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Biochemistry_Lippincott_383	Biochemistry_Lippinco	neonatal-onset form, early death. The gene for the α subunit is X linked, and because both males and females may be affected, the deficiency is classified as X-linked dominant. Although there is no proven treatment for PDHC deficiency, dietary restriction of carbohydrate and supplementation with thiamine may reduce symptoms in select patients.	Biochemistry_Lippinco. neonatal-onset form, early death. The gene for the α subunit is X linked, and because both males and females may be affected, the deficiency is classified as X-linked dominant. Although there is no proven treatment for PDHC deficiency, dietary restriction of carbohydrate and supplementation with thiamine may reduce symptoms in select patients.
Biochemistry_Lippincott_384	Biochemistry_Lippinco	Leigh syndrome (subacute necrotizing encephalomyelopathy) is a rare, progressive, neurodegenerative disorder caused by defects in mitochondrial ATP production, primarily as a result of mutations in genes that encode proteins of the PDHC, the ETC, or ATP synthase. Both nuclear and mitochondrial DNA can be affected.	Biochemistry_Lippinco. Leigh syndrome (subacute necrotizing encephalomyelopathy) is a rare, progressive, neurodegenerative disorder caused by defects in mitochondrial ATP production, primarily as a result of mutations in genes that encode proteins of the PDHC, the ETC, or ATP synthase. Both nuclear and mitochondrial DNA can be affected.
Biochemistry_Lippincott_385	Biochemistry_Lippinco	5. Arsenic poisoning: As previously described (see p. 101), pentavalent arsenic (arsenate) can interfere with glycolysis at the glyceraldehyde 3phosphate step, thereby decreasing ATP production. However, arsenic poisoning is due primarily to inhibition of enzyme complexes that require lipoic acid as a coenzyme, including PDH, α-ketoglutarate dehydrogenase (see E. below), and branched-chain α-keto acid dehydrogenase (see p. 266). Arsenite (the trivalent form of arsenic) forms a stable complex with the thiol (−SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme. When it binds to lipoic acid in the PDHC, pyruvate (and, consequently, lactate) accumulates. As with PDHC deficiency, this particularly affects the brain, causing neurologic disturbances and death. B. Citrate synthesis	Biochemistry_Lippinco. 5. Arsenic poisoning: As previously described (see p. 101), pentavalent arsenic (arsenate) can interfere with glycolysis at the glyceraldehyde 3phosphate step, thereby decreasing ATP production. However, arsenic poisoning is due primarily to inhibition of enzyme complexes that require lipoic acid as a coenzyme, including PDH, α-ketoglutarate dehydrogenase (see E. below), and branched-chain α-keto acid dehydrogenase (see p. 266). Arsenite (the trivalent form of arsenic) forms a stable complex with the thiol (−SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme. When it binds to lipoic acid in the PDHC, pyruvate (and, consequently, lactate) accumulates. As with PDHC deficiency, this particularly affects the brain, causing neurologic disturbances and death. B. Citrate synthesis
Biochemistry_Lippincott_386	Biochemistry_Lippinco	B. Citrate synthesis The irreversible condensation of acetyl CoA and OAA to form citrate (a tricarboxylic acid) is catalyzed by citrate synthase, the initiating enzyme of the TCA cycle (Fig. 9.4). This aldol condensation has a highly negative change in standard free energy ([∆G0] see p. 70), which strongly favors citrate formation. The enzyme is inhibited by citrate (product inhibition). Substrate availability is another means of regulation for citrate synthase. The binding of OAA greatly increases the enzyme’s affinity for acetyl CoA. [Note: Citrate, in addition to being an intermediate in the TCA cycle, is a source of acetyl CoA for the cytosolic synthesis of fatty acids (see p. 183) and cholesterol (see p. 220). Citrate also inhibits phosphofructokinase-1 (PFK-1), the rate-limiting enzyme of glycolysis (see p. 99), and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis, see p. 183).] C. Citrate isomerization	Biochemistry_Lippinco. B. Citrate synthesis The irreversible condensation of acetyl CoA and OAA to form citrate (a tricarboxylic acid) is catalyzed by citrate synthase, the initiating enzyme of the TCA cycle (Fig. 9.4). This aldol condensation has a highly negative change in standard free energy ([∆G0] see p. 70), which strongly favors citrate formation. The enzyme is inhibited by citrate (product inhibition). Substrate availability is another means of regulation for citrate synthase. The binding of OAA greatly increases the enzyme’s affinity for acetyl CoA. [Note: Citrate, in addition to being an intermediate in the TCA cycle, is a source of acetyl CoA for the cytosolic synthesis of fatty acids (see p. 183) and cholesterol (see p. 220). Citrate also inhibits phosphofructokinase-1 (PFK-1), the rate-limiting enzyme of glycolysis (see p. 99), and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis, see p. 183).] C. Citrate isomerization
Biochemistry_Lippincott_387	Biochemistry_Lippinco	C. Citrate isomerization Citrate is isomerized to isocitrate through hydroxyl group migration catalyzed by aconitase (aconitate hydratase), an iron-sulfur protein (see Fig. 9.4). [Note: Aconitase is inhibited by fluoroacetate, a plant toxin that is used as a pesticide. Fluoroacetate is converted to fluoroacetyl CoA that condenses with OAA to form fluorocitrate, a potent inhibitor of aconitase.] D. Oxidative decarboxylation of isocitrate Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate to α-ketoglutarate, yielding the first of three NADH molecules produced by the cycle and the first release of CO2 (see Fig. 9.4). This is one of the rate-limiting steps of the TCA cycle. The enzyme is allosterically activated by ADP (a low-energy signal) and Ca2+ and is inhibited by ATP and NADH, levels of which are elevated when the cell has abundant energy stores. E. Oxidative decarboxylation of α-ketoglutarate	Biochemistry_Lippinco. C. Citrate isomerization Citrate is isomerized to isocitrate through hydroxyl group migration catalyzed by aconitase (aconitate hydratase), an iron-sulfur protein (see Fig. 9.4). [Note: Aconitase is inhibited by fluoroacetate, a plant toxin that is used as a pesticide. Fluoroacetate is converted to fluoroacetyl CoA that condenses with OAA to form fluorocitrate, a potent inhibitor of aconitase.] D. Oxidative decarboxylation of isocitrate Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate to α-ketoglutarate, yielding the first of three NADH molecules produced by the cycle and the first release of CO2 (see Fig. 9.4). This is one of the rate-limiting steps of the TCA cycle. The enzyme is allosterically activated by ADP (a low-energy signal) and Ca2+ and is inhibited by ATP and NADH, levels of which are elevated when the cell has abundant energy stores. E. Oxidative decarboxylation of α-ketoglutarate
Biochemistry_Lippincott_388	Biochemistry_Lippinco	The irreversible conversion of α-ketoglutarate to succinyl CoA is catalyzed by the α-ketoglutarate dehydrogenase complex, a protein aggregate of multiple copies of three enzymes (Fig. 9.5). The mechanism of this oxidative decarboxylation is very similar to that used for the conversion of pyruvate to acetyl CoA by the PDHC. The reaction releases the second CO2 and produces the second NADH of the cycle. The coenzymes required are TPP, lipoic acid, FAD, NAD+, and CoA. Each functions as part of the catalytic mechanism in a way analogous to that described for the PDHC (see p. 110). The large negative ∆G0 of the reaction favors formation of succinyl CoA, a high-energy thioester similar to acetyl CoA. The αketoglutarate dehydrogenase complex is inhibited by its products, NADH and succinyl CoA, and activated by Ca2+ . However, it is not regulated by phosphorylation/dephosphorylation reactions as described for the PDHC. [Note: α-Ketoglutarate is also produced by the oxidative deamination (see	Biochemistry_Lippinco. The irreversible conversion of α-ketoglutarate to succinyl CoA is catalyzed by the α-ketoglutarate dehydrogenase complex, a protein aggregate of multiple copies of three enzymes (Fig. 9.5). The mechanism of this oxidative decarboxylation is very similar to that used for the conversion of pyruvate to acetyl CoA by the PDHC. The reaction releases the second CO2 and produces the second NADH of the cycle. The coenzymes required are TPP, lipoic acid, FAD, NAD+, and CoA. Each functions as part of the catalytic mechanism in a way analogous to that described for the PDHC (see p. 110). The large negative ∆G0 of the reaction favors formation of succinyl CoA, a high-energy thioester similar to acetyl CoA. The αketoglutarate dehydrogenase complex is inhibited by its products, NADH and succinyl CoA, and activated by Ca2+ . However, it is not regulated by phosphorylation/dephosphorylation reactions as described for the PDHC. [Note: α-Ketoglutarate is also produced by the oxidative deamination (see
Biochemistry_Lippincott_389	Biochemistry_Lippinco	activated by Ca2+ . However, it is not regulated by phosphorylation/dephosphorylation reactions as described for the PDHC. [Note: α-Ketoglutarate is also produced by the oxidative deamination (see p. 252) and transamination of the amino acid glutamate (see p. 250).]	Biochemistry_Lippinco. activated by Ca2+ . However, it is not regulated by phosphorylation/dephosphorylation reactions as described for the PDHC. [Note: α-Ketoglutarate is also produced by the oxidative deamination (see p. 252) and transamination of the amino acid glutamate (see p. 250).]
Biochemistry_Lippincott_390	Biochemistry_Lippinco	F. Succinyl coenzyme A cleavage Succinate thiokinase (also called succinyl CoA synthetase, named for the reverse reaction) cleaves the high-energy thioester bond of succinyl CoA (see Fig. 9.5). This reaction is coupled to phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). GTP and ATP are energetically interconvertible by the nucleoside diphosphate kinase reaction: The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation (see p. 102). [Note: Succinyl CoA is also produced from propionyl CoA derived from the metabolism of fatty acids with an odd number of carbon atoms (see p. 193) and from the metabolism of several amino acids (see pp. 265–266). It can be converted to pyruvate for gluconeogenesis (see p. 118) or used in heme synthesis (see p. 278).] G. Succinate oxidation	Biochemistry_Lippinco. F. Succinyl coenzyme A cleavage Succinate thiokinase (also called succinyl CoA synthetase, named for the reverse reaction) cleaves the high-energy thioester bond of succinyl CoA (see Fig. 9.5). This reaction is coupled to phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). GTP and ATP are energetically interconvertible by the nucleoside diphosphate kinase reaction: The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation (see p. 102). [Note: Succinyl CoA is also produced from propionyl CoA derived from the metabolism of fatty acids with an odd number of carbon atoms (see p. 193) and from the metabolism of several amino acids (see pp. 265–266). It can be converted to pyruvate for gluconeogenesis (see p. 118) or used in heme synthesis (see p. 278).] G. Succinate oxidation
Biochemistry_Lippincott_391	Biochemistry_Lippinco	G. Succinate oxidation Succinate is oxidized to fumarate by succinate dehydrogenase, as its coenzyme FAD is reduced to FADH2 (see Fig. 9.5). Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane. As such, it functions as Complex II of the ETC (see p. 75). [Note: FAD, rather than NAD+, is the electron acceptor because the reducing power of succinate is not sufficient to reduce NAD+.] H. Fumarate hydration Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (fumarate hydratase, see Fig. 9.5). [Note: Fumarate is also produced by the urea cycle (see p. 255), in purine synthesis (see Fig. 22.7 on p. 294), and during catabolism of the amino acids phenylalanine and tyrosine (see p. 263).] I. Malate oxidation	Biochemistry_Lippinco. G. Succinate oxidation Succinate is oxidized to fumarate by succinate dehydrogenase, as its coenzyme FAD is reduced to FADH2 (see Fig. 9.5). Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane. As such, it functions as Complex II of the ETC (see p. 75). [Note: FAD, rather than NAD+, is the electron acceptor because the reducing power of succinate is not sufficient to reduce NAD+.] H. Fumarate hydration Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (fumarate hydratase, see Fig. 9.5). [Note: Fumarate is also produced by the urea cycle (see p. 255), in purine synthesis (see Fig. 22.7 on p. 294), and during catabolism of the amino acids phenylalanine and tyrosine (see p. 263).] I. Malate oxidation
Biochemistry_Lippincott_392	Biochemistry_Lippinco	I. Malate oxidation Malate is oxidized to OAA by malate dehydrogenase (Fig. 9.6). This reaction produces the third and final NADH of the cycle. The ∆G0 of the reaction is positive, but the reaction is driven in the direction of OAA by the highly exergonic citrate synthase reaction. [Note: OAA is also produced by the transamination of the amino acid aspartic acid (see p. 250).] III. ENERGY PRODUCED BY THE CYCLE	Biochemistry_Lippinco. I. Malate oxidation Malate is oxidized to OAA by malate dehydrogenase (Fig. 9.6). This reaction produces the third and final NADH of the cycle. The ∆G0 of the reaction is positive, but the reaction is driven in the direction of OAA by the highly exergonic citrate synthase reaction. [Note: OAA is also produced by the transamination of the amino acid aspartic acid (see p. 250).] III. ENERGY PRODUCED BY THE CYCLE
Biochemistry_Lippincott_393	Biochemistry_Lippinco	III. ENERGY PRODUCED BY THE CYCLE Four pairs of electrons are transferred during one turn of the TCA cycle: three pairs reducing three NAD+ to NADH and one pair reducing FAD to FADH2. Oxidation of one NADH by the ETC leads to formation of three ATP, whereas oxidation of FADH2 produces two ATP (see p. 77). The total yield of ATP from the oxidation of one acetyl CoA is shown in Figure 9.7. Figure 9.8 summarizes the reactions of the TCA cycle. [Note: The cycle does not involve the net consumption or production of intermediates. Two carbons entering as acetyl CoA are balanced by two CO2 exiting.] dinucleotides; GDP and GTP = guanosine di-and triphosphates; Pi = inorganic phosphate. IV. CYCLE REGULATION	Biochemistry_Lippinco. III. ENERGY PRODUCED BY THE CYCLE Four pairs of electrons are transferred during one turn of the TCA cycle: three pairs reducing three NAD+ to NADH and one pair reducing FAD to FADH2. Oxidation of one NADH by the ETC leads to formation of three ATP, whereas oxidation of FADH2 produces two ATP (see p. 77). The total yield of ATP from the oxidation of one acetyl CoA is shown in Figure 9.7. Figure 9.8 summarizes the reactions of the TCA cycle. [Note: The cycle does not involve the net consumption or production of intermediates. Two carbons entering as acetyl CoA are balanced by two CO2 exiting.] dinucleotides; GDP and GTP = guanosine di-and triphosphates; Pi = inorganic phosphate. IV. CYCLE REGULATION
Biochemistry_Lippincott_394	Biochemistry_Lippinco	IV. CYCLE REGULATION In contrast to glycolysis, which is regulated primarily by PFK-1, the TCA cycle is controlled by the regulation of several enzymes (see Fig. 9.8). The most important of these regulated enzymes are those that catalyze reactions with highly negative ∆G0: citrate synthase, isocitrate dehydrogenase, and the αketoglutarate dehydrogenase complex. Reducing equivalents needed for oxidative phosphorylation are generated by the PDHC and the TCA cycle, and both processes are upregulated in response to a decrease in the ATP/ADP ratio. V. CHAPTER SUMMARY	Biochemistry_Lippinco. IV. CYCLE REGULATION In contrast to glycolysis, which is regulated primarily by PFK-1, the TCA cycle is controlled by the regulation of several enzymes (see Fig. 9.8). The most important of these regulated enzymes are those that catalyze reactions with highly negative ∆G0: citrate synthase, isocitrate dehydrogenase, and the αketoglutarate dehydrogenase complex. Reducing equivalents needed for oxidative phosphorylation are generated by the PDHC and the TCA cycle, and both processes are upregulated in response to a decrease in the ATP/ADP ratio. V. CHAPTER SUMMARY
Biochemistry_Lippincott_395	Biochemistry_Lippinco	Pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex (PDHC), producing acetyl coenzyme A (CoA), which is the major fuel for the tricarboxylic acid (TCA) cycle (Fig. 9.9). The multienzyme PDHC requires five coenzymes: thiamine pyrophosphate, lipoic acid, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD+), and CoA. The PDHC is regulated by covalent modification of E1 (pyruvate decarboxylase) by PDH kinase and PDH phosphatase: Phosphorylation inhibits E1. PDH kinase is allosterically activated by ATP, acetyl CoA, and NADH and inhibited by pyruvate. The phosphatase is activated by calcium (Ca2+). E1 deficiency is the most common biochemical cause of congenital lactic acidosis. The brain is particularly affected in this X-linked dominant disorder. Arsenic poisoning causes inactivation of the PDHC by binding to lipoic acid. In the TCA cycle, citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase, which is inhibited	Biochemistry_Lippinco. Pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex (PDHC), producing acetyl coenzyme A (CoA), which is the major fuel for the tricarboxylic acid (TCA) cycle (Fig. 9.9). The multienzyme PDHC requires five coenzymes: thiamine pyrophosphate, lipoic acid, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD+), and CoA. The PDHC is regulated by covalent modification of E1 (pyruvate decarboxylase) by PDH kinase and PDH phosphatase: Phosphorylation inhibits E1. PDH kinase is allosterically activated by ATP, acetyl CoA, and NADH and inhibited by pyruvate. The phosphatase is activated by calcium (Ca2+). E1 deficiency is the most common biochemical cause of congenital lactic acidosis. The brain is particularly affected in this X-linked dominant disorder. Arsenic poisoning causes inactivation of the PDHC by binding to lipoic acid. In the TCA cycle, citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase, which is inhibited
Biochemistry_Lippincott_396	Biochemistry_Lippinco	Arsenic poisoning causes inactivation of the PDHC by binding to lipoic acid. In the TCA cycle, citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase, which is inhibited by product. Citrate is isomerized to isocitrate by aconitase (aconitate hydratase). Isocitrate is oxidatively decarboxylated by isocitrate dehydrogenase to α-ketoglutarate, producing carbon dioxide (CO2) and NADH. The enzyme is inhibited by ATP and NADH and activated by adenosine diphosphate (ADP) and Ca2+ . α-Ketoglutarate is oxidatively decarboxylated to succinyl CoA by the α-ketoglutarate dehydrogenase complex, producing CO2 and NADH. The enzyme is very similar to the PDHC and uses the same coenzymes. The α-ketoglutarate dehydrogenase complex is activated by Ca2+ and inhibited by NADH and succinyl CoA but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and guanosine triphosphate (GTP). This is an	Biochemistry_Lippinco. Arsenic poisoning causes inactivation of the PDHC by binding to lipoic acid. In the TCA cycle, citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase, which is inhibited by product. Citrate is isomerized to isocitrate by aconitase (aconitate hydratase). Isocitrate is oxidatively decarboxylated by isocitrate dehydrogenase to α-ketoglutarate, producing carbon dioxide (CO2) and NADH. The enzyme is inhibited by ATP and NADH and activated by adenosine diphosphate (ADP) and Ca2+ . α-Ketoglutarate is oxidatively decarboxylated to succinyl CoA by the α-ketoglutarate dehydrogenase complex, producing CO2 and NADH. The enzyme is very similar to the PDHC and uses the same coenzymes. The α-ketoglutarate dehydrogenase complex is activated by Ca2+ and inhibited by NADH and succinyl CoA but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and guanosine triphosphate (GTP). This is an
Biochemistry_Lippincott_397	Biochemistry_Lippinco	succinyl CoA but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and guanosine triphosphate (GTP). This is an example of substrate-level phosphorylation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. Fumarate is hydrated to malate by fumarase (fumarate hydratase), and malate is oxidized to OAA by malate dehydrogenase, producing NADH. Three NADH and one FADH2 are produced by one round of the TCA cycle. The generation of acetyl CoA by the oxidation of pyruvate via the PDHC also produces an NADH. Oxidation of the NADH and FADH2 by the ETC yields 14 ATP. The terminal phosphate of the GTP produced by substrate-level phosphorylation in the TCA cycle can be transferred to ADP by nucleoside diphosphate kinase, yielding another ATP. Therefore, a total of 15 ATP are produced from the complete mitochondrial oxidation of pyruvate to CO2.	Biochemistry_Lippinco. succinyl CoA but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and guanosine triphosphate (GTP). This is an example of substrate-level phosphorylation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. Fumarate is hydrated to malate by fumarase (fumarate hydratase), and malate is oxidized to OAA by malate dehydrogenase, producing NADH. Three NADH and one FADH2 are produced by one round of the TCA cycle. The generation of acetyl CoA by the oxidation of pyruvate via the PDHC also produces an NADH. Oxidation of the NADH and FADH2 by the ETC yields 14 ATP. The terminal phosphate of the GTP produced by substrate-level phosphorylation in the TCA cycle can be transferred to ADP by nucleoside diphosphate kinase, yielding another ATP. Therefore, a total of 15 ATP are produced from the complete mitochondrial oxidation of pyruvate to CO2.
Biochemistry_Lippincott_398	Biochemistry_Lippinco	NAD(H) = nicotinamide adenine dinucleotide; FAD(H2) = flavin adenine dinucleotide; GDP and GTP = guanosine di-and triphosphates; ADP = adenosine diphosphate; Pi = inorganic phosphate. Choose the ONE best answer. .1. The conversion of pyruvate to acetyl coenzyme A and carbon dioxide: A. involves the participation of lipoic acid. B. is activated when pyruvate decarboxylase of the pyruvate dehydrogenase complex (PDHC) is phosphorylated by PDH kinase in the presence of ATP. C. is reversible. D. occurs in the cytosol. E. requires the coenzyme biotin.	Biochemistry_Lippinco. NAD(H) = nicotinamide adenine dinucleotide; FAD(H2) = flavin adenine dinucleotide; GDP and GTP = guanosine di-and triphosphates; ADP = adenosine diphosphate; Pi = inorganic phosphate. Choose the ONE best answer. .1. The conversion of pyruvate to acetyl coenzyme A and carbon dioxide: A. involves the participation of lipoic acid. B. is activated when pyruvate decarboxylase of the pyruvate dehydrogenase complex (PDHC) is phosphorylated by PDH kinase in the presence of ATP. C. is reversible. D. occurs in the cytosol. E. requires the coenzyme biotin.
Biochemistry_Lippincott_399	Biochemistry_Lippinco	C. is reversible. D. occurs in the cytosol. E. requires the coenzyme biotin. Correct answer = A. Lipoic acid is an intermediate acceptor of the acetyl group formed in the reaction. [Note: Lipoic acid linked to a lysine residue in E2 functions as a “swinging arm” that allows interaction with E1 and E3.] The PDHC catalyzes an irreversible reaction that is inhibited when the decarboxylase component (E1) is phosphorylated. The PDHC is located in the mitochondrial matrix. Biotin is utilized by carboxylases, not decarboxylases. .2. Which one of the following conditions decreases the oxidation of acetyl coenzyme A by the citric acid cycle? A. A high availability of calcium B. A high acetyl CoA/CoA ratio C. A low ATP/ADP ratio D. A low NAD+/NADH ratio Correct answer = D. A low NAD+/NADH (oxidized to reduced nicotinamide adenine dinucleotide) ratio limits the rates of the NAD+-requiring dehydrogenases. High availability of calcium and substrate (acetyl coenzyme	Biochemistry_Lippinco. C. is reversible. D. occurs in the cytosol. E. requires the coenzyme biotin. Correct answer = A. Lipoic acid is an intermediate acceptor of the acetyl group formed in the reaction. [Note: Lipoic acid linked to a lysine residue in E2 functions as a “swinging arm” that allows interaction with E1 and E3.] The PDHC catalyzes an irreversible reaction that is inhibited when the decarboxylase component (E1) is phosphorylated. The PDHC is located in the mitochondrial matrix. Biotin is utilized by carboxylases, not decarboxylases. .2. Which one of the following conditions decreases the oxidation of acetyl coenzyme A by the citric acid cycle? A. A high availability of calcium B. A high acetyl CoA/CoA ratio C. A low ATP/ADP ratio D. A low NAD+/NADH ratio Correct answer = D. A low NAD+/NADH (oxidized to reduced nicotinamide adenine dinucleotide) ratio limits the rates of the NAD+-requiring dehydrogenases. High availability of calcium and substrate (acetyl coenzyme
Biochemistry_Lippincott_400	Biochemistry_Lippinco	A) and a low ATP/ADP (adenosine tri-to diphosphate) ratio stimulate the cycle. .3. The following is the sum of three steps in the citric acid cycle. A + B + FAD + H2O → C + FADH2 + NADH Choose the lettered answer that corresponds to the missing “A,” “B,” and “C” in the equation. Correct answer = B. Succinate + NAD+ + FAD + H2O → oxaloacetate + NADH + FADH2. .4. A 1-month-old male shows neurologic problems and lactic acidosis. Enzyme assay for pyruvate dehydrogenase complex (PDHC) activity on extracts of cultured skin fibroblasts showed 5% of normal activity with a low concentration of thiamine pyrophosphate (TPP) but 80% of normal activity when the assay contained a thousand-fold higher concentration of TPP. Which one of the following statements concerning this patient is correct? A. Administration of thiamine is expected to reduce his serum lactate level and improve his clinical symptoms. B. A high-carbohydrate diet would be expected to be beneficial for this patient.	Biochemistry_Lippinco. A) and a low ATP/ADP (adenosine tri-to diphosphate) ratio stimulate the cycle. .3. The following is the sum of three steps in the citric acid cycle. A + B + FAD + H2O → C + FADH2 + NADH Choose the lettered answer that corresponds to the missing “A,” “B,” and “C” in the equation. Correct answer = B. Succinate + NAD+ + FAD + H2O → oxaloacetate + NADH + FADH2. .4. A 1-month-old male shows neurologic problems and lactic acidosis. Enzyme assay for pyruvate dehydrogenase complex (PDHC) activity on extracts of cultured skin fibroblasts showed 5% of normal activity with a low concentration of thiamine pyrophosphate (TPP) but 80% of normal activity when the assay contained a thousand-fold higher concentration of TPP. Which one of the following statements concerning this patient is correct? A. Administration of thiamine is expected to reduce his serum lactate level and improve his clinical symptoms. B. A high-carbohydrate diet would be expected to be beneficial for this patient.
Biochemistry_Lippincott_401	Biochemistry_Lippinco	A. Administration of thiamine is expected to reduce his serum lactate level and improve his clinical symptoms. B. A high-carbohydrate diet would be expected to be beneficial for this patient. C. Citrate production from aerobic glycolysis is expected to be increased. D. PDH kinase, a regulatory enzyme of the PDHC, is expected to be active.	Biochemistry_Lippinco. A. Administration of thiamine is expected to reduce his serum lactate level and improve his clinical symptoms. B. A high-carbohydrate diet would be expected to be beneficial for this patient. C. Citrate production from aerobic glycolysis is expected to be increased. D. PDH kinase, a regulatory enzyme of the PDHC, is expected to be active.
Biochemistry_Lippincott_402	Biochemistry_Lippinco	C. Citrate production from aerobic glycolysis is expected to be increased. D. PDH kinase, a regulatory enzyme of the PDHC, is expected to be active. Correct answer = A. The patient appears to have a thiamine-responsive PDHC deficiency. The pyruvate decarboxylase (E1) component of the PDHC fails to bind thiamine pyrophosphate at low concentration but shows significant activity at a high concentration of the coenzyme. This mutation, which affects the Km (Michaelis constant) of the enzyme for the coenzyme, is present in some, but not all, cases of PDHC deficiency. Because the PDHC is an integral part of carbohydrate metabolism, a diet low in carbohydrates would be expected to blunt the effects of the enzyme deficiency. Aerobic glycolysis generates pyruvate, the substrate of the PDHC. Decreased activity of the complex decreases production of acetyl coenzyme A, a substrate for citrate synthase. Because PDH kinase is allosterically inhibited by pyruvate, it is inactive.	Biochemistry_Lippinco. C. Citrate production from aerobic glycolysis is expected to be increased. D. PDH kinase, a regulatory enzyme of the PDHC, is expected to be active. Correct answer = A. The patient appears to have a thiamine-responsive PDHC deficiency. The pyruvate decarboxylase (E1) component of the PDHC fails to bind thiamine pyrophosphate at low concentration but shows significant activity at a high concentration of the coenzyme. This mutation, which affects the Km (Michaelis constant) of the enzyme for the coenzyme, is present in some, but not all, cases of PDHC deficiency. Because the PDHC is an integral part of carbohydrate metabolism, a diet low in carbohydrates would be expected to blunt the effects of the enzyme deficiency. Aerobic glycolysis generates pyruvate, the substrate of the PDHC. Decreased activity of the complex decreases production of acetyl coenzyme A, a substrate for citrate synthase. Because PDH kinase is allosterically inhibited by pyruvate, it is inactive.
Biochemistry_Lippincott_403	Biochemistry_Lippinco	.5. Which coenzyme–cosubstrate is used by dehydrogenases in both glycolysis and the tricarboxylic acid cycle? Oxidized nicotinamide adenine dinucleotide (NAD+) is used by glyceraldehyde 3-phosphate dehydrogenase of glycolysis and by isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase of the tricarboxylic acid cycle. [Note: E3 of the pyruvate dehydrogenase complex requires oxidized flavin adenine dinucleotide (FAD) and NAD+.] For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW	Biochemistry_Lippinco. .5. Which coenzyme–cosubstrate is used by dehydrogenases in both glycolysis and the tricarboxylic acid cycle? Oxidized nicotinamide adenine dinucleotide (NAD+) is used by glyceraldehyde 3-phosphate dehydrogenase of glycolysis and by isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase of the tricarboxylic acid cycle. [Note: E3 of the pyruvate dehydrogenase complex requires oxidized flavin adenine dinucleotide (FAD) and NAD+.] For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW
Biochemistry_Lippincott_404	Biochemistry_Lippinco	Some tissues, such as the brain, red blood cells (RBC), kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for <24 hours in the absence of dietary intake of carbohydrate (see p. 125). During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is made from noncarbohydrate precursors. The formation of glucose does not occur by a simple reversal of glycolysis, because the overall equilibrium of glycolysis strongly favors pyruvate formation (that is, the change in standard free energy [∆G0] is negative). Instead, glucose is synthesized de novo by a special pathway, gluconeogenesis, which requires both mitochondrial and cytosolic enzymes. [Note: Deficiencies of gluconeogenic enzymes cause hypoglycemia.] During an overnight fast, ~90% of gluconeogenesis occurs in the liver, with the remaining ~10%	Biochemistry_Lippinco. Some tissues, such as the brain, red blood cells (RBC), kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for <24 hours in the absence of dietary intake of carbohydrate (see p. 125). During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is made from noncarbohydrate precursors. The formation of glucose does not occur by a simple reversal of glycolysis, because the overall equilibrium of glycolysis strongly favors pyruvate formation (that is, the change in standard free energy [∆G0] is negative). Instead, glucose is synthesized de novo by a special pathway, gluconeogenesis, which requires both mitochondrial and cytosolic enzymes. [Note: Deficiencies of gluconeogenic enzymes cause hypoglycemia.] During an overnight fast, ~90% of gluconeogenesis occurs in the liver, with the remaining ~10%
Biochemistry_Lippincott_405	Biochemistry_Lippinco	mitochondrial and cytosolic enzymes. [Note: Deficiencies of gluconeogenic enzymes cause hypoglycemia.] During an overnight fast, ~90% of gluconeogenesis occurs in the liver, with the remaining ~10% occurring in the kidneys. However, during prolonged fasting, the kidneys become major glucose-producing organs, contributing ~40% of the total glucose production. [Note: The small intestine can also make glucose.] Figure 10.1 shows the relationship of gluconeogenesis to other essential pathways of energy metabolism.	Biochemistry_Lippinco. mitochondrial and cytosolic enzymes. [Note: Deficiencies of gluconeogenic enzymes cause hypoglycemia.] During an overnight fast, ~90% of gluconeogenesis occurs in the liver, with the remaining ~10% occurring in the kidneys. However, during prolonged fasting, the kidneys become major glucose-producing organs, contributing ~40% of the total glucose production. [Note: The small intestine can also make glucose.] Figure 10.1 shows the relationship of gluconeogenesis to other essential pathways of energy metabolism.
Biochemistry_Lippincott_406	Biochemistry_Lippinco	carbon dioxide. II. SUBSTRATES Gluconeogenic precursors are molecules that can be used to produce a net synthesis of glucose. The most important gluconeogenic precursors are glycerol, lactate, and α-keto acids obtained from the metabolism of glucogenic amino acids. [Note: All but two amino acids (leucine and lysine) are glucogenic (see p. 262).] A. Glycerol Glycerol is released during the hydrolysis of triacylglycerols (TAG) in adipose tissue (see p. 190) and is delivered by the blood to the liver. Glycerol is phosphorylated by glycerol kinase to glycerol 3-phosphate, B. Lactate Lactate from anaerobic glycolysis is released into the blood by exercising skeletal muscle and by cells that lack mitochondria such as RBC. In the Cori cycle, this lactate is taken up by the liver and oxidized to pyruvate that is converted to glucose, which is released back into the circulation (Fig. 10.2). C. Amino acids	Biochemistry_Lippinco. carbon dioxide. II. SUBSTRATES Gluconeogenic precursors are molecules that can be used to produce a net synthesis of glucose. The most important gluconeogenic precursors are glycerol, lactate, and α-keto acids obtained from the metabolism of glucogenic amino acids. [Note: All but two amino acids (leucine and lysine) are glucogenic (see p. 262).] A. Glycerol Glycerol is released during the hydrolysis of triacylglycerols (TAG) in adipose tissue (see p. 190) and is delivered by the blood to the liver. Glycerol is phosphorylated by glycerol kinase to glycerol 3-phosphate, B. Lactate Lactate from anaerobic glycolysis is released into the blood by exercising skeletal muscle and by cells that lack mitochondria such as RBC. In the Cori cycle, this lactate is taken up by the liver and oxidized to pyruvate that is converted to glucose, which is released back into the circulation (Fig. 10.2). C. Amino acids
Biochemistry_Lippincott_407	Biochemistry_Lippinco	C. Amino acids Amino acids produced by hydrolysis of tissue proteins are the major sources of glucose during a fast. Their metabolism generates α-keto acids, such as pyruvate that is converted to glucose, or α-ketoglutarate that can enter the tricarboxylic acid (TCA) cycle and form oxaloacetate (OAA), a direct precursor of phosphoenolpyruvate (PEP). [Note: Acetyl coenzyme A (CoA) and compounds that give rise only to acetyl CoA (for example, acetoacetate, lysine, and leucine) cannot give rise to a net synthesis of glucose. This is because of the irreversible nature of the pyruvate dehydrogenase complex (PDHC), which converts pyruvate to acetyl CoA (see p. 109). These compounds give rise instead to ketone bodies (see p. 195) and are termed ketogenic.] III. REACTIONS	Biochemistry_Lippinco. C. Amino acids Amino acids produced by hydrolysis of tissue proteins are the major sources of glucose during a fast. Their metabolism generates α-keto acids, such as pyruvate that is converted to glucose, or α-ketoglutarate that can enter the tricarboxylic acid (TCA) cycle and form oxaloacetate (OAA), a direct precursor of phosphoenolpyruvate (PEP). [Note: Acetyl coenzyme A (CoA) and compounds that give rise only to acetyl CoA (for example, acetoacetate, lysine, and leucine) cannot give rise to a net synthesis of glucose. This is because of the irreversible nature of the pyruvate dehydrogenase complex (PDHC), which converts pyruvate to acetyl CoA (see p. 109). These compounds give rise instead to ketone bodies (see p. 195) and are termed ketogenic.] III. REACTIONS
Biochemistry_Lippincott_408	Biochemistry_Lippinco	195) and are termed ketogenic.] III. REACTIONS Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. However, three glycolytic reactions are irreversible and must be circumvented by four alternate reactions that energetically favor the synthesis of glucose. These irreversible reactions, which together are unique to gluconeogenesis, are described below. A. Pyruvate carboxylation The first roadblock to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyruvate by pyruvate kinase (PK). In gluconeogenesis, pyruvate is carboxylated by pyruvate carboxylase (PC) to OAA, which is converted to PEP by PEPcarboxykinase (PEPCK) (Fig. 10.3). mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate.	Biochemistry_Lippinco. 195) and are termed ketogenic.] III. REACTIONS Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. However, three glycolytic reactions are irreversible and must be circumvented by four alternate reactions that energetically favor the synthesis of glucose. These irreversible reactions, which together are unique to gluconeogenesis, are described below. A. Pyruvate carboxylation The first roadblock to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyruvate by pyruvate kinase (PK). In gluconeogenesis, pyruvate is carboxylated by pyruvate carboxylase (PC) to OAA, which is converted to PEP by PEPcarboxykinase (PEPCK) (Fig. 10.3). mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate.
Biochemistry_Lippincott_409	Biochemistry_Lippinco	mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate. 1. Biotin: PC requires the coenzyme biotin (see p. 385) covalently bound to the ε-amino group of a lysine residue in the enzyme (see Fig. 10.3). ATP hydrolysis drives formation of an enzyme–biotin–carbon dioxide (CO2) intermediate, which then carboxylates pyruvate to form OAA. [Note: HCO3− provides the CO2.] The PC reaction occurs in the mitochondria of liver and kidney cells and has two purposes: to allow production of PEP, an important substrate for gluconeogenesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted. Muscle cells also contain PC but use the OAA product only for the replenishment (anaplerotic) purpose and do not synthesize glucose. [Note: Pyruvate carrier protein moves pyruvate from the cytosol into mitochondria.]	Biochemistry_Lippinco. mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate. 1. Biotin: PC requires the coenzyme biotin (see p. 385) covalently bound to the ε-amino group of a lysine residue in the enzyme (see Fig. 10.3). ATP hydrolysis drives formation of an enzyme–biotin–carbon dioxide (CO2) intermediate, which then carboxylates pyruvate to form OAA. [Note: HCO3− provides the CO2.] The PC reaction occurs in the mitochondria of liver and kidney cells and has two purposes: to allow production of PEP, an important substrate for gluconeogenesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted. Muscle cells also contain PC but use the OAA product only for the replenishment (anaplerotic) purpose and do not synthesize glucose. [Note: Pyruvate carrier protein moves pyruvate from the cytosol into mitochondria.]
Biochemistry_Lippincott_410	Biochemistry_Lippinco	PC is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (p. 183), propionyl CoA carboxylase (p. 194), and methylcrotonyl CoA carboxylase (p. 266). 2. Allosteric regulation: PC is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which increased synthesis of OAA is required. For example, this occurs during fasting, when OAA is used for gluconeogenesis in the liver and kidneys. Conversely, at low levels of acetyl CoA, PC is largely inactive, and pyruvate is primarily oxidized by the PDHC to acetyl CoA that can be further oxidized by the TCA cycle (see p. 109). B. Oxaloacetate transport to the cytosol	Biochemistry_Lippinco. PC is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (p. 183), propionyl CoA carboxylase (p. 194), and methylcrotonyl CoA carboxylase (p. 266). 2. Allosteric regulation: PC is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which increased synthesis of OAA is required. For example, this occurs during fasting, when OAA is used for gluconeogenesis in the liver and kidneys. Conversely, at low levels of acetyl CoA, PC is largely inactive, and pyruvate is primarily oxidized by the PDHC to acetyl CoA that can be further oxidized by the TCA cycle (see p. 109). B. Oxaloacetate transport to the cytosol
Biochemistry_Lippincott_411	Biochemistry_Lippinco	For gluconeogenesis to continue, OAA must be converted to PEP by PEPCK. PEP production in the cytosol requires transport of OAA out of mitochondria. However, there is no OAA transporter in the inner mitochondrial membrane, and OAA is first reduced to malate by mitochondrial malate dehydrogenase (MD). Malate is transported into the cytosol and reoxidized to OAA by cytosolic MD as nicotinamide adenine dinucleotide (NAD+) is reduced to NADH (see Fig. 10.3). The NADH is used in the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3phosphate by glyceraldehyde 3-phosphate dehydrogenase (see p. 101), a reaction common to glycolysis and gluconeogenesis. [Note: When abundant, lactate is oxidized to pyruvate as NAD+ is reduced. The pyruvate is transported into mitochondria and carboxylated by PC to OAA, which can be converted to PEP by the mitochondrial isozyme of PEPCK. PEP is transported to the cytosol. OAA can also be converted to aspartate that is transported into the cytosol.]	Biochemistry_Lippinco. For gluconeogenesis to continue, OAA must be converted to PEP by PEPCK. PEP production in the cytosol requires transport of OAA out of mitochondria. However, there is no OAA transporter in the inner mitochondrial membrane, and OAA is first reduced to malate by mitochondrial malate dehydrogenase (MD). Malate is transported into the cytosol and reoxidized to OAA by cytosolic MD as nicotinamide adenine dinucleotide (NAD+) is reduced to NADH (see Fig. 10.3). The NADH is used in the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3phosphate by glyceraldehyde 3-phosphate dehydrogenase (see p. 101), a reaction common to glycolysis and gluconeogenesis. [Note: When abundant, lactate is oxidized to pyruvate as NAD+ is reduced. The pyruvate is transported into mitochondria and carboxylated by PC to OAA, which can be converted to PEP by the mitochondrial isozyme of PEPCK. PEP is transported to the cytosol. OAA can also be converted to aspartate that is transported into the cytosol.]
Biochemistry_Lippincott_412	Biochemistry_Lippinco	C. Cytosolic oxaloacetate decarboxylation OAA is decarboxylated and phosphorylated to PEP in the cytosol by PEPCK. The reaction is driven by hydrolysis of guanosine triphosphate ([GTP] see Fig. 10.3). The combined actions of PC and PEPCK provide an energetically favorable pathway from pyruvate to PEP. PEP is then acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate. The pairing of carboxylation with decarboxylation drives reactions that would otherwise be energetically unfavorable. This strategy is also used in fatty acid (FA) synthesis (see p. 184). D. Fructose 1,6-bisphosphate dephosphorylation	Biochemistry_Lippinco. C. Cytosolic oxaloacetate decarboxylation OAA is decarboxylated and phosphorylated to PEP in the cytosol by PEPCK. The reaction is driven by hydrolysis of guanosine triphosphate ([GTP] see Fig. 10.3). The combined actions of PC and PEPCK provide an energetically favorable pathway from pyruvate to PEP. PEP is then acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate. The pairing of carboxylation with decarboxylation drives reactions that would otherwise be energetically unfavorable. This strategy is also used in fatty acid (FA) synthesis (see p. 184). D. Fructose 1,6-bisphosphate dephosphorylation
Biochemistry_Lippincott_413	Biochemistry_Lippinco	D. Fructose 1,6-bisphosphate dephosphorylation Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase, found in the liver and kidneys, bypasses the irreversible phosphofructokinase-1 (PFK-1) reaction of glycolysis and provides an energetically favorable pathway for the formation of fructose 6-phosphate (Fig. 10.4). This reaction is an important regulatory site of gluconeogenesis. 1. Regulation by intracellular energy levels: Fructose 1,6-bisphosphatase is inhibited by a rise in the adenosine monophosphate (AMP)/ATP ratio, which signals a low-energy state in the cell. Conversely, low AMP and high ATP levels stimulate gluconeogenesis, an energy-requiring pathway. 2.	Biochemistry_Lippinco. D. Fructose 1,6-bisphosphate dephosphorylation Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase, found in the liver and kidneys, bypasses the irreversible phosphofructokinase-1 (PFK-1) reaction of glycolysis and provides an energetically favorable pathway for the formation of fructose 6-phosphate (Fig. 10.4). This reaction is an important regulatory site of gluconeogenesis. 1. Regulation by intracellular energy levels: Fructose 1,6-bisphosphatase is inhibited by a rise in the adenosine monophosphate (AMP)/ATP ratio, which signals a low-energy state in the cell. Conversely, low AMP and high ATP levels stimulate gluconeogenesis, an energy-requiring pathway. 2.
Biochemistry_Lippincott_414	Biochemistry_Lippinco	2. Regulation by fructose 2,6-bisphosphate: Fructose 1,6-bisphosphatase is inhibited by fructose 2,6-bisphosphate, an allosteric effector whose concentration is influenced by the insulin/glucagon ratio. When glucagon is high, the effector is not made by hepatic PFK-2 (see p. 99), and thus, the phosphatase is active (Fig. 10.5). [Note: The signals that inhibit (low energy, high fructose 2,6-bisphosphate) or activate (high energy, low fructose 2,6-bisphosphate) gluconeogenesis have the opposite effect on glycolysis, providing reciprocal control of the pathways that synthesize and oxidize glucose (see p. 100).] E. Glucose 6-phosphate dephosphorylation	Biochemistry_Lippinco. 2. Regulation by fructose 2,6-bisphosphate: Fructose 1,6-bisphosphatase is inhibited by fructose 2,6-bisphosphate, an allosteric effector whose concentration is influenced by the insulin/glucagon ratio. When glucagon is high, the effector is not made by hepatic PFK-2 (see p. 99), and thus, the phosphatase is active (Fig. 10.5). [Note: The signals that inhibit (low energy, high fructose 2,6-bisphosphate) or activate (high energy, low fructose 2,6-bisphosphate) gluconeogenesis have the opposite effect on glycolysis, providing reciprocal control of the pathways that synthesize and oxidize glucose (see p. 100).] E. Glucose 6-phosphate dephosphorylation
Biochemistry_Lippincott_415	Biochemistry_Lippinco	Glucose 6-phosphate hydrolysis by glucose 6-phosphatase bypasses the irreversible hexokinase/glucokinase reaction and provides an energetically favorable pathway for the formation of free glucose (Fig. 10.6). The liver is the primary organ that produces free glucose from glucose 6-phosphate. This process requires a complex of two proteins found only in gluconeogenic tissue: glucose 6-phosphate translocase, which transports glucose 6-phosphate across the endoplasmic reticular (ER) membrane, and glucose 6-phosphatase, which removes the phosphate, producing free glucose (see Fig. 10.6). [Note: These ER membrane proteins are also required for the final step of glycogen degradation (see p. 130). Glycogen storage diseases Ia and Ib, caused by deficiencies in the phosphatase and the translocase, respectively, are characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for	Biochemistry_Lippinco. Glucose 6-phosphate hydrolysis by glucose 6-phosphatase bypasses the irreversible hexokinase/glucokinase reaction and provides an energetically favorable pathway for the formation of free glucose (Fig. 10.6). The liver is the primary organ that produces free glucose from glucose 6-phosphate. This process requires a complex of two proteins found only in gluconeogenic tissue: glucose 6-phosphate translocase, which transports glucose 6-phosphate across the endoplasmic reticular (ER) membrane, and glucose 6-phosphatase, which removes the phosphate, producing free glucose (see Fig. 10.6). [Note: These ER membrane proteins are also required for the final step of glycogen degradation (see p. 130). Glycogen storage diseases Ia and Ib, caused by deficiencies in the phosphatase and the translocase, respectively, are characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for
Biochemistry_Lippincott_416	Biochemistry_Lippinco	respectively, are characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for moving the free glucose into the cytosol and then into blood.	Biochemistry_Lippinco. respectively, are characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for moving the free glucose into the cytosol and then into blood.
Biochemistry_Lippincott_417	Biochemistry_Lippinco	F. Summary of the reactions of glycolysis and gluconeogenesis Of the 11 reactions required to convert pyruvate to free glucose, 7 are catalyzed by reversible glycolytic enzymes (Fig. 10.7). The 3 irreversible reactions (catalyzed by hexokinase/glucokinase, PFK-1, and PK) are circumvented by reactions catalyzed by glucose 6-phosphatase, fructose 1,6-bisphosphatase, PC, and PEPCK. In gluconeogenesis, the equilibria of the reversible glycolytic reactions are pushed toward glucose synthesis as a result of the essentially irreversible formation of PEP, fructose 6-phosphate, and glucose by the gluconeogenic enzymes. [Note: The stoichiometry of gluconeogenesis from two pyruvate molecules couples the cleavage of six high-energy phosphate bonds and the oxidation of two NADH with the formation of one glucose molecule (see Fig. 10.7).] IV. REGULATION	Biochemistry_Lippinco. F. Summary of the reactions of glycolysis and gluconeogenesis Of the 11 reactions required to convert pyruvate to free glucose, 7 are catalyzed by reversible glycolytic enzymes (Fig. 10.7). The 3 irreversible reactions (catalyzed by hexokinase/glucokinase, PFK-1, and PK) are circumvented by reactions catalyzed by glucose 6-phosphatase, fructose 1,6-bisphosphatase, PC, and PEPCK. In gluconeogenesis, the equilibria of the reversible glycolytic reactions are pushed toward glucose synthesis as a result of the essentially irreversible formation of PEP, fructose 6-phosphate, and glucose by the gluconeogenic enzymes. [Note: The stoichiometry of gluconeogenesis from two pyruvate molecules couples the cleavage of six high-energy phosphate bonds and the oxidation of two NADH with the formation of one glucose molecule (see Fig. 10.7).] IV. REGULATION
Biochemistry_Lippincott_418	Biochemistry_Lippinco	IV. REGULATION The moment-to-moment regulation of gluconeogenesis is determined primarily by the circulating level of glucagon and by the availability of gluconeogenic substrates. In addition, slow adaptive changes in enzyme amount result from an alteration in the rate of enzyme synthesis or degradation or both. [Note: Hormonal control of the glucoregulatory system is presented in Chapter 23.] This peptide hormone from pancreatic islet α cells (see p. 313) stimulates gluconeogenesis by three mechanisms. 1. Changes in allosteric effectors: Glucagon lowers hepatic fructose 2,6bisphosphate, resulting in fructose 1,6-bisphosphatase activation and PFK-1 inhibition, thereby favoring gluconeogenesis over glycolysis (see Fig. 10.5). [Note: See pp. 99–100 for the role of fructose 2,6bisphosphate in glycolysis regulation.] 2.	Biochemistry_Lippinco. IV. REGULATION The moment-to-moment regulation of gluconeogenesis is determined primarily by the circulating level of glucagon and by the availability of gluconeogenic substrates. In addition, slow adaptive changes in enzyme amount result from an alteration in the rate of enzyme synthesis or degradation or both. [Note: Hormonal control of the glucoregulatory system is presented in Chapter 23.] This peptide hormone from pancreatic islet α cells (see p. 313) stimulates gluconeogenesis by three mechanisms. 1. Changes in allosteric effectors: Glucagon lowers hepatic fructose 2,6bisphosphate, resulting in fructose 1,6-bisphosphatase activation and PFK-1 inhibition, thereby favoring gluconeogenesis over glycolysis (see Fig. 10.5). [Note: See pp. 99–100 for the role of fructose 2,6bisphosphate in glycolysis regulation.] 2.
Biochemistry_Lippincott_419	Biochemistry_Lippinco	Covalent modification of enzyme activity: Glucagon binds its G protein– coupled receptor (see p. 95) and, via an elevation in cyclic AMP (cAMP) level and cAMP-dependent protein kinase A activity, stimulates the conversion of hepatic PK to its inactive (phosphorylated) form. This decreases PEP conversion to pyruvate, which has the effect of diverting PEP to gluconeogenesis (Fig. 10.8). the enzyme. [Note: Only the hepatic isozyme is subject to covalent regulation.] AMP. 3. Induction of enzyme synthesis: Glucagon increases transcription of the gene for PEPCK via the transcription factor cAMP response element– binding (CREB) protein, thereby increasing the availability of this enzyme as levels of its substrate rise during fasting. [Note: Cortisol (a glucocorticoid) also increases expression of the gene, whereas insulin decreases expression.] B. Substrate availability	Biochemistry_Lippinco. Covalent modification of enzyme activity: Glucagon binds its G protein– coupled receptor (see p. 95) and, via an elevation in cyclic AMP (cAMP) level and cAMP-dependent protein kinase A activity, stimulates the conversion of hepatic PK to its inactive (phosphorylated) form. This decreases PEP conversion to pyruvate, which has the effect of diverting PEP to gluconeogenesis (Fig. 10.8). the enzyme. [Note: Only the hepatic isozyme is subject to covalent regulation.] AMP. 3. Induction of enzyme synthesis: Glucagon increases transcription of the gene for PEPCK via the transcription factor cAMP response element– binding (CREB) protein, thereby increasing the availability of this enzyme as levels of its substrate rise during fasting. [Note: Cortisol (a glucocorticoid) also increases expression of the gene, whereas insulin decreases expression.] B. Substrate availability
Biochemistry_Lippincott_420	Biochemistry_Lippinco	B. Substrate availability The availability of gluconeogenic precursors, particularly glucogenic amino acids, significantly influences the rate of glucose synthesis. Decreased insulin levels favor mobilization of amino acids from muscle protein to provide the carbon skeletons for gluconeogenesis. The ATP and NADH coenzymes required for gluconeogenesis are primarily provided by FA oxidation. C. Allosteric activation by acetyl CoA Allosteric activation of hepatic PC by acetyl CoA occurs during fasting. As a result of increased TAG hydrolysis in adipose tissue, the liver is flooded with FA (see p. 330). The rate of formation of acetyl CoA by β-oxidation of these FA exceeds the capacity of the liver to oxidize it to CO2 and water. As a result, acetyl CoA accumulates and activates PC. [Note: Acetyl CoA inhibits the PDHC (by activating PDH kinase; see p. 111). Thus, this single compound can divert pyruvate toward gluconeogenesis and away from the TCA cycle (Fig. 10.9).]	Biochemistry_Lippinco. B. Substrate availability The availability of gluconeogenic precursors, particularly glucogenic amino acids, significantly influences the rate of glucose synthesis. Decreased insulin levels favor mobilization of amino acids from muscle protein to provide the carbon skeletons for gluconeogenesis. The ATP and NADH coenzymes required for gluconeogenesis are primarily provided by FA oxidation. C. Allosteric activation by acetyl CoA Allosteric activation of hepatic PC by acetyl CoA occurs during fasting. As a result of increased TAG hydrolysis in adipose tissue, the liver is flooded with FA (see p. 330). The rate of formation of acetyl CoA by β-oxidation of these FA exceeds the capacity of the liver to oxidize it to CO2 and water. As a result, acetyl CoA accumulates and activates PC. [Note: Acetyl CoA inhibits the PDHC (by activating PDH kinase; see p. 111). Thus, this single compound can divert pyruvate toward gluconeogenesis and away from the TCA cycle (Fig. 10.9).]
Biochemistry_Lippincott_421	Biochemistry_Lippinco	D. Allosteric inhibition by AMP Fructose 1,6-bisphosphatase is inhibited by AMP, a compound that activates PFK-1. This results in reciprocal regulation of glycolysis and gluconeogenesis seen previously with fructose 2,6-bisphosphate (see p. 121). [Note: Thus, elevated AMP stimulates energy-producing pathways and inhibits energy-requiring ones.] V. CHAPTER SUMMARY	Biochemistry_Lippinco. D. Allosteric inhibition by AMP Fructose 1,6-bisphosphatase is inhibited by AMP, a compound that activates PFK-1. This results in reciprocal regulation of glycolysis and gluconeogenesis seen previously with fructose 2,6-bisphosphate (see p. 121). [Note: Thus, elevated AMP stimulates energy-producing pathways and inhibits energy-requiring ones.] V. CHAPTER SUMMARY
Biochemistry_Lippincott_422	Biochemistry_Lippinco	Gluconeogenic precursors include glycerol released during triacylglycerol hydrolysis in adipose tissue, lactate released by cells that lack mitochondria and by exercising skeletal muscle, and α-keto acids (for example, αketoglutarate and pyruvate) derived from glucogenic amino acid metabolism (Fig. 10.10). Seven of the reactions of glycolysis are reversible and are used for gluconeogenesis in the liver and kidneys. Three reactions, catalyzed by pyruvate kinase, phosphofructokinase-1, and glucokinase/hexokinase, are physiologically irreversible and must be circumvented. Pyruvate is converted to oxaloacetate and then to phosphoenolpyruvate (PEP) by pyruvate carboxylase (PC) and PEPcarboxykinase (PEPCK ). PC requires biotin and ATP and is allosterically activated by acetyl coenzyme A. PEPCK requires guanosine triphosphate. Transcription of its gene is increased by glucagon and cortisol and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6phosphate by fructose	Biochemistry_Lippinco. Gluconeogenic precursors include glycerol released during triacylglycerol hydrolysis in adipose tissue, lactate released by cells that lack mitochondria and by exercising skeletal muscle, and α-keto acids (for example, αketoglutarate and pyruvate) derived from glucogenic amino acid metabolism (Fig. 10.10). Seven of the reactions of glycolysis are reversible and are used for gluconeogenesis in the liver and kidneys. Three reactions, catalyzed by pyruvate kinase, phosphofructokinase-1, and glucokinase/hexokinase, are physiologically irreversible and must be circumvented. Pyruvate is converted to oxaloacetate and then to phosphoenolpyruvate (PEP) by pyruvate carboxylase (PC) and PEPcarboxykinase (PEPCK ). PC requires biotin and ATP and is allosterically activated by acetyl coenzyme A. PEPCK requires guanosine triphosphate. Transcription of its gene is increased by glucagon and cortisol and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6phosphate by fructose