetyl-CoA + NADH + CO_2$ Energetically, the most important products are NADH, FADH2, and GTP, each of which can be used to generate ATP or, in the case of GTP, can be used directly to power various biological processes. Table 12.1 shows the numbers of each high-energy product formed per pyruvate molecule that enters the cycle. Because glycolysis produces two pyruvate molecules per glucose, each glucose molecule can produce double the number of high-energy products. **Table 12.1** High-energy products of pyruvate dehydrogenase in the citric acid cycle, per pyruvate or glucose. | Product | Number of products per pyruvate | Number of products per glucose | |---|---|---| | NADH | 4 | 8 | | FADH2 | 1 | 2 | | GTP | 1 | 2 | This lesson has focused on the citric acid cycle starting with the reaction between acetyl-CoA and oxaloacetate because this is the point of entry for metabolites derived from glucose. However, it is possible for other metabolites to enter the citric acid cycle at different points. For example, certain amino acids can be converted directly to a-ketoglutarate (discussed further in Lesson 13.2), which can participate in Steps 4–8 of the cycle to become oxaloacetate. In this case, two of the NADH-producing steps are skipped: conversion of pyruvate to acetyl-CoA and conversion of isocitrate to a-ketoglutarate. This is shown in Figure 12.15. **Figure 12.15** Entry into the cycle at a point other than acetyl-CoA alters the amount of NADH, FADH2, and GTP produced. Once oxaloacetate forms, it cannot continue through another round of the cycle unless it reacts with a new acetyl-CoA. Consequently, when a-ketoglutarate enters the cycle directly, it only provides enough energy to produce two NADH molecules instead of four (FADH2 and GTP production are not affected in this case). The energy for any further NADH production comes from the addition of new acetyl-CoA. In other words, for the citric acid cycle to continue from one round to the next, a constant influx of acetyl-CoA is required.