Lehninger Principles of Biochemistry PDF

16 The Citric Acid Cycle © 2021 Macmillan Learning  Cellular Respiration cellular respiration = process by which the pyruvate produced by glycolysis is further oxidized to H2O and CO2  Stage 1 of Cellular Respiration Stage 1: oxidation of fuels to acetyl-CoA –...

16 The Citric Acid Cycle © 2021 Macmillan Learning  Cellular Respiration cellular respiration = process by which the pyruvate produced by glycolysis is further oxidized to H2O and CO2  Stage 1 of Cellular Respiration Stage 1: oxidation of fuels to acetyl-CoA – generates ATP, NADH, FADH2  Stage 2 of Cellular Respiration Stage 2: oxidation of acetyl groups to CO2 in the citric acid cycle (tricarboxylic acid (TCA) cycle, Krebs cycle) – nearly universal pathway – generates NADH, FADH2, and one GTP  Stage 3 of Cellular Respiration Stage 3: electron transfer chain and oxidative phosphorylation – generates the vast majority of ATP from catabolism  Principle 1 (1 of 4) Pyruvate is the metabolite that links two central catabolic pathways, glycolysis and the citric acid cycle. It is therefore a logical point for regulation that determines the rate of catabolic activity and the partitioning of pyruvate among its possible uses.  Principle 2 (1 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways.  Principle 3 (1 of 4) The citric acid cycle is a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. Acetate groups (acetyl-CoA) from the catabolism of various fuels are used in the synthesis of such metabolites as amino acids, fatty acids, and sterols. The breakdown products of many amino acids and nucleotides are intermediates of the cycle, and they can be fed in or siphoned off as needed by the cell.  Principle 4 (1 of 5) The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. Some mutations that affect the reactions of the citric acid cycle lead to tumor formation.  Principle 5 (1 of 6) Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules. 16.1 Production of Acetyl- CoA (Activated Acetate)  Coenzyme A (CoA-SH) coenzyme A has a reactive thiol (–SH) group that is critical to its role as an acyl carrier – the –SH group forms a thioester with acetate in acetyl- CoA  Clicker Question 1 Coenzyme A: A. forms thioester acyl groups. B. has lipoic acid as one of its components. C. forms esters with relatively small standard free energies of hydrolysis. D. can have pyruvate linked to it through an ester linkage.  Clicker Question 1, Response Coenzyme A: A. forms thioester acyl groups. The —SH group of the mercaptoethylamine moiety forms a thioester with acetate in acetyl-coenzyme A (acetyl-CoA).  Principle 1 (2 of 4) Pyruvate is the metabolite that links two central catabolic pathways, glycolysis and the citric acid cycle. It is therefore a logical point for regulation that determines the rate of catabolic activity and the partitioning of pyruvate among its possible uses.  Principle 5 (2 of 6) Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules.  Principle 4 (2 of 5) The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. Some mutations that affect the reactions of the citric acid cycle lead to tumor formation. Pyruvate Is Oxidized to Acetyl-CoA and CO2 mitochondrial pyruvate carrier (MPC) = an H+-coupled pyruvate-specific symporter in the inner mitochondrial membrane pyruvate dehydrogenase (PDH) complex = highly ordered cluster of enzymes and cofactors that oxidizes pyruvate in the mitochondrial matrix to acetyl-CoA and CO2 – the series of chemical intermediates remain bound to the enzyme subunits – regulation results in precisely regulated flux  Clicker Question 2 Pyruvate is produced in glycolysis and used by the citric acid cycle in the mitochondrial matrix. How does pyruvate get into the matrix? A. It moves through the membrane by simple diffusion. B. Diffusion is facilitated through a specific uniport. C. It transforms into acetate, which moves through a facilitated transporter. D. A transporter is not needed because pyruvate from glycolysis is already in the matrix. E. It moves through the malate shuttle system.  Clicker Question 2, Response Pyruvate is produced in glycolysis and used by the citric acid cycle in the mitochondrial matrix. How does pyruvate get into the matrix? B. Diffusion is facilitated through a specific uniport. In eukaryotes, pyruvate may diffuse into mitochondria, first through large openings in the outer mitochondrial membrane and then into the matrix via an H+-coupled pyruvate-specific symporter in the inner mitochondrial membrane, the mitochondrial pyruvate carrier (MPC).  The PDH Complex Catalyzes an Oxidative Decarboxylation oxidative decarboxylation = an irreversible oxidation process in which the carboxyl group is removed, forming CO2 The PDH Complex Employs Three Enzymes and Five Coenzymes to Oxidize Pyruvate three enzymes: five coenzymes: – pyruvate – thiamine dehydrogenase, E1 pyrophosphate (TPP) – dihydrolipoyl – lipoate transacetylase, E2 – coenzyme A (CoA, – dihydrolipoyl CoA-SH) dehydrogenase, E3 – flavin adenine dinucleotide (FAD) – nicotinamide adenine dinucleotide (NAD)  Lipoate lipoate = coenzyme with two thiol groups that can undergo reversible oxidation to a disulfide bond (–S–S–) – serves as an electron (hydrogen) carrier and an acyl carrier – covalently linked to E2 via a lysine residue  Clicker Question 3 Which vitamin is a coenzyme to the pyruvate dehydrogenase (PDH) complex? A. pantothenate B. thiamine pyrophosphate C. niacin D. riboflavin E. All of the answers are correct.  Clicker Question 3, Response Which vitamin is a coenzyme to the pyruvate dehydrogenase (PDH) complex? E. All of the answers are correct. Four different vitamins required in human nutrition are vital components of the PDH complex: thiamine (TPP), pantothenate (CoA), riboflavin (FAD), and niacin (NAD).  The PDH Complex Enzymes the PDH complex contains multiple copies of: – pyruvate dehydrogenase (E1) – dihydrolipoyl transacetylase (E2) – dihydrolipoyl dehydrogenase (E3) an E2 core (of 24-60 copies) is surrounded by multiple and variable numbers of E1 and E3 copies  Principle 5 (3 of 6) Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules.  The E1-E2-E3 Structure of the PDH Complex the structure of the PDH complex is similar to other enzymes that catalyze oxidations: – α-ketoglutarate dehydrogenase – branched-chain α-keto acid dehydrogenase in a given species, E3 is identical in all three complexes similarities reflect a common evolutionary origin – they are paralogs  Principle 5 (4 of 6) Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules.  The PDH Complex Channels Its Intermediates through Five Reactions  Oxidative Decarboxylation of Pyruvate - Steps 1 and 2 pyruvate dehydrogenase, E1, with bound TPP catalyzes: – step 1: decarboxylation of pyruvate to the hydroethyl derivate rate-limiting step – step 2: oxidation of the hydroethyl derivate to an acetyl group electrons and the acetyl group are transferred from TPP to the lipoyllysyl group of E2  Oxidative Decarboxylation of Pyruvate - Steps 3-5 dihydrolipoyl transacetylase, E2, catalyzes: – step 3: esterification of the acetyl moiety to one of the lipoyl –SH groups, followed by transesterification to CoA to form acetyl-CoA dihydrolipoyl dehydrogenase, E3, catalyzes: – step 4: electron transfer to regenerate the oxidized form of the lipoyllysyl group – step 5: electron transfer to regenerate the oxidized FAD cofactor, forming NADH  Clicker Question 4 Which two chemical mechanisms change pyruvate to acetyl-CoA in the pyruvate dehydrogenase complex? A. dehydrogenation and oxidation B. decarboxylation and condensation C. condensation and dehydrogenation D. dehydrogenation and decarboxylation E. condensation and oxidation  Clicker Question 4, Response Which two chemical mechanisms change pyruvate to acetyl-CoA in the pyruvate dehydrogenase complex? D. dehydrogenation and decarboxylation The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA.  Principle 5 (5 of 6) Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules. The Five-Reaction Sequence of the PDH Complex Is An Example of Substrate Channeling substrate channeling = the passage of intermediates from one enzyme directly to another enzyme without release the long lipoyllysyl arm of E2 channels the substrate from the active site of E1 to E2 to E3 – tethers intermediates to the enzyme complex – increases the efficiency of the overall reaction – minimizes side reactions  Clicker Question 5 What is the advantage to having an enzyme complex, as in pyruvate dehydrogenase (PDH) complex? A. Multiple steps can be regulated at one point. B. Products do not need to diffuse to become substrates for the next enzymatic reaction. C. Products cannot be scavenged by other enzymes or pathways. D. Conservation of energy drives the reactions. E. All of the answers are correct.  Clicker Question 5, Response What is the advantage to having an enzyme complex, as in pyruvate dehydrogenase (PDH) complex? E. All of the answers are correct. Substrate channeling in this five-reaction sequence keeps the local concentration of the substrate of E2 very high and prevents theft of the activated acetyl group by other enzymes. It also allows the energy of oxidation to drive the formation of a high-energy thioester of acetate and provides a single point for the regulation of multiple steps. 16.2 Reactions of the Citric Acid Cycle  Principle 2 (2 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways.  Reactions of the Citric Acid Cycle one oxaloacetate molecule can theoretically oxidize an infinite number of acetyl groups energy from the four oxidations is conserved as NADH and FADH2 In Eukaryotes, the Mitochondrion Is the Site of Energy-Yielding Oxidative Reactions and ATP Synthesis isolated mitochondria contain all enzyme, coenzymes, and proteins needed for: – the citric acid cycle – electron transfer and ATP synthesis by oxidative phosphorylation – oxidation of fatty acids and amino acids to acetyl-CoA – oxidative degradation of amino acids to citric acid cycle intermediates  Principle 2 (3 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways. The Sequence of Reactions in the Citric Acid Cycle Makes Chemical Sense complete oxidation of acetyl-CoA to CO2 extracts the maximum potential energy direct oxidation to yield CO2 and CH4 is not biochemically feasible because organisms cannot oxidize CH4  Principle 2 (4 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways.  The Chemical Logic of the Citric Acid Cycle carbonyl groups are more chemically reactive than a methylene group or methane each step of the cycle involves either: – an energy-conserving oxidation – placing functional groups in position to facilitate oxidation or oxidative decarboxylation  Clicker Question 6 Which statement regarding the citric acid cycle is false? A. Part of the chemical logic behind it involves the conversion of the relatively unreactive methyl group of acetyl-CoA to a more reactive methylene group. B. The carbon atoms that feed into the cycle as acetyl-CoA do not leave as CO2 during their first turn in the cycle. C. It is found in the mitochondria of eukaryotes. D. Its role is strictly limited to energy conservation during the catabolism of the acetyl group.  Clicker Question 6, Response Which statement regarding the citric acid cycle is false? A. Its role is strictly limited to energy conservation during the catabolism of the acetyl group. Although the citric acid cycle is central to energy-yielding metabolism, its role is not limited to energy conservation.  The Citric Acid Cycle Has Eight Steps citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield: – CO2 – NADH – FADH2 – GTP or ATP  Formation of Citrate citrate synthase = catalyzes the condensation of acetyl- CoA with oxaloacetate to form citrate – involves the formation of a transient intermediate, citroyl-CoA – large, negative ∆G′° is needed because [oxaloacetate] is normally very low  Structure of Citrate Synthase binding of oxaloacetate creates a binding site for acetyl-CoA induced fit decreases the likelihood of premature cleavage of the thioester bond of acetyl-CoA Mechanism of Citrate Synthase: Acid/Base Catalysis Mechanism of Citrate Synthase: Acid/Base Catalysis, Continued  Clicker Question 7 The citrate synthase step of the citric acid cycle: A. is freely reversible under physiological conditions. B. is an example of a Ping-Pong enzyme mechanism. C. does not involve ATP hydrolysis. D. is considered to be both the first and last step of the cycle.  Clicker Question 7, Response The citrate synthase step of the citric acid cycle: C. does not involve ATP hydrolysis. Unlike synthetase enzymes, synthases do not require nucleotide triphosphates such as ATP. The hydrolysis of a high-energy thioester in a citroyl-CoA intermediate makes the forward reaction catalyzed by citrate synthase highly exergonic. Mechanism of Citrate Synthase: Thioester Hydrolysis  Formation of Isocitrate via Cis-Aconitate aconitase (aconitate hydratase) = catalyzes the reversible transformation of citrate to isocitrate through the intermediate cis- aconitate – addition of H2O to cis-aconitate is stereospecific – low [isocitrate] pulls the reaction forward  Iron-Sulfur Center in Aconitase iron-sulfur center = acts both in the binding of the substrate to the active site and in the catalytic addition or removal of H2O  Oxidation of Isocitrate to α-Ketoglutarate and CO2 isocitrate dehydrogenase = catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate – Mn2+ interacts with the carbonyl group of the oxalosuccinate and stabilizes the transiently-formed enol – specific isozymes for NADP+ (cytosolic and mitochondrial) or NAD+ (mitochondrial)  Clicker Question 8 Which statement regarding isocitrate dehydrogenase is false? A. It has a Mn2+ cofactor. B. In eukaryotes, there is an NAD+-dependent form in mitochondria and an NAD+-independent form found in both mitochondria and the cytosol. C. It catalyzes a reversible reaction under physiological conditions. D. It catalyzes an oxidative decarboxylation.  Clicker Question 8, Response Which statement regarding isocitrate dehydrogenase is false? C. It catalyzes a reversible reaction under physiological conditions. Isocitrate dehydrogenase catalyzes an irreversible oxidation process in which a carboxyl group is removed from isocitrate as a molecule of CO2.  Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2 α-ketoglutarate dehydrogenase complex = catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl- CoA and CO2 – energy of oxidation is conserved in the thioester bond of succinyl-CoA  Principle 2 (5 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways.  A Conserved Mechanism for Oxidative Decarboxylation pathways use the same five cofactors, similar multienzyme complexes, and the same enzymatic mechanism – have homologous E1 and E2 and identical E3 – example of gene duplication and divergent evolution  Clicker Question 9 Which two other enzyme complexes have an E1—E2—E3 structure similar to that of the pyruvate dehydrogenase (PDH) complex? A. α-ketoglutarate dehydrogenase and branched-chain α-keto acid dehydrogenase B. α-ketoglutarate dehydrogenase and lactate dehydrogenase C. branched-chain α-keto acid dehydrogenase and lactate dehydrogenase D. pyruvate carboxylase and pyruvate decarboxylase  Clicker Question 9, Response Which two other enzyme complexes have an E1—E2—E3 structure similar to that of the pyruvate dehydrogenase (PDH) complex? A. α-ketoglutarate dehydrogenase and branched-chain α-keto acid dehydrogenase The remarkable similarity in protein structure, cofactor requirements, and reaction mechanisms of these three complexes doubtless reflects a common evolutionary origin; they are paralogs.  Conversion of Succinyl-CoA to Succinate succinyl-CoA synthetase (succinic thiokinase) = catalyzes the breakage of the thioester bond of succinyl- CoA to form succinate – energy released drives the synthesis of a phosphoanhydride bond in either GTP or ATP (substrate level phosphorylation)  The Succinyl-CoA Synthetase Reaction (1 of 2) enzyme molecule becomes phosphorylated at a His residue in the active site phosphoryl group is then transferred to ADP or GDP to form ATP or GTP – animal cells have specific isozymes for ADP and GDP  The Succinyl-CoA Synthetase Reaction (2 of 2) power helices place the partial positive charges of the helix dipole near the phosphate group of the α chain phosphorylated His246 to stabilize the phosphoenzyme intermediate  Nucleoside Diphosphate Kinase nucleoside diphosphate kinase = catalyzes the reversible conversion of GTP and ATP GTP + ADP ⇌ GDP + ATP ∆G′° = 0 kJ/mol net result of the activity of either isozyme of succinyl-CoA synthetase is the conservation of energy as ATP  Clicker Question 10 Which enzyme of the citric acid cycle is capable of a substrate- level phosphorylation? A. citrate synthase B. aconitase C. succinate dehydrogenase D. isocitrate dehydrogenase E. succinyl-CoA synthetase  Clicker Question 10, Response Which enzyme of the citric acid cycle is capable of a substrate- level phosphorylation? E. succinyl-CoA synthetase The formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of α‐ketoglutarate is a substrate-level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by phosphoglycerate kinase and pyruvate kinase. Oxidation of Succinate to Fumarate succinate dehydrogenase = flavoprotein that catalyzes the reversible oxidation of succinate to fumarate – integral protein of the mitochondrial inner membrane in eukaryotes – contains three iron-sulfur clusters and covalently bound FAD Malonate Is a Strong Competitive Inhibitor of Succinate Dehydrogenase malonate = an analog of succinate – not normally present in cells – addition to mitochondria in vitro blocks citric acid cycle activity  Clicker Question 11 Which citric acid cycle reaction produces FADH2? A. succinyl-CoA synthetase B. aconitase C. α-ketoglutarate dehydrogenase complex D. isocitrate dehydrogenase E. succinate dehydrogenase  Clicker Question 11, Response Which citric acid cycle reaction produces FADH2? E. succinate dehydrogenase The flavoprotein succinate dehydrogenase oxidizes succinate while reducing FAD to FADH2.  Hydration of Fumarate to Malate fumarase = catalyzes the reversible hydration of fumarate to L-malate – transition state is a carbanion Fumarase Is Highly Stereospecific in the forward direction, fumarase catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate in the reverse direction, fumarase is equally stereospecific Oxidation of Malate to Oxaloacetate L-malate dehydrogenase = catalyzes the oxidation of L-malate to oxaloacetate, coupled to the reduction of NAD+ – low [oxaloacetate] pulls the reaction forward – regenerates oxaloacetate for citrate synthesis  Clicker Question 12 Which statement regarding the citric acid cycle is false? A. The PDH complex is considered to be an enzyme of the citric acid cycle. B. Succinate dehydrogenase is an integral membrane protein. C. For the complete conversion of glucose to CO2, approximately 32 ATP can be synthesized. D. Succinyl-CoA synthetase and succinic thiokinase are two names for the same enzyme.  Clicker Question 12, Response Which statement regarding the citric acid cycle is false? A. The PDH complex is considered to be an enzyme of the citric acid cycle. Although the PDH complex links glycolysis and the citric acid cycle, it is not considered to be part of either pathway.  Principle 2 (6 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways.  The Energy of Oxidations in the Cycle Is Efficiently Conserved energy released by oxidation is conserved in the production of: – 3 NADH – 1 FADH2 – 1 GTP (or ATP)  Clicker Question 13 How many reducing equivalents are transferred to electron carriers after one turn of the citric acid cycle? A. 4 B. 6 C. 8 D. 10 E. 16  Clicker Question 13, Response How many reducing equivalents are transferred to electron carriers after one turn of the citric acid cycle? C. 8 One turn of the citric acid cycle generates three NADH and one FADH2 with two electrons each.  Principle 2 (7 of 7) The reactions of the citric acid cycle follow a chemical logic. In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways.  Electrons from NADH and FADH2 Enter the Respiratory Chain the citric acid cycle directly generates only one ATP per turn the large flow of electrons into the respiratory chain via NADH and FADH2 leads to formation of almost 10 times more ATP during oxidative phosphorylation – each NADH drives formation of ~2.5 ATP – each FADH2 drives formation of ~1.5 ATP  Clicker Question 14 Which statement regarding the energetics of the citric acid cycle is true? A. The cycle in animal cells directly produces one ATP instead of one GTP. B. All of the energy-producing capacity of the cycle occurs in the first four reactions; the remaining reactions serve to regenerate oxaloacetate. C. Production of FADH2 by the citric acid cycle represents the energic capacity to synthesize about 1.5 ATP. D. Production of 3 NADH by the citric acid cycle represents the energic capacity to synthesize about 2.5 ATP.  Clicker Question 14, Response Which statement regarding the energetics of the citric acid cycle is true? C. Production of FADH2 by the citric acid cycle represents the energic capacity to synthesize about 1.5 ATP. In oxidative phosphorylation, passage of two electrons from FADH2 to O2 yields about 1.5 ATP.  Clicker Question 15 Using currently accepted P/O ratios, what is the total ATP potential yield from one acetyl-CoA in the citric acid cycle? A. 8 B. 10 C. 24 D. 32 E. 106  Clicker Question 15, Response Using currently accepted P/O ratios, what is the total ATP potential yield from one acetyl-CoA in the citric acid cycle? B. 10 The oxidation of one acetyl-CoA in the citric acid cycle yields three NADH (3×2.5 = 7.5) , one FADH2 (1×1.5), and one GTP/ATP. Stoichiometry of Coenzyme Reduction and ATP Formation in Aerobic Oxidation of Glucose Table 16-1 Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis, the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation Reaction Number of ATP or reduced Number of ATP ultimately coenzyme directly formed formed Glucose → glucose 6-phosphate −1 ATP −1 Fructose 6-phosphate → fructose 1,6-bisphosphate −1 ATP −1 2 Glyceraldehyde 3-phosphate → 2 1,3-biphosphoglycerate 2 NADH 3 or 5 2 1,3-Bisphosphoglycerate → 2 3-phosphoglycerate 2 ATP 2 2 Phosphoenolpyruvate → 2 pyruvate 2 ATP 2 2 Pyruvate → 2 acetyl-CoA 2 NADH 5 2 Isocitrate → 2 α-ketoglutarate 2 NADH 5 2 α-Ketoglutarate → 2 succinyl-CoA 2 NADH 5 2 Succinyl-CoA → 2 succinate 2 ATP (or 2 GTP) 2 2 Succinate → 2 fumarate 2 FADH2 3 2 Malate → 2 oxaloacetate 2 NADH 5 Total 30-32  Clicker Question 16 How many NADH molecules are generated from the complete oxidation of one glucose? A. 5 B. 7 C. 10 D. 16 E. 32  Clicker Question 16, Response How many NADH molecules are generated from the complete oxidation of one glucose? C. 10 Glycolysis produces two NADH and two pyruvates. The two pyruvates yield two NADH via pyruvate dehydrogenase and two acetyl-CoA. Each acetyl-CoA generates three NADH in the citric acid cycle. 16.3 The Hub of Intermediary Metabolism  Principle 3 (2 of 4) The citric acid cycle is a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. Acetate groups (acetyl-CoA) from the catabolism of various fuels are used in the synthesis of such metabolites as amino acids, fatty acids, and sterols. The breakdown products of many amino acids and nucleotides are intermediates of the cycle, and they can be fed in or siphoned off as needed by the cell.  The Citric Acid Cycle Accepts Carbon Skeletons the cycle accepts 3-, 4-, and 5-carbon skeletons the breakdown of amino acids yields carbon skeletons: – deaminated aspartate yields oxaloacetate – deaminated glutamate yields α-ketoglutarate  Principle 3 (3 of 4) The citric acid cycle is a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. Acetate groups (acetyl-CoA) from the catabolism of various fuels are used in the synthesis of such metabolites as amino acids, fatty acids, and sterols. The breakdown products of many amino acids and nucleotides are intermediates of the cycle, and they can be fed in or siphoned off as needed by the cell. The Citric Acid Cycle Serves in Both Catabolic and Anabolic Processes amphibolic pathway = one that serves in both catabolic and anabolic processes animals cannot convert acetate or acetyl-CoA to glucose – in the citric acid cycle, there is no net conversion of acetate to oxaloacetate glyoxylate cycle = reaction sequence that converts acetate to carbohydrate – present in bacteria, plants, fungi, and protists  Clicker Question 17 The term amphibolic means: A. something that is both catabolic and anabolic. B. nothing; it is a made-up word. C. thermodynamic coupling of catabolic pathways to drive anabolic pathways. D. containing both polar and nonpolar functional groups.  Clicker Question 17, Response The term amphibolic means: A. something that is both catabolic and anabolic. The prefix amph- comes from the Greek word meaning “both kinds.” In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes.  Clicker Question 18 The glyoxylate cycle is remarkably similar to the citric acid cycle but differs in several important ways. Which important molecule is conserved by the glyoxylate cycle but NOT the citric acid cycle? A. acetyl-CoA B. malate C. citrate D. carbon dioxide E. NADH  Clicker Question 18, Response The glyoxylate cycle is remarkably similar to the citric acid cycle but differs in several important ways. Which important molecule is conserved by the glyoxylate cycle but NOT the citric acid cycle? D. carbon dioxide The citric acid cycle has a net carbon loss (2 CO2), whereas the glyoxylate cycle does not contain the two oxidative decarboxylations from the citric acid cycle. Conserving the carbon allows the cell to make cellulose and sucrose in high concentrations rapidly.  Principle 3 (4 of 4) The citric acid cycle is a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. Acetate groups (acetyl-CoA) from the catabolism of various fuels are used in the synthesis of such metabolites as amino acids, fatty acids, and sterols. The breakdown products of many amino acids and nucleotides are intermediates of the cycle, and they can be fed in or siphoned off as needed by the cell.  Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates when intermediates are shunted from the citric acid cycle to other pathways, they are replenished anaplerotic reactions = chemical reactions that replenish intermediates  Clicker Question 19 Anaplerotic reactions: A. are also known as cataplerotic reactions. B. are defined as those that have oxaloacetate as a product. C. never involve ATP hydrolysis. D. replenish all citric acid cycle intermediates.  Clicker Question 19, Response Anaplerotic reactions: C. replenish all citric acid cycle intermediates. The term anapleortic derives from the Greek wording meaning “to refill.” When the withdrawal of cycle intermediates for use in biosynthesis lowers the concentrations of citric acid cycle intermediates enough to slow the cycle, the intermediates are replenished by anaplerotic reactions.  Clicker Question 20 Which enzyme does NOT catalyze an anaplerotic reaction? A. pyruvate carboxylase B. PEP carboxykinase C. succinate carboxykinase D. PEP carboxylase E. malic enzyme  Clicker Question 20, Response Which enzyme does NOT catalyze an anaplerotic reaction? C. succinate carboxykinase Pyruvate carboxylase, PEP carboxykinase, and PEP carboxylase all replenish the oxaloacetate that is lost from the citric acid cycle to biosynthetic reactions. Malic enzyme replenishes malate. Succinate carboykinase is not an actual enzyme. Role of the Citric Acid Cycle in Anabolism  Clicker Question 21 The power of the citric acid cycle is partly in the ability to shuttle intermediates out for the synthesis of important groups of molecules. What group of molecules is produced from citrate? A. lipids and sterols B. nucleic acids (purines) C. a number of amino acids D. glucose and a few amino acids E. porphyrin rings for various molecules such as heme  Clicker Question 21, Response The power of the citric acid cycle is partly in the ability to shuttle intermediates out for the synthesis of important groups of molecules. What group of molecules is produced from citrate? A. lipids and sterols Citrate may be exported from the mitochondria and used as the starting material for synthesis of fatty acids and sterols in the cytosol.  Pyruvate Carboxylase pyruvate carboxylase = catalyzes the reversible carboxylation of pyruvate by HCO3− to form oxaloacetate – most important anaplerotic reaction in mammalian liver, kidney, and brown adipose tissue – requires energy from ATP – allosterically activated by acetyl-CoA  Biotin in Pyruvate Carboxylase Carries One-Carbon (CO2) Groups biotin = vitamin that acts as a specialized carrier of one- carbon groups as CO2 in many carboxylation reactions – serves as the prosthetic group of pyruvate carboxylase  Principle 5 (6 of 6) Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules. The Role of Biotin in the Pyruvate Carboxylase Reaction the two steps in carboxylation of pyruvate occur at separate active sites – the arm of biotin transfers activated carboxyl groups from the first active site to the second  Clicker Question 22 The release of carbon dioxide from the complete oxidation of pyruvate can pose problems for cells. Which molecule can easily be formed from carbon dioxide that can serve as a one- carbon donor and double as a biological buffer? A. biotin B. acetate C. glyceraldehyde 3-phosphate D. glycine E. bicarbonate  Clicker Question 22, Response The release of carbon dioxide from the complete oxidation of pyruvate can pose problems for cells. Which molecule can easily be formed from carbon dioxide that can serve as a one- carbon donor and double as a biological buffer? E. bicarbonate Only bicarbonate (HCO3−) serves both of these roles. Biotin can accept a CO2, but it is not a biological buffer nor the molecule into which CO2 is transformed. Acetate is a biological buffer, but it has two carbons and does not serve as a one-carbon donor. Biological Tethers Allow Flexibility all participate in substrate channeling through the flexible tethers that move intermediates from one active site to the next 16.4 Regulation of the Citric Acid Cycle  Principle 4 (3 of 5) The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. Some mutations that affect the reactions of the citric acid cycle lead to tumor formation.  Citric Acid Cycle Regulation regulation balances the supply of key intermediates with the demands of energy production and biosynthetic processes regulation occurs at several points: – PDH complex – citrate synthase – isocitrate dehydrogenase complex – α-ketoglutarate dehydrogenase complex  Clicker Question 23 The citric acid cycle is regulated at the reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. What do these three reactions have in common? A. They are all close to equilibrium. B. They are all strongly exergonic. C. They all reduce NAD+ to NADH. D. They all carry out substrate-level phosphorylation.  Clicker Question 23, Response The citric acid cycle is regulated at the reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. What do these three reactions have in common? B. They are all strongly exergonic. Recall from Chapter 13 that reactions far from equilibrium are common points of regulation. Each of these three strongly exergonic steps in the citric acid cycle can become the rate-limiting step under some circumstances.  Principle 1 (3 of 4) Pyruvate is the metabolite that links two central catabolic pathways, glycolysis and the citric acid cycle. It is therefore a logical point for regulation that determines the rate of catabolic activity and the partitioning of pyruvate among its possible uses.  Production of Acetyl-CoA by the PDH Complex Is Regulated by Allosteric and Covalent Mechanisms PDH complex activity is turned off when: – ample fatty acids and acetyl-CoA are available as fuel – [ATP]/[ADP] and [NADH]/[NAD+] ratios are high PDH complex activity is turned on when: – energy demands are high – the cell requires greater flux of acetyl-CoA into the citric acid cycle  Clicker Question 24 The pyruvate dehydrogenase (PDH) complex is NOT regulated by: A. ADP. B. fatty acids. C. covalent modification. D. acetyl-CoA.  Clicker Question 24, Response The pyruvate dehydrogenase (PDH) complex is NOT regulated by: A. ADP. The PDH complex is regulated by allosteric mechanisms and covalent modification (phosphorylation). ATP, acetyl- CoA, NADH, and fatty acids inhibit the activity of the PDH complex, whereas AMP, CoA, NAD+, and Ca2+ stimulate PDH complex activity. Regulation of Metabolite Flow Through the Citric Acid Cycle  Principle 1 (4 of 4) Pyruvate is the metabolite that links two central catabolic pathways, glycolysis and the citric acid cycle. It is therefore a logical point for regulation that determines the rate of catabolic activity and the partitioning of pyruvate among its possible uses.  Covalent Modification of the PDH Complex PDH kinase = inhibits the PDH complex by phosphorylation – allosterically activated by products of the complex – inhibited by substrates of the complex PDH phosphatase = reverses the inhibition by PDH kinase  Principle 4 (4 of 5) The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. Some mutations that affect the reactions of the citric acid cycle lead to tumor formation.  The Citric Acid Cycle Is Also Regulated at Three Exergonic Steps regulation occurs at strongly exergonic steps catalyzed by: – citrate synthase – isocitrate dehydrogenase complex – α-ketoglutarate dehydrogenase complex fluxes are affected by the concentrations of substrates and products: – end products ATP and NADH are inhibitory – NAD+ and ADP are stimulatory – long-chain fatty acids are inhibitory  Clicker Question 25 The citric acid cycle is regulated in a manner similar to glycolysis. Which molecule is an allosteric activator of BOTH of those pathways? A. ATP B. NAD+ C. ADP D. citrate E. NADH  Clicker Question 25, Response The citric acid cycle is regulated in a manner similar to glycolysis. Which molecule is an allosteric activator of BOTH of those pathways? C. ADP ADP allosterically activates the glycolytic enzyme phosphofructokinase-1 (PFK-1) as well as the citric acid cycle enzymes citrate synthase and isocitrate dehydrogenase.  Clicker Question 26 How is the regulation of glucose metabolism through glycolysis and the citric acid cycle coordinated? A. High ADP levels stimulate glycolysis and the citric acid cycle. B. High NADH levels inhibit glycolysis and the citric acid cycle. C. High citrate levels inhibit glycolysis. D. In muscle, Ca2+ release (a signal of muscle contraction) stimulates both glycogen phosphorylase and enzymes of the citric acid cycle. E. All of the answers are correct.  Clicker Question 26, Response How is the regulation of glucose metabolism through glycolysis and the citric acid cycle coordinated? E. All of the answers are correct. High levels of ADP and Ca2+ stimulate both glycolysis and citric acid cycle. Conversely, high levels of NADH inhibit both glycolysis and the citric acid cycle. High levels of citrate also inhibit glycolysis. Citric Acid Cycle Activity Changes in Tumors some mutations that affect the PDH complex or citric acid cycle enzymes are oncogenic: – downregulation of mitochondrial pyruvate carrier (MPC) – inactivation of PDH complex – inactivation of succinate dehydrogenase oncometabolites = stimulate tumor growth by acting though specific GPCRs in the plasma membrane  Clicker Question 27 Which statement regarding regulation of the citric acid cycle is false? A. Ca2+ is involved in citric acid cycle regulation in vertebrate muscle. B. Dysfunction of the citric acid cycle (e.g., due to mutations in enzymes of the cycle) is often associated with different types of cancer C. In addition to regulation of the PDH complex, regulation of the citric acid cycle occurs by regulation of the enzymes that catalyze the strongly exergonic steps. D. As [NADH]/[NAD+] increases, flux through the cycle increases.  Clicker Question 27, Response Which statement regarding regulation of the citric acid cycle is false? D. As [NADH]/[NAD+] increases, flux through the cycle increases. PDH complex activity is turned off when the cell’s [NADH]/[NAD+] ratio is high. Similarly, a high [NADH]/[NAD+] ratio inhibits the citric acid cycle reactions catalyzed by isocitrate and α-ketoglutarate dehydrogenase by mass action.  Lactate and Succinate Are Oncometabolites tumor cells accumulate lactate and succinate oncometabolites = stimulate tumor growth by acting though specific GPCRs in the plasma membrane  Principle 4 (5 of 5) The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. Some mutations that affect the reactions of the citric acid cycle lead to tumor formation.  Mutations in Citric Acid Cycle Enzymes mutations in succinate dehydrogenase and fumarase cause tumors, defining them as tumor suppressors many glial cell tumors have mutant NADPH-dependent isocitrate dehydrogenase – lose ability to convert isocitrate to α-ketoglutarate – gain ability to convert α- ketoglutarate to 2- hydroxyglutarate Certain Intermediates Are Channeled through Metabolons metabolons = integrated multienzyme complexes that are held together by noncovalent interactions malate dehydrogenase, citrate synthase, and aconitase likely constitute a metabolon  Clicker Question 28 Metabolons are: A. multienzyme complexes that ensure efficient passage of the product of one enzyme to the next enzyme in the pathway. B. products of one metabolic pathway that act as allosteric regulators of another pathway. C. metabolic intermediates in a pathway that act as allosteric regulators of that pathway. D. individual functional units of regulation for a metabolic pathway or cycle.  Clicker Question 28, Response Metabolons are: A. multienzyme complexes that ensure efficient passage of the product of one enzyme to the next enzyme in the pathway. Many reactions occur in metabolons, integrated multienzyme complexes held together by noncovalent interactions. Metabolons ensure efficient passage of the product of one enzyme reaction to the next enzyme in the pathway.