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The Tricarboxylic Acid Cycle The First of the Final Common Pathways Objectives: I. Describe that types of reactions available to the cell for breaking carbon-carbon bonds. II. Describe the entry of pyruvate into the Citric Acid Cycle. A. Its conversion into an acetate ion (acetate fragment) coupled to Coenzyme A. 1. Conversion of pyruvate into acetyl-CoA. III. Describe the pyruvate dehydrogenase complex. A. Subunit structure B. Necessary cosubstrates and coenzymes. 1. Vitamins that these cosubstrates and coenzymes are derived from. C. Reaction Mechanism IV. What is the “fuel” of the TCA Cycle? V. What are the products of one turn of the cycle? VI. Summarize the reactions of the citric acid cycle. A. How is the α-ketoglutarate dehydrogenase step similar to the reaction catalyzed by the pyruvate dehydrogenase complex? VII. State the reactants and products of the “first step” of the Citric Acid Cycle and the product(s) of the “last step” of the Citric Acid Cycle. VIII. Describe how the Tricarboxylic Acid Cycle provides precursors for energy generation under aerobic conditions. A. Generation of NADH and FADH2 IX. Given a step or the steps of the TCA cycle, indicate the type of reaction that has occurred and possible enzyme involved. X. Discuss the control points of the Tricarboxylic Acid Cycle A. Describe how TCA Cycle intermediate concentrations control the rate of the TCA Cycle. 1. Reaction(s) that increase the intermediate concentration. a) Describe the three most common ANAPLEUROTIC REACTIONS. b) Allosteric control of the Pyruvate Carboxylase reaction. 2. Intermediates that are removed from the cycle to decrease the rate of the pathway. a) Products formed from the intermediates removed. 3. Use of TCA Cycle intermediates as precursors for anabolic reactions. B. Describe how acetate (acetyl-CoA) availability controls the rate of the TCA Cycle. 1. Describe how the rate of the pyruvate dehydrogenase complex is controlled. 2. Allosteric effectors and their effects. 3. Control by reversible covalent modification. a) Allosteric effectors and their effects on Pyruvate Dehydrogenase Kinase and Pyruvate Dehydrogenase Phosphatase. C. Describe the allosteric enzymes within the Citric Acid Cycle. 1. Describe the allosteric modulators and their effects on the allosteric enzymes that control The Citric Acid Cycle. D. Discuss why The TCA Cycle is classified as an AMPHIBOLIC PATHWAY. XI. Ask yourself “What If Questions” 1 ©Kevin R. Siebenlist, 2016 Background The two Final Common Pathways are both oxidative pathways. Although cells are marvelous machines they are forced to function with a limited bag of tricks. Biological oxidations that require the cleavage of carbon-carbon bonds are accomplished in the cell by one of two strategies. The first type of carbon bond cleavage breaks the bond α to a carboxylic acid group provided that the α carbon is carrying a hydroxyl group or carbonyl (ketone) group. This is an α-cleavage reaction. The second reaction type requires a molecule with a carbonyl (ketone) or carboxylic acid group and it involves bond cleavage between the alpha (α) and beta (β) carbons. The first step of the reaction catalyzed by the Pyruvate Dehydrogenase Complex, the decarboxylation of pyruvate, is an oxidation reaction with an α-cleavage type. Two reactions during the TCA cycle are oxidations that result in carbon-carbon bond breakage. One of the reactions employs cleavage between the α- and β-carbons and the other reaction uses an α-cleavage. O C OH O C C C C C C O Cleavage Cleavage O O C O C C O O Metabolism of Pyruvate Pyruvate, produced by glycolysis or from other metabolic sources, is one of the key intermediates of cellular metabolism. It can serve as a precursor in many biosynthetic pathways. For example, pyruvate can serve as a precursor for glucose, oxaloacetate, and several of the amino acids. One of the reactions pyruvate takes part in is the reaction catalyzed by the Pyruvate Dehydrogenase Complex. This enzyme complex oxidatively decarboxylates pyruvate to acetate and then activates the acetate for subsequent reactions by coupling it to Coenzyme A (CoA or CoA-SH) forming Acetyl-Coenzyme A (Ac-CoA or Ac-S-CoA). Acetate carried by CoA (Acetyl-CoA) is also a key intermediate of metabolism. Acetyl-CoA is the precursor for fatty acid, cholesterol, and isoprenoid biosynthesis. Acetyl-CoA is also the “fuel” for the first of the two final common pathways. The TRICARBOXYLIC ACID CYCLE oxidizes the two carbon acetate fragment into two molecules of CO2 and stores the energy released by the oxidations as high energy electrons accepted by NAD → NADH and FAD → FADH2). The conversion of pyruvate to acetate by the Pyruvate Dehydrogenase Complex is an irreversible process in mammalian cells. Mammalian cells do not have the enzymatic machinery necessary to convert the two carbon acetate fragment into a three carbon fragment. At this point the cell must make a “decision”: Is the pyruvate from glycolysis going to be used as pyruvate for biosynthetic reactions or is it going to be converted to Ac-CoA? The “decision” is made by the energy charge of the cell, the concentrations of allosteric effectors controlling key metabolic enzymes, and the signal molecules present in the tissues. 2 ©Kevin R. Siebenlist, 2016 Once converted to Ac-CoA will the acetate fragment be used for biosynthetic reactions or will it be oxidized for energy production? The pyruvate dehydrogenase complex stands at this crossroads and controls the flux of pyruvate/acetyl-CoA through the various metabolic pathways. For this reason the Pyruvate Dehydrogenase Complex is one of the most tightly controlled enzyme complexes. At this point in the discussion of Biochemistry, the pyruvate obtained from glycolysis will be passed through the Pyruvate Dehydrogenase Complex and the resulting acetyl-CoA will serve as the “fuel” for the TRICARBOXYLIC ACID CYCLE. The other fates of pyruvate and acetyl-CoA will be discussed as time goes on. The Pyruvate Dehydrogenase Complex The Pyruvate Dehydrogenase Complex catalyzes the following overall reaction: Pyruvate + CoA + NAD+ → Acetyl-CoA + CO2 + NADH The Pyruvate Dehydrogenase Complex is a large multisubunit enzyme with a molecular mass of over 1,000,000 g/mole. It contains five different enzyme activities on four different polypeptide subunits. In mammals there are 20-30 copies of Pyruvate Dehydrogenase (E1); 60 copies of Dihydrolipoyl Transacetylase (E2); 20-30 copies of Dihydrolipoyl Dehydrogenase (E3); variable number of copies of Pyruvate Dehydrogenase Kinase and Pyruvate Dehydrogenase Phosphatase. The number of kinase and phosphatase subunits increase with starvation. The activities reside on the same polypeptide, similar to the arrangement of kinase and phosphatase activities on Phosphofructokinase-2. The enzyme complex requires two cosubstrates and three coenzymes for activity. From the overall reaction the complex requires the cosubstrates NAD+ {niacin} and Coenzyme A {pantothenic acid}. E1 contains Thiamine Pyrophosphate {thiamine} as a coenzyme. Each E2 subunit contains two Lipoic Acid molecules as covalently linked prosthetic groups. The lipoic acids are linked to specific lysine side chains by amide bonds. E3 contains Flavin Adenine Dinucleotide (FAD) {riboflavin}as a covalently linked coenzyme. Mechanism of Pyruvate Dehydrogenase Action The oxidative decarboxylation of pyruvate and the covalent attachment of the resulting acetate to coenzyme A is divided into numerous steps. The first three steps of the reaction are catalyzed by the Pyruvate Dehydrogenase (E1) subunit. The ketone group of pyruvate is covalently linked to the Thiamine Pyrophosphate prosthetic group of Pyruvate Dehydrogenase (E1) and this enzyme catalyzes the decarboxylation of pyruvate. The decarboxylation reaction is an α-cleavage reaction and it is non-oxidative; the carbon atom attached to the thiamine pyrophosphate has not undergone a change in oxidation state. Lipoic Acid covalently linked to Dihydrolipoyl Transacetylase (E2) swings into the active site of E1 and E1 catalyzes the oxidative transfer of the two carbon fragment from Thiamine Pyrophosphate to the oxidized lipoyl (Lipoic Acid) group of E2. During this transfer, the carbonyl group of the two carbon fragment is oxidized to a carboxylic acid group, the disulfide of Lipoic Acid is reduced, and the resulting acetate group is covalently linked to one of the -SH groups of the reduced Lipoic Acid by a high energy thioester bond. 3 ©Kevin R. Siebenlist, 2016 Dihydrolipoyl Transacetylase (E2) now catalyzes a transesterification reaction. The acetate group is transferred from the -SH group of Lipoic Acid to the -SH group of Coenzyme A to form acetyl-Coenzyme A and reduced Lipoic Acid. The high energy thioester bond between the acetate and lipoic acid is broken and the energy is conserved in the thioester bond between acetate and CoA. In the last step of the reaction the reduced lipoyl group of E2 swings to the active site of Dihydrolipoyl Dehydrogenase (E3) where the reduced Lipoic Acid is oxidized to the cyclic disulfide. The FAD on E3 is reduced to FADH2. To complete the reaction and regenerate the FAD for the next reaction cycle, the electrons on FADH2 are passed to NAD+ forming FAD and the last product, NADH. O H 3C HS-CoA H3C C S CoA O C S SH Dihydrolipoyl Transacetylase SH SH O C Pyruvate C C NH O O H 3C C NH E2 O O Thiamine Pyrophosphate (TPP) FAD HN C Pyruvate Dehydrogenase E1 OH H 3C C TPP O H 3C C O O TPP C H OH S Dihydrolipoyl Dehydrogenase E3 NADH FADH2 S NAD CO2 The Tricarboxylic Acid Cycle The TRICARBOXYLIC ACID CYCLE is the first of the two final common pathways responsible for the complete oxidation of metabolites to CO2 and H2O with the concomitant production of ATP. The second of the two final common pathways is the ELECTRON TRANSPORT / OXIDATIVE PHOSPHORYLATION (ET/ 4 ©Kevin R. Siebenlist, 2016 OXPHOS) pathway. Both of these pathways occur within the mitochondria of the cell. The TRICARBOXYLIC ACID CYCLE was elucidated by HANS KREBS in 1933. This pathway is also called the KREBS CYCLE in honor of Hans Krebs, the CITRIC ACID CYCLE, and/or the TCA CYCLE. Metabolic intermediates derived from carbohydrates, lipids, and/or amino acids enter the TRICARBOXYLIC ACID CYCLE where they are completely oxidized to CO2. Energy is released during the oxidative process. Some of the energy is stored as high energy electrons carried by NADH and FADH2, some is stored as GTP or ATP, and the remainder is released as heat. During the TCA cycle, the equivalent of the two carbon acetate fragment is oxidized to two molecules of CO2. The energy released during this oxidation is stored in three molecules of NADH, one molecule of FADH2, and one molecule of GTP or ATP. The overall reaction of the TCA cycle is: Acetyl-CoA + 3 NAD+ + FAD + GDP (or ADP) + PO4-3 + 2 H2O CoA + 3 NADH + FADH2 + GTP (or ATP) + 2 CO2 + 2 H+ The Pathway In the first reaction of the TCA cycle, acetyl-CoA, the activated form of acetate covalently linked to CoA, reacts with oxaloacetate to form citrate and CoA-SH. This is a lyase reaction as well as an aldol condensation. A hydrogen from the methyl group and the methylene carbon add across the carbon-oxygen double bond of oxaloacetate. This irreversible reaction is catalyzed by Citrate Synthase. The TCA cycle is an oxidative pathway. However, citrate does not contain an easily oxidizable group. During the second step of the TCA cycle, the citrate molecule is rearranged, isomerized to D-isocitrate. The tertiary hydroxyl group on carbon three of citrate is moved to carbon two becoming a secondary alcohol. This freely reversible reaction is catalyzed by the enzyme Aconitase. It is called Aconitase, after the enzyme bound intermediate cis-aconitate. O O C CH2 O C O C CH C The first oxidation reaction occurs at the third step of the cycle. D-Isocitrate is oxidatively O O + decarboxylated to yield α-ketoglutarate, with the concomitant reduction of NAD to NADH. This two step reaction involves oxidation of the secondary alcohol to a carbonyl group followed by an α-β type elimination (decarboxylation) that expels the carboxyl group on carbon 3 as CO2 (the central carboxylic acid group) resulting in the product α-ketoglutarate. This irreversible reaction is catalyzed by the enzyme Isocitrate Dehydrogenase. The α-ketoglutarate is now oxidized. The product of this reaction is succinyl-CoA. This reaction is irreversible and is catalyzed by the α-Ketoglutarate Dehydrogenase Complex. Oxidation of α-ketoglutarate to succinyl-CoA occurs by a mechanism analogous to the oxidation of pyruvate to acetyl-CoA, an αcleavage reaction followed by oxidation and coupling to CoA. The same five cosubstrates/coenzymes 5 ©Kevin R. Siebenlist, 2016 O O NADH O C C NAD+ H3C O O O 8 CH2 O HO Oxaloacetate OH HC O C O C O CH2 CH2 C C O O C 1 C C CoA-SH S Acetyl-CoA CH2 O CoA C O O O Citrate Malate H2O 1 - Citrate Synthase 7 2 2 - Aconitase O O O 3 - Isocitrate Dehydrogenase C C H HC C 5 - Succinyl-CoA Synthetase (Succinate Thiokinase) C O CH2 4 - α-Ketoglutarate Dehydrogenase Complex H HO 6 - Succinate Dehydrogenase O 6 8 - Malate Dehydrogenase O FADH2 O FAD O CH2 CH2 CH2 CH2 C 5 O O C CO2 O 4 CoA-SH CH2 3 NADH CO2 O O α -Ketoglutarate CH2 CoA-SH GDP+PO4–3 C O ADP+PO4–3 S CoA or GDP NAD+ C O Succinate GTP O O C C O C Nucleoside Diphosphate Kinase O CH Isocitrate 7 - Fumarase ATP O C C O Fumarate ADP O C GTP or ATP NAD+ NADH Succinyl-S-CoA 6 ©Kevin R. Siebenlist, 2016 (NAD+, Coenzyme A, thiamine pyrophosphate, lipoic acid, & FAD) are involved and two of the products are identical, CO2 and NADH. At this point in the cycle two CO2 molecules have been released, so in essence the acetate group that entered the cycle has been completely oxidized. Some of the energy released during the oxidation reactions has been stored in two molecules of NADH and some energy remains in succinyl-CoA. In the remaining steps of the pathway oxaloacetate is regenerated from succinyl-CoA and the energy released during this process is trapped in a molecule of GTP/ATP, a molecule of NADH, and a molecule of FADH2. The bond between succinate and CoA in succinyl-CoA is a high energy thioester bond. In animal cells the energy stored in this bond is used to drive the formation of a GTP or ATP from GDP or ADP and PO4–3. Succinate and the nucleoside triphosphate are the products of this reaction. This reversible reaction is catalyzed by Succinyl-CoA Synthetase. This reaction is reversible and the enzyme was named by Krebs for the reverse reaction. A better, more correct, name for this enzyme is Succinate Thiokinase. The mitochondria contains two isoforms of this enzyme, one uses GDP/GTP pair and the other is specific for the ADP/ATP pair. Mechanistically, the enzyme employs phosphate to cleave the high energy thioester bond releasing CoA and forming a high energy mixed anhydride between the phosphate and the carboxyl group of succinate. The high energy phosphate of this mixed anhydride is then passed from the intermediate to GDP (ADP) forming GTP (ATP) and succinate. GTP (ATP) formation in this reaction is another example of substrate level phosphorylation. The GTP is used by the mitochondria for the synthesis of its DNA, RNA, and proteins. Alternatively it can be (is) converted to ATP by the action of Nucleosidediphosphate Kinase (NDK) and made available to the entire cell. NDK ⎯⎯⎯ ⎯⎯ → ADP + NTP ATP + NDP ← ⎯ In the sixth step of this cyclic pathway a “trans” double bond is introduced into succinate to form fumarate. This reaction is catalyzed by the Succinate Dehydrogenase Complex. The electrons removed from succinate are initially accepted by a covalently bound FAD to form FADH2. To regenerate the enzyme these electrons must be passed to a second electron acceptor. In the case of the Succinate Dehydrogenase Complex, the secondary electron acceptor is the diffusible cosubstrate, Coenzyme Q (Ubiquinone or CoQ). Electrons on the enzyme bound FADH2 are passed to CoQ forming FAD and reducing Coenzyme Q to Coenzyme QH2 (CoQH2). Several steps are required for the movement of the electrons from FADH2 to Coenzyme Q. These additional steps will be examined when the ELECTRON TRANSPORT / OXIDATIVE PHOSPHORYLATION (ET/OXPHOS) pathway is discussed. Fumarate is now reversibly hydrated to form malate. Water is added across the “trans” double bond in a reaction catalyzed by the enzyme Fumarase (Fumarate Hydratase). Fumarase catalyzes a lyase type reaction. In the last step of this cyclic pathway, the secondary alcohol group on malate is oxidized to a carbonyl group forming oxaloacetate. This reversible reaction is catalyzed by Malate Dehydrogenase. NAD+ is the electron acceptor for this reaction and is reduced to NADH. Malate formation is favored in this reversible reaction. The reaction is pulled toward oxaloacetate formation by the constant removal of oxaloacetate during the Citrate Synthase reaction. With the (re)formation of oxaloacetate the cyclic pathway is completed. 7 ©Kevin R. Siebenlist, 2016 Regulation of The Citric Acid Cycle Control by Intermediate Concentration The CITRIC ACID CYCLE is a CATALYTIC CYCLE. Since the intermediates of the pathway are not consumed during the cycle, low concentrations of TCA cycle intermediates (i.e., oxaloacetate) can catalyze the complete oxidation of innumerable two carbon acetate fragments. One way to control the flux of metabolites through the cycle is to control the level of TCA Cycle intermediates in the mitochondrial matrix. The concentrations of TCA cycle intermediates can be increased by reactions called ANAPLEUROTIC REACTIONS. One of the three important anapleurotic reactions is the formation of oxaloacetate from pyruvate. This reaction is catalyzed by the enzyme Pyruvate Carboxylase. Pyruvate Carboxylase is a mitochondrial enzyme and the reaction, the addition of CO2 (HCO3–) to pyruvate to form oxaloacetate, occurs in the mitochondrial matrix. Pyruvate Carboxylase, like most carboxylase enzymes, contains Biotin as a covalently linked prosthetic group. This enzyme is an allosteric enzyme with an absolute allosteric requirement for acetyl-CoA. Acetyl-CoA is a positive allosteric effector for the enzyme. When the concentration of acetyl-CoA in the mitochondrial matrix is low this enzyme is for the most part inactive. As the concentration of acetyl-CoA increases the activity of this enzyme increases dramatically. Thus, when acetyl-CoA levels exceed the oxaloacetate supply, allosteric activation of Pyruvate Carboxylase by acetylCoA raises oxaloacetate levels, so that a greater amount of acetyl-CoA can enter the TCA cycle and be metabolized. CO2 (HCO3–) + ADP + PO4–3 ATP + H2 O O H3C C O Biotin O O C C O Pyruvate Carboxylase O C H2 C O C O A second ANAPLEUROTIC REACTION is catalyzed by Phosphoenolpyruvate Carboxykinase. (PEP Carboxykinase). This is a mitochondrial enzyme. The reversible reaction takes phosphoenolpyruvate (PEP), CO2 and GDP and forms oxaloacetate and GTP. Some of the energy stored in PEP is used to phosphorylate GDP to GTP and the remaining energy is used to form the new carbon-carbon bond. O O P O O H 2C C CO2 + GDP GTP O O O C C Phosphoenolpyruvate O 8 O C H2 C O C O Oxaloacetate ©Kevin R. Siebenlist, 2016 The third ANAPLEUROTIC REACTION is catalyzed by Malic Enzyme. The mitochondrial isoenzyme forms malate by reductively carboxylating pyruvate. HCO3– is the source of the carboxyl group and in the mitochondria, electrons are donated by NADH. There is a cytoplasmic isoform of Malic Enzyme. This isoenzyme favors the formation of pyruvate from malate by oxidative decarboxylation using NADP as the oxidizing agent. – HCO2 + NADH O H 3C C O O OH O Malic Enzyme C Pyruvate NAD C O H2 C C H Malate O C O NADP NADPH + CO2 The level of TCA cycle intermediates can be / are decreased by a variety of means. Intermediates of the TCA cycle are drawn off and used as precursors in many anabolic pathways. For example: oxaloacetate → aspartate (and related amino acids)+ carbohydrates + nucleotides citrate → fatty acids + steroids + isoprenes α-ketoglutarate → glutamate + glutamine (and related amino acids) + nucleotides succinyl-CoA → porphyrins (porphyrins + Fe+2 = heme) Since the TCA cycle is central to many catabolic and anabolic pathways, the TCA cycle is more correctly termed an AMPHIBOLIC PATHWAY. AMPHI means BOTH. The TCA cycle is both a catabolic and anabolic pathway. Control at the Pyruvate Dehydrogenase Complex The TCA cycle is also controlled by the availability of “fuel” for the pathway. Fuel for the TCA cycle is the acetate fragment carried by CoA. The flow of pyruvate, from glycolysis, to acetyl-CoA is tightly controlled at the pyruvate dehydrogenase complex. This enzyme complex is under allosteric control and it is controlled by reversible covalent modification. The allosteric controls are at Dihydrolipoyl Transacetylase, (E2) and Dihydrolipoyl Dehydrogenase, (E3). Dihydrolipoyl Transacetylase, the E2 subunit, is allosterically activated by Coenzyme A and allosterically inhibited by Acetyl-CoA. The substrate and product of the reaction, respectively. Dihydrolipoyl Dehydrogenase, the E3 subunit, is allosterically activated by NAD+ and allosterically inhibited by NADH. The substrate and product of the reaction, respectively. 9 ©Kevin R. Siebenlist, 2016 Control by reversible covalent modification involves the fourth subunit, the Pyruvate Dehydrogenase Kinase / Pyruvate Dehydrogenase Phosphatase of the Pyruvate Dehydrogenase Complex. The Pyruvate Dehydrogenase Kinase transfers phosphate from ATP to the E1 subunit, (Pyruvate Dehydrogenase), and the Pyruvate Dehydrogenase Phosphatase removes the phosphate. When E1 is phosphorylated by the kinase, the enzyme is inhibited, when it is dephosphorylated by the phosphatase E1 is activated. ATP, NADH, Acetyl-CoA — Pyruvate Dehydrogenase Phosphatase Catalyzes the Removal of PO4–3 Acetyl-CoA — + Insulin, PEP, AMP, Ca+2 ,Mg+2 E1 (—) O3PO Pyruvate, ADP Ca2+, Mg2+ — Pyruvate Dehydrogenase Kinase + E2 NADH — E3 + + NAD Coenzyme A Catalyzes the Addition of PO4–3 ATP, NADH Acetyl-CoA The kinase / phosphatase of the Pyruvate Dehydrogenase Complex are under allosteric control. Pyruvate Dehydrogenase Kinase subunits are allosterically activated by elevated levels of NADH and Acetyl-CoA. The products of the Pyruvate Dehydrogenase Complex activate the Pyruvate Dehydrogenase Kinase, the active Protein Kinase phosphorylates the Pyruvate Dehydrogenase (E1) subunit, and phosphorylation of E1 inhibits that activity of this subunit and thereby slows (inhibits) the activity of the enzyme complex. The Pyruvate Dehydrogenase Kinase is inhibited by ADP, Pyruvate, Ca+2, and Mg+2. The Pyruvate Dehydrogenase Phosphatase is likewise highly regulated. It is stimulated by insulin, 10 ©Kevin R. Siebenlist, 2016 Phosphoenolpyruvate, AMP. Muscle-specific isoforms of the enzyme are also stimulated by elevated concentrations of Ca+2 and Mg+2. It is inhibited by ATP, NADH and Acetyl-CoA. Control by Allosteric Enzymes Within the TCA Cycle Finally, the Krebs cycle is controlled by allosteric enzymes within the pathway. Three enzymes within the TCA cycle catalyze irreversible reactions and are sites for control. These three enzymes are Citrate Synthase, Isocitrate Dehydrogenase, and the α-Ketoglutarate Dehydrogenase Complex. + ADP Citrate Synthase – ATP & NADH (Allosteric Inhibitors) Citrate & Succinyl-CoA (Competitive Feedback Inhibitors) + Ca+2, NAD, & ADP Isocitrate Dehydrogenase – NADH & ATP + Ca+2 -Ketoglutarate Dehydrogenase – NADH & Succinyl-CoA The mechanism for the control of Citrate Synthase is complex and still open to question. In vitro ATP and NADH allosterically inhibit the enzyme. Citrate and Succinyl-CoA act as Competitive Feedback Inhibitors, competing with the normal substrates for binding at the substrate / active site. ADP acts as an allosteric activator. However, it is unclear whether these “effectors” ever achieve a concentration sufficiently high in the mitochondrial matrix to inhibit or activate the enzyme. Isocitrate Dehydrogenase is allosterically activated by Ca+2, NAD and ADP, and allosterically inhibited by NADH and ATP. α-Ketoglutarate Dehydrogenase Complex is allosterically activated by elevated levels of Ca+2 and is allosterically inhibited by NADH and succinyl-CoA. 11 ©Kevin R. Siebenlist, 2016