Biochemistry_Lippinco

Biochemistry_Lippinco. The major source of acetyl CoA for the TCA cycle is the oxidative decarboxylation of pyruvate by the multienzyme pyruvate dehydrogenase complex (PDH complex, or PDHC). However, the PDHC (described below) is not a component of the TCA cycle. Pyruvate, the end product of aerobic glycolysis, is transported from the cytosol into the mitochondrial matrix by the pyruvate mitochondrial carrier of the inner mitochondrial membrane. In the matrix, the PDHC converts pyruvate to acetyl CoA. [Note: Fatty acid oxidation is another source of acetyl CoA (see p. 192).] 1. PDHC component enzymes: The PDHC is a protein aggregate of multiple copies of three enzymes, pyruvate decarboxylase ([E1] sometimes called pyruvate dehydrogenase), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Each catalyzes a part of the overall reaction (Fig. 9.2). Their physical association links the reactions in proper sequence without the release of intermediates. In addition to the enzymes


Biochemistry_Lippinco

Biochemistry_Lippinco. (E3). Each catalyzes a part of the overall reaction (Fig. 9.2). Their physical association links the reactions in proper sequence without the release of intermediates. In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the PDHC also contains two regulatory enzymes, pyruvate dehydrogenase kinase (PDH kinase) and pyruvate dehydrogenase phosphatase (PDH phosphatase).


Biochemistry_Lippinco

Biochemistry_Lippinco. dioxide; TPP = thiamine pyrophosphate; L = lipoic acid; CoA = coenzyme A; FAD(H2) and NAD(H) = flavin and nicotinamide adenine dinucleotides; ~ = high-energy bond. 2. Coenzymes: The PDHC contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions shown in Figure 9.2. E1 requires thiamine pyrophosphate (TPP), E2 requires lipoic acid and CoA, and E3 requires FAD and NAD+. [Note: TPP, lipoic acid, and FAD are tightly bound to the enzymes and function as coenzymes–prosthetic groups (see p. 54).] Deficiencies of thiamine or niacin can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) if the PDHC is inactive. Wernicke-Korsakoff, an encephalopathy-psychosis syndrome due to thiamine deficiency, may be seen with alcohol abuse (see p. 383).


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Regulation: Covalent modifications by the two regulatory enzymes of the PDHC alternately activate and inactivate E1. PDH kinase phosphorylates and inactivates E1, whereas PDH phosphatase dephosphorylates and activates E1 (Fig. 9.3). The kinase itself is allosterically activated by ATP, acetyl CoA, and NADH. Therefore, in the presence of these high-energy products, the PDHC is turned off. [Note: It is actually the rise in the ATP/ADP (adenosine diphosphate), NADH/NAD+, or acetyl CoA/CoA ratios that affects enzymic activity.] Pyruvate is a potent inhibitor of PDH kinase. Therefore, if pyruvate concentrations are elevated, E1 will be maximally active. Calcium (Ca2+) is a strong activator of PDH phosphatase, stimulating E1 activity. This is particularly important in skeletal muscle, where Ca2+ release during contraction stimulates the PDHC and, thus, energy production. [Note: Although covalent regulation by the kinase and phosphatase is primary, the PDHC is also subject to product


Biochemistry_Lippinco

Biochemistry_Lippinco. Ca2+ release during contraction stimulates the PDHC and, thus, energy production. [Note: Although covalent regulation by the kinase and phosphatase is primary, the PDHC is also subject to product (NADH and acetyl CoA) inhibition.] 4. Deficiency: A deficiency of the α subunits of the tetrameric E1 component of the PDHC, although very rare, is the most common biochemical cause of congenital lactic acidosis. The deficiency results in a decreased ability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactate via lactate dehydrogenase (see p. 103). This creates particular problems for the brain, which relies on the TCA cycle for most of its energy and is particularly sensitive to acidosis. Symptoms are variable and include neurodegeneration, muscle spasticity, and, in the 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


Biochemistry_Lippinco

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Biochemistry_Lippinco. 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 1,6-bisphosphatase. This enzyme is inhibited by a high adenosine monophosphate (AMP)/ATP ratio. It is also inhibited by fructose 2,6-bisphosphate, the primary allosteric activator of glycolysis. Glucose 6-phosphate is dephosphorylated to glucose by glucose 6phosphatase. This enzyme of the endoplasmic reticular membrane catalyzes the final step in gluconeogenesis and in glycogen degradation. Its deficiency results in severe, fasting hypoglycemia.


Biochemistry_Lippinco

Biochemistry_Lippinco. dioxide. Choose the ONE best answer. 0.1. Which one of the following statements concerning gluconeogenesis is correct? A. It is an energy-producing (exergonic) process. B. It is important in maintaining blood glucose during a 2-day fast. C. It is inhibited by a fall in the insulin/glucagon ratio. D. It occurs in the cytosol of muscle cells. E. It uses carbon skeletons provided by fatty acid degradation.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. It is inhibited by a fall in the insulin/glucagon ratio. D. It occurs in the cytosol of muscle cells. E. It uses carbon skeletons provided by fatty acid degradation. Correct answer = B. During a 2-day fast, glycogen stores are depleted, and gluconeogenesis maintains blood glucose. This is an energy-requiring (endergonic) pathway (both ATP and GTP get hydrolyzed) that occurs primarily in the liver, with the kidneys becoming major glucose producers in prolonged fasting. Gluconeogenesis uses both mitochondrial and cytosolic enzymes and is stimulated by a fall in the insulin/glucagon ratio. Fatty acid degradation yields acetyl coenzyme A (CoA), which cannot be converted to glucose. This is because there is no net gain of carbons from acetyl CoA in the tricarboxylic acid cycle, and the pyruvate dehydrogenase complex is physiologically irreversible. It is the carbon skeletons of most amino acids that are glucogenic.


Biochemistry_Lippinco

Biochemistry_Lippinco. 0.2. Which reaction in the diagram below would be inhibited in the presence of large amounts of avidin, an egg white protein that binds and sequesters biotin? Correct answer = C. Pyruvate is carboxylated to oxaloacetate by pyruvate carboxylase, a biotin-requiring enzyme. B (pyruvate dehydrogenase complex) requires thiamine pyrophosphate, lipoic acid, flavin and nicotinamide adenine dinucleotides (FAD and NAD+), and coenzyme A; D (transaminase) requires pyridoxal phosphate; E (lactate dehydrogenase) requires NADH. 0.3. Which one of the following reactions is unique to gluconeogenesis? A. 1,3-Bisphosphoglycerate → 3-phosphoglycerate B. Lactate → pyruvate C. Oxaloacetate → phosphoenolpyruvate D. Phosphoenolpyruvate → pyruvate Correct answer = C. The other reactions are common to both gluconeogenesis and glycolysis. 0.4. Use the chart below to show the effect of adenosine monophosphate (AMP) and fructose 2,6-bisphosphate on the listed enzymes of gluconeogenesis and glycolysis.


Biochemistry_Lippinco

Biochemistry_Lippinco. 0.4. Use the chart below to show the effect of adenosine monophosphate (AMP) and fructose 2,6-bisphosphate on the listed enzymes of gluconeogenesis and glycolysis. Both fructose 2,6-bisphosphate and adenosine monophosphate inhibit fructose 1,6-bisphosphatase of gluconeogenesis and activate phosphofructokinase-1 of glycolysis. This results in reciprocal regulation of the two pathways. 0.5. The metabolism of ethanol by alcohol dehydrogenase produces reduced nicotinamide adenine dinucleotide (NADH) from the oxidized (NAD+) form. What effect is the fall in the NAD+/NADH ratio expected to have on gluconeogenesis? Explain.


Biochemistry_Lippinco

Biochemistry_Lippinco. The increase in NADH as ethanol is oxidized decreases the availability of oxaloacetate (OAA) because the reversible oxidation of malate to OAA by malate dehydrogenase of the tricarboxylic acid cycle is driven in the reverse direction by NADH. Additionally, the reversible reduction of pyruvate to lactate by lactate dehydrogenase is driven to lactate by NADH. Thus, two important gluconeogenic substrates, OAA and pyruvate, decrease as a result of the increase in NADH during ethanol metabolism. Consequently, gluconeogenesis decreases. 0.6. Given that acetyl coenzyme A cannot be a substrate for gluconeogenesis, why is its production in fatty acid oxidation essential for gluconeogenesis? Acetyl coenzyme A inhibits the pyruvate dehydrogenase complex and activates pyruvate carboxylase, pushing pyruvate to gluconeogenesis and away from oxidation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. A constant source of blood glucose is an absolute requirement for human life. Glucose is the greatly preferred energy source for the brain and the required energy source for cells with few or no mitochondria such as mature red blood cells. Glucose is also essential as an energy source for exercising muscle, where it is the substrate for anaerobic glycolysis. Blood glucose can be obtained from three primary sources: the diet, glycogen degradation, and gluconeogenesis. Dietary intake of glucose and glucose precursors, such as starch (a polysaccharide), disaccharides, and monosaccharides, is sporadic and, depending on the diet, is not always a reliable source of blood glucose. In contrast, gluconeogenesis (see p. 117) can provide sustained synthesis of glucose, but it is somewhat slow in responding to a falling blood glucose level. Therefore, the body has developed mechanisms for storing a supply of glucose in a rapidly mobilized form, namely, glycogen. In the absence of a dietary source


Biochemistry_Lippinco

Biochemistry_Lippinco. to a falling blood glucose level. Therefore, the body has developed mechanisms for storing a supply of glucose in a rapidly mobilized form, namely, glycogen. In the absence of a dietary source of glucose, this sugar is rapidly released into the blood from liver glycogen. Similarly, muscle glycogen is extensively degraded in exercising muscle to provide that tissue with an important energy source. When glycogen stores are depleted, specific tissues synthesize glucose de novo, using glycerol, lactate, pyruvate, and amino acids as carbon sources for gluconeogenesis (see Chapter 10). Figure 11.1 shows the reactions of glycogen synthesis and degradation as part of the essential pathways of energy metabolism.


Biochemistry_Lippinco

Biochemistry_Lippinco. II. STRUCTURE AND FUNCTION The main stores of glycogen are found in skeletal muscle and liver, although most other cells store small amounts of glycogen for their own use. The function of muscle glycogen is to serve as a fuel reserve for the synthesis of ATP during muscle contraction. That of liver glycogen is to maintain the blood glucose concentration, particularly during the early stages of a fast (Fig. 11.2; also see p. 329). [Note: Liver glycogen can maintain blood glucose for <24 hours.] A. Amounts in liver and muscle


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Amounts in liver and muscle Approximately 400 g of glycogen make up 1%–2% of the fresh weight of resting muscle, and ~100 g of glycogen make up to 10% of the fresh weight of a well-fed adult liver. What limits the production of glycogen at these levels is not clear. However, in some glycogen storage diseases (GSD) (see Fig. 11.8), the amount of glycogen in the liver and/or muscle can be significantly higher. [Note: In the body, muscle mass is greater than liver mass. Consequently, most of the body’s glycogen is found in skeletal muscle.] B. Structure


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Structure Glycogen is a branched-chain polysaccharide made exclusively from α-Dglucose. The primary glycosidic bond is an α(1→4) linkage. After an average of 8–14 glucosyl residues, there is a branch containing an α(1→6) linkage (Fig. 11.3). A single glycogen molecule can contain up to 55,000 glucosyl residues. These polymers of glucose exist as large, spherical, cytoplasmic granules (particles) that also contain most of the enzymes necessary for glycogen synthesis and degradation. C. Glycogen store fluctuation


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Glycogen store fluctuation Liver glycogen stores increase during the well-fed state (see p. 323) and are depleted during a fast (see p. 329). Muscle glycogen is not affected by short periods of fasting (a few days) and is only moderately decreased in prolonged fasting (weeks). Muscle glycogen is synthesized to replenish muscle stores after they have been depleted following strenuous exercise. [Note: Glycogen synthesis and degradation go on continuously. The difference between the rates of these two processes determines the levels of stored glycogen during specific physiologic states.] III. SYNTHESIS (GLYCOGENESIS) Glycogen is synthesized from molecules of α-D-glucose. The process occurs in the cytosol and requires energy supplied by ATP (for the phosphorylation of glucose) and uridine triphosphate (UTP).


Biochemistry_Lippinco

Biochemistry_Lippinco. Glycogen is synthesized from molecules of α-D-glucose. The process occurs in the cytosol and requires energy supplied by ATP (for the phosphorylation of glucose) and uridine triphosphate (UTP). A. Uridine diphosphate glucose synthesis α-D-Glucose attached to uridine diphosphate (UDP) is the source of all the glucosyl residues that are added to the growing glycogen molecule. UDP-glucose (Fig. 11.4) is synthesized from glucose 1-phosphate and UTP by UDP–glucose pyrophosphorylase (Fig. 11.5). Pyrophosphate (PPi), the second product of the reaction, is hydrolyzed to two inorganic phosphates (Pi) by pyrophosphatase. The hydrolysis is exergonic, insuring that the UDP–glucose pyrophosphorylase reaction proceeds in the direction of UDP-glucose production. [Note: Glucose 1-phosphate is generated from glucose 6-phosphate by phosphoglucomutase. Glucose 1,6-bisphosphate is an obligatory intermediate in this reversible reaction (Fig. 11.6).] B. Primer requirement and synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. Glycogen synthase makes the α(1→4) linkages in glycogen. This enzyme cannot initiate chain synthesis using free glucose as an acceptor of a molecule of glucose from UDP-glucose. Instead, it can only elongate already existing chains of glucose and, therefore, requires a primer. A fragment of glycogen can serve as a primer. In the absence of a fragment, the homodimeric protein glycogenin can serve as an acceptor of glucose from UDP-glucose (see Fig. 11.5). The side-chain hydroxyl group of tyrosine-194 in the protein is the site at which the initial glucosyl unit is attached. Because the reaction is catalyzed by glycogenin itself via autoglucosylation, glycogenin is an enzyme. Glycogenin then catalyzes the transfer of at least four molecules of glucose from UDP-glucose, producing a short, α(1→4)-linked glucosyl chain. This short chain serves as a primer that is able to be elongated by glycogen synthase, which is recruited by glycogenin, as described in C. below. [Note: Glycogenin stays


Biochemistry_Lippinco

Biochemistry_Lippinco. glucosyl chain. This short chain serves as a primer that is able to be elongated by glycogen synthase, which is recruited by glycogenin, as described in C. below. [Note: Glycogenin stays associated with and forms the core of a glycogen granule.]


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Elongation by glycogen synthase Elongation of a glycogen chain involves the transfer of glucose from UDP-glucose to the nonreducing end of the growing chain, forming a new glycosidic bond between the anomeric hydroxyl group of carbon 1 of the activated glucose and carbon 4 of the accepting glucosyl residue (see Fig. 11.5). [Note: The nonreducing end of a carbohydrate chain is one in which the anomeric carbon of the terminal sugar is linked by a glycosidic bond to another molecule, making the terminal sugar nonreducing (see p. 84).] The enzyme responsible for making the α(1→4) linkages in glycogen is glycogen synthase. [Note: The UDP released when the new α(1→4) glycosidic bond is made can be phosphorylated to UTP by nucleoside diphosphate kinase (UDP + ATP ⇄ UTP + ADP; see p. 296).] D. Branch formation


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Branch formation If no other synthetic enzyme acted on the chain, the resulting structure would be a linear (unbranched) chain of glucosyl residues attached by α(1→4) linkages. Such a compound is found in plant tissues and is called amylose. In contrast, glycogen has branches located, on average, eight glucosyl residues apart, resulting in a highly branched, tree-like structure (see Fig. 11.3) that is far more soluble than the unbranched amylose. Branching also increases the number of nonreducing ends to which new glucosyl residues can be added (and also, as described in IV. below, from which these residues can be removed), thereby greatly accelerating the rate at which glycogen synthesis can occur and dramatically increasing the size of the glycogen molecule. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Branch synthesis: Branches are made by the action of the branching enzyme, amylo-α(1→4)→α(1→6)-transglycosylase. This enzyme removes a set of six to eight glucosyl residues from the nonreducing end of the glycogen chain, breaking an α(1→4) bond to another residue on the chain, and attaches it to a nonterminal glucosyl residue by an α(1→6) linkage, thus functioning as a 4:6 transferase. The resulting new, nonreducing end (see “i” in Fig. 11.5), as well as the old nonreducing end from which the six to eight residues were removed (see “o” in Fig. 11.5), can now be further elongated by glycogen synthase. 2. Additional branch synthesis: After elongation of these two ends has been accomplished, their terminal six to eight glucosyl residues can be removed and used to make additional branches. IV. DEGRADATION (GLYCOGENOLYSIS)


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. DEGRADATION (GLYCOGENOLYSIS) The degradative pathway that mobilizes stored glycogen in liver and skeletal muscle is not a reversal of the synthetic reactions. Instead, a separate set of cytosolic enzymes is required. When glycogen is degraded, the primary product is glucose 1-phosphate, obtained by breaking α(1→4) glycosidic bonds. In addition, free glucose is released from each α(1→6)–linked glucosyl residue (branch point). A. Chain shortening Glycogen phosphorylase sequentially cleaves the α(1→4) glycosidic bonds between the glucosyl residues at the nonreducing ends of the glycogen chains by simple phosphorolysis (producing glucose 1-phosphate) until four glucosyl units remain on each chain at a branch point (Fig. 11.7). The resulting structure is called a limit dextrin, and phosphorylase cannot degrade it any further (Fig. 11.8). [Note: Phosphorylase requires pyridoxal phosphate (a derivative of vitamin B6; see p. 382) as a coenzyme.] B. Branch removal


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Branch removal Branches are removed by the two enzymic activities of a single bifunctional protein, the debranching enzyme (see Fig. 11.8). First, oligoα(1→4)→α(1→4)-glucantransferase activity removes the outer three of the four glucosyl residues remaining at a branch. It next transfers them to the nonreducing end of another chain, lengthening it accordingly. Thus, an α(1→4) bond is broken and an α(1→4) bond is made, and the enzyme functions as a 4:4 transferase. Next, the remaining glucose residue attached in an α(1→6) linkage is removed hydrolytically by amylo-α(1→6)glucosidase activity, releasing free (nonphosphorylated) glucose. The glucosyl chain is now available again for degradation by glycogen phosphorylase until four glucosyl units in the next branch are reached. C. Glucose 1-phosphate isomerization to glucose 6phosphate


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Glucose 1-phosphate isomerization to glucose 6phosphate Glucose 1-phosphate, produced by glycogen phosphorylase, is isomerized in the cytosol to glucose 6-phosphate by phosphoglucomutase (see Fig. 11.6). In the liver, glucose 6-phosphate is transported into the endoplasmic reticulum (ER) by glucose 6-phosphate translocase. There, it is dephosphorylated to glucose by glucose 6-phosphatase (the same enzyme used in the last step of gluconeogenesis; see p. 121). The glucose is then transported from the ER to the cytosol. Hepatocytes release glycogen-derived glucose into the blood to help maintain blood glucose levels until the gluconeogenic pathway is actively producing glucose. [Note: Muscle lacks glucose 6-phosphatase. Consequently, glucose 6-phosphate cannot be dephosphorylated and sent into the blood. Instead, it enters glycolysis, providing energy needed for muscle contraction.] D. Lysosomal degradation


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Lysosomal degradation A small amount (1%–3%) of glycogen is degraded by the lysosomal enzyme, acid α(1→4)-glucosidase (acid maltase). The purpose of this autophagic pathway is unknown. However, a deficiency of this enzyme causes accumulation of glycogen in vacuoles in the lysosomes, resulting in the serious GSD type II: Pompe disease (see Fig. 11.8). [Note: Pompe disease is the only GSD that is a lysosomal storage disease.] Lysosomal storage diseases are genetic disorders characterized by the accumulation of abnormal amounts of carbohydrates or lipids primarily due to their decreased lysosomal degradation resulting from decreased activity or amount of lysosomal acid hydrolases. V. GLYCOGENESIS AND GLYCOGENOLYSIS REGULATION


Biochemistry_Lippinco

Biochemistry_Lippinco. V. GLYCOGENESIS AND GLYCOGENOLYSIS REGULATION Because of the importance of maintaining blood glucose levels, the synthesis and degradation of its glycogen storage form are tightly regulated. In the liver, glycogenesis accelerates during periods when the body has been well fed, whereas glycogenolysis accelerates during periods of fasting. In skeletal muscle, glycogenolysis occurs during active exercise, and glycogenesis begins as soon as the muscle is again at rest. Regulation of synthesis and degradation is accomplished on two levels. First, glycogen synthase and glycogen phosphorylase are hormonally regulated (by covalent phosphorylation/dephosphorylation) to meet the needs of the body as a whole. Second, these same enzymes are allosterically regulated (by effector molecules) to meet the needs of a particular tissue. A. Covalent activation of glycogenolysis


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Covalent activation of glycogenolysis The binding of hormones, such as glucagon or epinephrine, to plasma membrane G protein–coupled receptors ([GPCR] see p. 94) signals the need for glycogen to be degraded, either to elevate blood glucose levels or to provide energy for exercising muscle. 1. Protein kinase A activation: Binding of glucagon or epinephrine to their specific hepatocyte GPCR, or of epinephrine to a specific myocyte GPCR, results in the G protein–mediated activation of adenylyl cyclase. This enzyme catalyzes the synthesis of cyclic adenosine monophosphate (cAMP), which activates cAMP-dependent protein kinase A (PKA). cAMP binds the two regulatory subunits of tetrameric PKA, releasing two individual catalytic subunits that are active (Fig. 11.9; also see p. 95). PKA then phosphorylates several enzymes of glycogen metabolism, affecting their activity. [Note: When cAMP is removed, the inactive tetramer reforms.] on next page.) catalytic subunit. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Phosphorylase kinase activation: Phosphorylase kinase exists in two forms: an inactive “b” form and an active “a” form. Active PKA phosphorylates the inactive “b” form of phosphorylase kinase, producing the active “a” form (see Fig. 11.9). 3. Glycogen phosphorylase activation: Glycogen phosphorylase also exists in a dephosphorylated, inactive “b” form and a phosphorylated, active “a” form. Phosphorylase kinase a is the only enzyme that phosphorylates glycogen phosphorylase b to its active “a” form, which then begins glycogenolysis (see Fig. 11.9). 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Signal amplification: The cascade of reactions described above activates glycogenolysis. The large number of sequential steps serves to amplify the effect of the hormonal signal (that is, a few hormone molecules binding to their GPCR result in a number of PKA molecules being activated that can each activate many phosphorylase kinase molecules). This causes the production of many active glycogen phosphorylase a molecules that can degrade glycogen. 5.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5. Phosphorylated state maintenance: The phosphate groups added to phosphorylase kinase and phosphorylase in response to cAMP are maintained because the enzyme that hydrolytically removes the phosphate, protein phosphatase-1 (PP1), is inactivated by inhibitor proteins that are also phosphorylated and activated in response to cAMP (see Fig. 11.9). [Note: PP1 is activated by a signal cascade initiated by insulin (see Fig. 27.7 on p. 311). Because insulin also activates the phosphodiesterase that degrades cAMP, it opposes the effects of glucagon and epinephrine.] B. Covalent inhibition of glycogenesis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Covalent inhibition of glycogenesis The regulated enzyme in glycogenesis, glycogen synthase, also exists in two forms, the active “a” form and the inactive “b” form. However, in contrast to phosphorylase kinase and phosphorylase, the active form of glycogen synthase is dephosphorylated, whereas the inactive form is phosphorylated at several sites on the enzyme, with the level of inactivation proportional to the degree of phosphorylation (Fig. 11.10). Phosphorylation is catalyzed by several different protein kinases in response to cAMP (for example, PKA and phosphorylase kinase) or other signaling mechanisms (see C. below). Glycogen synthase b can be reconverted to the “a” form by PP1. Figure 11.11 summarizes the covalent regulation of glycogen metabolism. = regulatory subunit; C = catalytic subunit; ADP = adenosine diphosphate. C. Allosteric regulation of glycogenesis and glycogenolysis


Biochemistry_Lippinco

Biochemistry_Lippinco. In addition to hormonal signals, glycogen synthase and glycogen phosphorylase respond to the levels of metabolites and energy needs of the cell. Glycogenesis is stimulated when glucose and energy levels are high, whereas glycogenolysis is increased when glucose and energy levels are low. This allosteric regulation allows a rapid response to the needs of a cell and can override the effects of hormone-mediated covalent regulation. [Note: The “a” and “b” forms of the allosteric enzymes of glycogen metabolism are each in an equilibrium between the R (relaxed, more active) and T (tense, less active) conformations (see p. 28). The binding of effectors shifts the equilibrium and alters enzymic activity without directly altering the covalent modification.] 1. Regulation in the well-fed state: In the well-fed state, glycogen synthase b in both liver and muscle is allosterically activated by glucose 6phosphate, which is present in elevated concentrations (Fig. 11.12). In contrast, glycogen


Biochemistry_Lippinco

Biochemistry_Lippinco. In the well-fed state, glycogen synthase b in both liver and muscle is allosterically activated by glucose 6phosphate, which is present in elevated concentrations (Fig. 11.12). In contrast, glycogen phosphorylase a is allosterically inhibited by glucose 6-phosphate, as well as by ATP, a high-energy signal. [Note: In liver, but not muscle, free glucose is also an allosteric inhibitor of glycogen phosphorylase a.] (A) and muscle (B). P = phosphate; AMP = adenosine monophosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Glycogenolysis activation by AMP: Muscle glycogen phosphorylase (myophosphorylase), but not the liver isozyme, is active in the presence of the high AMP concentrations that occur under extreme conditions of anoxia and ATP depletion. AMP binds to glycogen phosphorylase b, causing its activation without phosphorylation (see Fig. 11.9). [Note: Recall that AMP also activates phosphofructokinase-1 of glycolysis (see p. 99), allowing glucose from glycogenolysis to be oxidized.] 3. Glycogenolysis activation by calcium: Calcium (Ca2+) is released into the sarcoplasm in muscle cells (myocytes) in response to neural stimulation and in the liver in response to epinephrine binding to α1 adrenergic receptors. The Ca2+ binds to calmodulin (CaM), the most widely distributed member of a family of small, Ca2+-binding proteins. The binding of four molecules of Ca2+ to CaM triggers a conformational change such that the activated Ca2+–CaM complex binds to and activates protein molecules, often


Biochemistry_Lippinco

Biochemistry_Lippinco. of small, Ca2+-binding proteins. The binding of four molecules of Ca2+ to CaM triggers a conformational change such that the activated Ca2+–CaM complex binds to and activates protein molecules, often enzymes, that are inactive in the absence of this complex (Fig. 11.13). Thus, CaM functions as an essential subunit of many complex proteins. One such protein is the tetrameric phosphorylase kinase, whose “b” form is activated by the binding of Ca2+ to its δ subunit (CaM) without the need for the kinase to be phosphorylated by PKA. [Note: Epinephrine at β-adrenergic receptors signals through a rise in cAMP, not Ca2+ (see p. 131).] a.


Biochemistry_Lippinco

Biochemistry_Lippinco. Muscle phosphorylase kinase activation: During muscle contraction, there is a rapid and urgent need for ATP. It is supplied by the degradation of muscle glycogen to glucose 6-phosphate, which enters glycolysis. Nerve impulses cause membrane depolarization, which promotes Ca2+ release from the sarcoplasmic reticulum into the sarcoplasm of myocytes. The Ca2+ binds the CaM subunit, and the complex activates muscle phosphorylase kinase b (see Fig. 11.9). b. Liver phosphorylase kinase activation: During physiologic stress, epinephrine is released from the adrenal medulla and signals the need for blood glucose. This glucose initially comes from hepatic glycogenolysis. Binding of epinephrine to hepatocyte α1-adrenergic


Biochemistry_Lippinco

Biochemistry_Lippinco. GPCR activates a phospholipid-dependent cascade (see p. 205) that results in movement of Ca2+ from the ER into the cytoplasm. A Ca2+– CaM complex forms and activates hepatic phosphorylase kinase b. [Note: The released Ca2+ also helps to activate protein kinase C that can phosphorylate (therefore, inactivate) glycogen synthase a.] VI. GLYCOGEN STORAGE DISEASES


Biochemistry_Lippinco

Biochemistry_Lippinco. VI. GLYCOGEN STORAGE DISEASES GSD are a group of genetic diseases caused by defects in enzymes required for glycogen degradation or, more rarely, glycogen synthesis. They result either in formation of glycogen that has an abnormal structure or in the accumulation of excessive amounts of normal glycogen in specific tissues as a result of impaired degradation. A particular enzyme may be defective in a single tissue, such as the liver (resulting in hypoglycemia) or muscle (causing muscle weakness), or the defect may be more generalized, affecting a variety of tissues, such as the heart and kidneys. Severity ranges from fatal in early childhood to mild disorders that are not life threatening. Some of the more prevalent GSD are illustrated in Figure 11.8. [Note: Only GSD II is lysosomal because glycogen metabolism occurs primarily in the cytosol.] VII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. The main stores of glycogen in the body are found in skeletal muscle, where they serve as a fuel reserve for the synthesis of ATP during muscle contraction, and in the liver, where they are used to maintain the blood glucose concentration, particularly during the early stages of a fast. Glycogen is a highly branched polymer of α-D-glucose. The primary glycosidic bond is an α(1→4) linkage. After about 8–14 glucosyl residues, there is a branch containing an α(1→6) linkage. Uridine diphosphate (UDP)-glucose, the building block of glycogen, is synthesized from glucose 1-phosphate and UTP by UDP–glucose pyrophosphorylase (Fig. 11.14). Glucose from UDP-glucose is transferred to the nonreducing ends of glycogen chains by primer-requiring glycogen synthase, which makes the α(1→4) linkages. The primer is made by glycogenin. Branches are formed by amylo-α(1→4)→α(1→6)-transglycosylase (a 4:6 transferase), which transfers a set of six to eight glucosyl residues from the nonreducing end of the


Biochemistry_Lippinco

Biochemistry_Lippinco. primer is made by glycogenin. Branches are formed by amylo-α(1→4)→α(1→6)-transglycosylase (a 4:6 transferase), which transfers a set of six to eight glucosyl residues from the nonreducing end of the glycogen chain (breaking an α(1→4) linkage), and making an α(1→6) linkage to another residue in the chain. Pyridoxal phosphate–requiring glycogen phosphorylase cleaves the α(1→4) bonds between glucosyl residues at the nonreducing ends of the glycogen chains, producing glucose 1-phosphate. This sequential degradation continues until four glucosyl units remain before a branch point. The resulting structure is called a limit dextrin that is degraded by the bifunctional debranching enzyme. Oligo-α(1→4)→α(1→4)-glucantransferase (a 4:4 transferase) activity removes the outer three of the four glucosyl residues at a branch and transfers them to the nonreducing end of another chain, where they can be released as glucose 1-phosphate by glycogen phosphorylase. The remaining single glucose residue


Biochemistry_Lippinco

Biochemistry_Lippinco. residues at a branch and transfers them to the nonreducing end of another chain, where they can be released as glucose 1-phosphate by glycogen phosphorylase. The remaining single glucose residue attached in an α(1→6) linkage is removed hydrolytically by the amylo-α(1→6) glucosidase activity of debranching enzyme, releasing free glucose. Glucose 1-phosphate is converted to glucose 6-phosphate by phosphoglucomutase. In muscle, glucose 6phosphate enters glycolysis. In liver, the phosphate is removed by glucose 6-phosphatase (an enzyme of the endoplasmic reticular membrane), releasing free glucose that can be used to maintain blood glucose levels at the beginning of a fast. A deficiency of the phosphatase causes glycogen storage disease Ia (von Gierke disease) and results in an inability of the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body


Biochemistry_Lippinco

Biochemistry_Lippinco. the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body needs by the same hormonal signals (namely, an elevated insulin level results in overall increased glycogenesis and decreased glycogenolysis, whereas an elevated glucagon, or epinephrine, level causes the opposite effects). Key enzymes are phosphorylated by a family of protein kinases, some of which are dependent on cyclic adenosine monophosphate (cAMP), a compound increased by glucagon and epinephrine. Phosphate groups are removed by protein phosphatase-1 (active when its inhibitor is inactive in response to elevated insulin levels). In addition to this covalent regulation, glycogen synthase, phosphorylase kinase, and phosphorylase are allosterically regulated to meet tissues’ needs. In the well-fed state, glycogen synthase is activated by glucose 6-phosphate, but glycogen phosphorylase


Biochemistry_Lippinco

Biochemistry_Lippinco. phosphorylase kinase, and phosphorylase are allosterically regulated to meet tissues’ needs. In the well-fed state, glycogen synthase is activated by glucose 6-phosphate, but glycogen phosphorylase is inhibited by glucose 6-phosphate as well as by ATP. In the liver, free glucose also serves as an allosteric inhibitor of glycogen phosphorylase. The rise in calcium in muscle during exercise and in liver in response to epinephrine activates phosphorylase kinase by binding to the enzyme’s calmodulin subunit. This allows the enzyme to activate glycogen phosphorylase, thereby causing glycogen degradation. AMP activates glycogen phosphorylase (myophosphorylase) in muscle.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. For Questions 11.1–11.4, match the deficient enzyme to the clinical finding in selected glycogen storage diseases (GSD). 1.1. Exercise intolerance, with no rise in blood lactate during exercise Correct answer = E. Myophosphorylase (the muscle isozyme of glycogen phosphorylase) deficiency (or, McArdle disease) prevents glycogen degradation in muscle, depriving muscle of glycogen-derived glucose, resulting in decreased glycolysis and its anaerobic product, lactate. Correct answer = D. 4:6 Transferase (branching enzyme) deficiency (or, Andersen disease), a defect in glycogen synthesis, results in glycogen with fewer branches and decreased solubility. Correct answer = B. Acid maltase [acid α(1→4)-glucosidase] deficiency (or, Pompe disease) prevents degradation of any glycogen brought into lysosomes. A variety of tissues are affected, with the most severe pathology resulting from heart damage.


Biochemistry_Lippinco

Biochemistry_Lippinco. Correct answer = A. Glucose 6-phosphatase deficiency (or, von Gierke disease) prevents the liver from releasing free glucose into the blood, causing severe fasting hypoglycemia, lactic acidemia, hyperuricemia, and hyperlipidemia. 1.2. Fatal, progressive cirrhosis and glycogen with longer-than-normal outer chains 1.3. Generalized accumulation of glycogen, severe hypotonia, and death from heart failure 1.4. Severe fasting hypoglycemia, lactic acidemia, hyperuricemia, and hyperlipidemia 1.5. Epinephrine and glucagon have which one of the following effects on hepatic glycogen metabolism? A. Both glycogen phosphorylase and glycogen synthase are activated by phosphorylation but at significantly different rates. B. Glycogen phosphorylase is inactivated by the resulting rise in calcium, whereas glycogen synthase is activated. C. Glycogen phosphorylase is phosphorylated and active, whereas glycogen synthase is phosphorylated and inactive. D. The net synthesis of glycogen is increased.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Glycogen phosphorylase is phosphorylated and active, whereas glycogen synthase is phosphorylated and inactive. D. The net synthesis of glycogen is increased. Correct answer = C. Epinephrine and glucagon both cause increased glycogen degradation and decreased synthesis in the liver through covalent modification (phosphorylation) of key enzymes of glycogen metabolism. Glycogen phosphorylase is phosphorylated and active (“a” form), whereas glycogen synthase is phosphorylated and inactive (“b” form). Glucagon does not cause a rise in calcium. 1.6. In contracting skeletal muscle, a sudden elevation of the sarcoplasmic calcium concentration will result in: A. activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A. B. conversion of cAMP to AMP by phosphodiesterase. C. direct activation of glycogen synthase b. D. direct activation of phosphorylase kinase b. E. inactivation of phosphorylase kinase a by the action of protein phosphatase-1.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. direct activation of glycogen synthase b. D. direct activation of phosphorylase kinase b. E. inactivation of phosphorylase kinase a by the action of protein phosphatase-1. Correct answer = D. Calcium (Ca2+) released from the sarcoplasmic reticulum during exercise binds to the calmodulin subunit of phosphorylase kinase, thereby allosterically activating the dephosphorylated “b” form of this enzyme. The other choices are not caused by an elevation of cytosolic Ca2+ . [Note: Ca2+ also activates hepatic phosphorylase kinase b.] 1.7. Explain why the hypoglycemia seen with type Ia glycogen storage disease (glucose 6-phosphatase deficiency) is severe, whereas that seen with type VI (liver phosphorylase deficiency) is mild.


Biochemistry_Lippinco

Biochemistry_Lippinco. With type Ia, the liver is unable to generate free glucose either from glycogenolysis or gluconeogenesis because both processes produce glucose 6phosphate. With type VI, the liver is still able to produce free glucose from gluconeogenesis, but glycogenolysis is inhibited. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Glucose is the most common monosaccharide consumed by humans, and its metabolism has already been discussed. Two other monosaccharides, fructose and galactose, also occur in significant amounts in the diet (primarily in disaccharides) and make important contributions to energy metabolism. In addition, galactose is an important component of glycosylated proteins. Figure 12.1 shows the metabolism of fructose and galactose as part of the essential pathways of energy metabolism. II. FRUCTOSE METABOLISM


Biochemistry_Lippinco

Biochemistry_Lippinco. II. FRUCTOSE METABOLISM About 10% of the calories in the Western diet are supplied by fructose (~55 g/day). The major source of fructose is the disaccharide sucrose, which, when cleaved in the intestine, releases equimolar amounts of fructose and glucose. Fructose is also found as a free monosaccharide in many fruits, in honey, and in high-fructose corn syrup (typically, 55% fructose and 45% glucose), which is used to sweeten soft drinks and many foods (see p. 364). Fructose transport into cells is not insulin dependent (unlike that of glucose into certain tissues; see p. 97), and, in contrast to glucose, fructose does not promote the secretion of insulin. A. Phosphorylation


Biochemistry_Lippinco

Biochemistry_Lippinco. For fructose to enter the pathways of intermediary metabolism, it must first be phosphorylated (Fig. 12.2). This can be accomplished by either hexokinase or fructokinase. Hexokinase phosphorylates glucose in most cells of the body (see p. 98), and several additional hexoses can serve as substrates for this enzyme. However, it has a low affinity (that is, a high Michaelis constant [Km]; see p. 59) for fructose. Therefore, unless the intracellular concentration of fructose becomes unusually high, the normal presence of saturating concentrations of glucose means that little fructose is phosphorylated by hexokinase. Fructokinase provides the primary mechanism for fructose phosphorylation (see Fig. 12.2). The enzyme has a low Km for fructose and a high Vmax ([maximal velocity] see p. 57). It is found in the liver (which processes most of the dietary fructose), kidneys, and the small intestine and converts fructose to fructose 1-phosphate, using ATP as the phosphate donor. [Note: These


Biochemistry_Lippinco

Biochemistry_Lippinco. It is found in the liver (which processes most of the dietary fructose), kidneys, and the small intestine and converts fructose to fructose 1-phosphate, using ATP as the phosphate donor. [Note: These three tissues also contain aldolase B, discussed in section B.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Fructose 1-phosphate cleavage Fructose 1-phosphate is not phosphorylated to fructose 1,6-bisphosphate as is fructose 6-phosphate (see p. 99) but is cleaved by aldolase B (also called fructose 1-phosphate aldolase) to two trioses, dihydroxyacetone phosphate (DHAP) and glyceraldehyde. [Note: Humans express three aldolase isozymes (the products of three different genes): aldolase A in most tissues; aldolase B in the liver, kidneys, and small intestine; and aldolase C in the brain. All cleave fructose 1,6-bisphosphate produced during glycolysis to DHAP and glyceraldehyde 3-phosphate (see p. 101), but only aldolase B cleaves fructose 1-phosphate.] DHAP can be used in glycolysis or gluconeogenesis, whereas glyceraldehyde can be metabolized by a number of pathways, as illustrated in Figure 12.3. C. Kinetics


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Kinetics The rate of fructose metabolism is more rapid than that of glucose because triose production from fructose 1-phosphate bypasses phosphofructokinase-1, the major rate-limiting step in glycolysis (see p. 99). D. Disorders


Biochemistry_Lippinco

Biochemistry_Lippinco. A deficiency of one of the key enzymes required for the entry of fructose into metabolic pathways can result in either a benign condition as a result of fructokinase deficiency (essential fructosuria) or a severe disturbance of liver and kidney metabolism as a result of aldolase B deficiency (hereditary fructose intolerance [HFI]), which occurs in ~1:20,000 live births (see Fig. 12.3). The first symptoms of HFI appear when a baby is weaned from lactose-containing milk and begins ingesting food containing sucrose or fructose. Fructose 1-phosphate accumulates, resulting in a drop in the level of inorganic phosphate (Pi) and, therefore, of ATP production. As ATP falls, adenosine monophosphate (AMP) rises. The AMP is degraded, causing hyperuricemia (and lactic acidemia; see p. 299). The decreased availability of hepatic ATP decreases gluconeogenesis (causing hypoglycemia with vomiting) and protein synthesis (causing a decrease in blood-clotting factors and other essential proteins). Renal


Biochemistry_Lippinco

Biochemistry_Lippinco. availability of hepatic ATP decreases gluconeogenesis (causing hypoglycemia with vomiting) and protein synthesis (causing a decrease in blood-clotting factors and other essential proteins). Renal reabsorption of Pi is also decreased. [Note: The drop in Pi also inhibits glycogenolysis (see p. 128).] Diagnosis of HFI can be made on the basis of fructose in the urine, enzyme assay using liver cells, or by DNA-based testing (see Chapter 34). With HFI, sucrose, as well as fructose, must be removed from the diet to prevent liver failure and possible death. [Note: Individuals with HFI display an aversion to sweets and, consequently, have an absence of dental caries.]


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Mannose conversion to fructose 6-phosphate Mannose, the C-2 epimer of glucose (see p. 84), is an important component of glycoproteins (see p. 166). Hexokinase phosphorylates mannose, producing mannose 6-phosphate, which, in turn, is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. [Note: Most intracellular mannose is synthesized from fructose or is preexisting mannose produced by the degradation of glycoproteins and salvaged by hexokinase. Dietary carbohydrates contain little mannose.] F. Glucose conversion to fructose via sorbitol Most sugars are rapidly phosphorylated following their entry into cells. Therefore, they are trapped within the cells, because organic phosphates cannot freely cross membranes without specific transporters. An alternate mechanism for metabolizing a monosaccharide is to convert it to a polyol (sugar alcohol) by the reduction of an aldehyde group, thereby producing an additional hydroxyl group.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Sorbitol synthesis: Aldose reductase reduces glucose, producing sorbitol (or, glucitol; Fig. 12.4), but the Km is high. This enzyme is found in many tissues, including the retina, lens, kidneys, peripheral nerves, ovaries, and seminal vesicles. A second enzyme, sorbitol dehydrogenase, can oxidize sorbitol to fructose in cells of the liver, ovaries, and seminal vesicles (see Fig. 12.4). The two-reaction pathway from glucose to fructose in the seminal vesicles benefits sperm cells, which use fructose as a major carbohydrate energy source. The pathway from sorbitol to fructose in the liver provides a mechanism by which any available sorbitol is converted into a substrate that can enter glycolysis.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Hyperglycemia and sorbitol metabolism: Because insulin is not required for the entry of glucose into cells of the retina, lens, kidneys, and peripheral nerves, large amounts of glucose may enter these cells during times of hyperglycemia (for example, in uncontrolled diabetes). Elevated intracellular glucose concentrations and an adequate supply of reduced nicotinamide adenine dinucleotide phosphate (NADPH) cause aldose reductase to produce a significant increase in the amount of sorbitol, which cannot pass efficiently through cell membranes and, therefore, remains trapped inside the cell (see Fig. 12.4). This is exacerbated when sorbitol dehydrogenase is low or absent (for example, in cells of the retina, lens, kidneys, and peripheral nerves). As a result, sorbitol accumulates in these cells, causing strong osmotic effects and cell swelling due to water influx and retention. Some of the pathologic alterations associated with diabetes can be partly attributed to this osmotic stress,


Biochemistry_Lippinco

Biochemistry_Lippinco. cells, causing strong osmotic effects and cell swelling due to water influx and retention. Some of the pathologic alterations associated with diabetes can be partly attributed to this osmotic stress, including cataract formation, peripheral neuropathy, and microvascular problems leading to nephropathy and retinopathy (see p. 345). [Note: Use of NADPH in the aldose reductase reaction decreases the generation of reduced glutathione, an important antioxidant (see p. 148), and may be related to diabetic complications.]


Biochemistry_Lippinco

Biochemistry_Lippinco. III. GALACTOSE METABOLISM The major dietary source of galactose is lactose (galactosyl β-1,4-glucose) obtained from milk and milk products. [Note: The digestion of lactose by βgalactosidase (lactase) of the intestinal mucosal cell membrane was discussed on p. 87.] Some galactose can also be obtained by lysosomal degradation of glycoproteins and glycolipids. Like fructose (and mannose), the transport of galactose into cells is not insulin dependent. A. Phosphorylation Like fructose, galactose must be phosphorylated before it can be further metabolized. Most tissues have a specific enzyme for this purpose, galactokinase, which produces galactose 1-phosphate (Fig. 12.5). As with other kinases, ATP is the phosphate donor. B. Uridine diphosphate–galactose formation


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Uridine diphosphate–galactose formation Galactose 1-phosphate cannot enter the glycolytic pathway unless it is first converted to uridine diphosphate (UDP)-galactose (Fig. 12.6). This occurs in an exchange reaction, in which UDP-glucose reacts with galactose 1phosphate, producing UDP-galactose and glucose 1-phosphate (see Fig. 12.5). The reaction is catalyzed by galactose 1-phosphate uridylyltransferase (GALT). [Note: The glucose 1-phosphate product can be isomerized to glucose 6-phosphate, which can enter glycolysis or be dephosphorylated.] C. UDP-galactose conversion to UDP-glucose For UDP-galactose to enter the mainstream of glucose metabolism, it must first be isomerized to its C-4 epimer, UDP-glucose, by UDP-hexose 4epimerase. This “new” UDP-glucose (produced from the original UDPgalactose) can participate in biosynthetic reactions (for example, glycogenesis) as well as in the GALT reaction. [Note: See Fig. 12.5 for a summary of the interconversions.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. UDP-galactose in biosynthetic reactions UDP-galactose can serve as the donor of galactose units in a number of synthetic pathways, including synthesis of lactose (see IV. below), glycoproteins (see p. 166), glycolipids (see p. 210), and glycosaminoglycans (see p. 158). [Note: If galactose is not provided by the diet (for example, when it cannot be released from lactose owing to a lack of β-galactosidase in people who are lactose intolerant), all tissue requirements for UDP-galactose can be met by the action of UDP-hexose 4-epimerase on UDP-glucose, which is efficiently produced from glucose 1-phosphate and uridine triphosphate (see Fig. 12.5).] E. Disorders


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Disorders GALT is severely deficient in individuals with classic galactosemia (see Fig. 12.5). In this disorder, galactose 1-phosphate and, therefore, galactose accumulate. Physiologic consequences are similar to those found in HFI (see p. 138), but a broader spectrum of tissues is affected. The accumulated galactose is shunted into side pathways such as that of galactitol production. This reaction is catalyzed by aldose reductase, the same enzyme that reduces glucose to sorbitol (see p. 139). GALT deficiency is part of the newborn screening panel. Treatment of galactosemia requires removal of galactose and lactose from the diet. [Note: Deficiencies in galactokinase and the epimerase result in less severe disorders of galactose metabolism, although cataracts are common (see Fig. 12.5).] IV. LACTOSE SYNTHESIS


Biochemistry_Lippinco

Biochemistry_Lippinco. Lactose is a disaccharide that consists of a molecule of β-galactose attached by a β(1→4) linkage to glucose. Therefore, lactose is galactosyl β(1→4)-glucose. Because lactose (milk sugar) is made by lactating (milk-producing) mammary glands, milk and other dairy products are the dietary sources of lactose. Lactose is synthesized in the Golgi by lactose synthase (UDP-galactose:glucose galactosyltransferase), which transfers galactose from UDP-galactose to glucose, releasing UDP (Fig. 12.7). This enzyme is composed of two proteins, A and B. Protein A is a β-D-galactosyltransferase and is found in a number of body tissues. In tissues other than the lactating mammary gland, this enzyme transfers galactose from UDP-galactose to N-acetyl-D-glucosamine, forming the same β(1→4) linkage found in lactose, and producing N-acetyllactosamine, a component of the structurally important N-linked glycoproteins (see p. 167). In contrast, protein B is found only in lactating mammary glands. It is


Biochemistry_Lippinco

Biochemistry_Lippinco. in lactose, and producing N-acetyllactosamine, a component of the structurally important N-linked glycoproteins (see p. 167). In contrast, protein B is found only in lactating mammary glands. It is αlactalbumin, and its synthesis is stimulated by the peptide hormone prolactin. Protein B forms a complex with the enzyme, protein A, changing the specificity of that transferase (by decreasing the Km for glucose) so that lactose, rather than


Biochemistry_Lippinco

Biochemistry_Lippinco. N-acetyllactosamine, is produced (see Fig. 12.7). V. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. The major source of fructose is the disaccharide sucrose, which, when cleaved, releases equimolar amounts of fructose and glucose (Fig. 12.8). Transport of fructose into cells is insulin independent. Fructose is first phosphorylated to fructose 1-phosphate by fructokinase and then cleaved by aldolase B to dihydroxyacetone phosphate and glyceraldehyde. These enzymes are found in the liver, kidneys, and small intestine. A deficiency of fructokinase causes a benign condition (essential fructosuria), whereas a deficiency of aldolase B causes hereditary fructose intolerance (HFI), in which severe hypoglycemia and liver failure lead to death if fructose (and sucrose) is not removed from the diet. Mannose, an important component of glycoproteins, is phosphorylated by hexokinase to mannose 6phosphate, which is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. Glucose can be reduced to sorbitol (glucitol) by aldose reductase in many tissues, including the lens, retina,


Biochemistry_Lippinco

Biochemistry_Lippinco. which is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. Glucose can be reduced to sorbitol (glucitol) by aldose reductase in many tissues, including the lens, retina, peripheral nerves, kidneys, ovaries, and seminal vesicles. In the liver, ovaries, and seminal vesicles, a second enzyme, sorbitol dehydrogenase, can oxidize sorbitol to produce fructose. Hyperglycemia results in the accumulation of sorbitol in those cells lacking sorbitol dehydrogenase. The resulting osmotic events cause cell swelling and may contribute to the cataract formation, peripheral neuropathy, nephropathy, and retinopathy seen in diabetes. The major dietary source of galactose is lactose. The transport of galactose into cells is insulin independent. Galactose is first phosphorylated by galactokinase (a deficiency results in cataracts) to galactose 1phosphate. This compound is converted to uridine diphosphate (UDP)galactose by galactose 1-phosphate uridylyltransferase (GALT), with the


Biochemistry_Lippinco

Biochemistry_Lippinco. (a deficiency results in cataracts) to galactose 1phosphate. This compound is converted to uridine diphosphate (UDP)galactose by galactose 1-phosphate uridylyltransferase (GALT), with the nucleotide supplied by UDP-glucose. A deficiency of this enzyme causes classic galactosemia. Galactose 1-phosphate accumulates, and excess galactose is converted to galactitol by aldose reductase. This causes liver damage, brain damage, and cataracts. Treatment requires removal of galactose (and lactose) from the diet. For UDP-galactose to enter the mainstream of glucose metabolism, it must first be isomerized to UDP-glucose by UDP-hexose 4-epimerase. This enzyme can also be used to produce UDP-galactose from UDP-glucose when the former is required for glycoprotein and glycolipid synthesis. Lactose is a disaccharide of galactose and glucose. Milk and other dairy products are the dietary sources of lactose. Lactose is synthesized by lactose synthase from UDP-galactose and glucose in the lactating


Biochemistry_Lippinco

Biochemistry_Lippinco. a disaccharide of galactose and glucose. Milk and other dairy products are the dietary sources of lactose. Lactose is synthesized by lactose synthase from UDP-galactose and glucose in the lactating mammary gland. The enzyme has two subunits, protein A (which is a galactosyltransferase found in most cells where it synthesizes N-acetyllactosamine) and protein B (α-lactalbumin, which is found only in lactating mammary glands, and whose synthesis is stimulated by the peptide hormone prolactin). When both subunits are present, the transferase produces lactose.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 2.1. A nursing female with classic galactosemia is on a galactose-free diet. She is able to produce lactose in breast milk because: A. galactose can be produced from fructose by isomerization. B. galactose can be produced from a glucose metabolite by epimerization. C. hexokinase can efficiently phosphorylate galactose to galactose 1phosphate. D. the enzyme affected in galactosemia is activated by a hormone produced in the mammary gland. Correct answer = B. Uridine diphosphate (UDP)-glucose is converted to UDPgalactose by UDP-hexose 4-epimerase, thereby providing the appropriate form of galactose for lactose synthesis. Isomerization of fructose to galactose does not occur in the human body. Galactose is not converted to galactose 1phosphate by hexokinase. A galactose-free diet provides no galactose. Galactosemia is the result of an enzyme (galactose 1-phosphate uridylyltransferase) deficiency.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.2. A 5-month-old boy is brought to his physician because of vomiting, night sweats, and tremors. History revealed that these symptoms began after fruit juices were introduced to his diet as he was being weaned off breast milk. The physical examination was remarkable for hepatomegaly. Tests on the baby’s urine were positive for reducing sugar but negative for glucose. The infant most likely suffers from a deficiency of: A. aldolase B. B. fructokinase. C. galactokinase. D. β-galactosidase.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. aldolase B. B. fructokinase. C. galactokinase. D. β-galactosidase. Correct answer = A. The symptoms suggest hereditary fructose intolerance, a deficiency in aldolase B. Deficiencies in fructokinase or galactokinase result in relatively benign conditions characterized by elevated levels of fructose or galactose in the blood and urine. Deficiency in β-galactosidase (lactase) results in a decreased ability to degrade lactose (milk sugar). Congenital lactase deficiency is quite rare and would have presented much earlier in this baby (and with different symptoms). Typical lactase deficiency (adult hypolactasia) presents at a later age. 2.3. Lactose synthesis is essential in the production of milk by mammary glands. In lactose synthesis: A. galactose from galactose 1-phosphate is transferred to glucose by galactosyltransferase (protein A), generating lactose. B. protein A is used exclusively in lactose synthesis.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. galactose from galactose 1-phosphate is transferred to glucose by galactosyltransferase (protein A), generating lactose. B. protein A is used exclusively in lactose synthesis. C. α-lactalbumin (protein B) regulates the specificity of protein A by decreasing its affinity for glucose. D. protein B expression is stimulated by prolactin. Correct answer = D. α-Lactalbumin (protein B) expression is increased by the hormone prolactin. Uridine diphosphate–galactose is the form used by the galactosyltransferase (protein A). Protein A is also involved in the synthesis of the amino sugar N-acetyllactosamine. Protein B decreases the Michaelis constant (Km) and, so, increases the affinity of protein A for glucose.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.4. A 3-month-old girl is developing cataracts. Other than not having a social smile or being able to track objects visually, all other aspects of the girl’s examination are normal. Tests on the baby’s urine are positive for reducing sugar but negative for glucose. Which enzyme is most likely deficient in this girl? A. Aldolase B B. Fructokinase C. Galactokinase D. Galactose 1-phosphate uridylyltransferase


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Aldolase B B. Fructokinase C. Galactokinase D. Galactose 1-phosphate uridylyltransferase Correct answer = C. The girl is deficient in galactokinase and is unable to appropriately phosphorylate galactose. Galactose accumulates in the blood (and urine). In the lens of the eye, galactose is reduced by aldose reductase to galactitol, a sugar alcohol, which causes osmotic effects that result in cataract formation. Deficiency of galactose 1-phosphate uridylyltransferase also results in cataracts but is characterized by liver damage and neurologic effects. Fructokinase deficiency is a benign condition. Aldolase B deficiency is severe, with effects on several tissues. Cataracts are not typically seen. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. I. OVERVIEW The pentose phosphate pathway (or, hexose monophosphate shunt) occurs in the cytosol. It includes an irreversible oxidative phase, followed by a series of reversible sugar–phosphate interconversions (Fig. 13.1). In the oxidative phase, carbon 1 of a glucose 6-phosphate molecule is released as carbon dioxide (CO2), and one pentose sugar-phosphate plus two reduced nicotinamide adenine dinucleotide phosphates (NADPH) are produced. The rate and direction of the reversible reactions are determined by the supply of and demand for intermediates of the pathway. The pentose phosphate pathway provides a major portion of the body’s NADPH, which functions as a biochemical reductant. It also produces ribose 5-phosphate, required for nucleotide biosynthesis (see p. 293), and provides a mechanism for the conversion of pentose sugars to triose and hexose intermediates of glycolysis. No ATP is directly consumed or produced in the pathway. II. IRREVERSIBLE OXIDATIVE REACTIONS


Biochemistry_Lippinco

Biochemistry_Lippinco. II. IRREVERSIBLE OXIDATIVE REACTIONS The oxidative portion of the pentose phosphate pathway consists of three irreversible reactions that lead to the formation of ribulose 5-phosphate, CO2, and two molecules of NADPH for each molecule of glucose 6-phosphate oxidized (Fig. 13.2). This portion of the pathway is particularly important in the liver, lactating mammary glands, and adipose tissue for the NADPH-dependent biosynthesis of fatty acids (see p. 186); in the testes, ovaries, placenta, and adrenal cortex for the NADPH-dependent biosynthesis of steroid hormones (see p. 237); and in red blood cells (RBC) for the NADPH-dependent reduction of glutathione (see p. 148). A. Glucose 6-phosphate dehydrogenation


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Glucose 6-phosphate dehydrogenation Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconolactone as the coenzyme NADP+ gets reduced to NADPH. This initial reaction is the committed, rate-limiting, and regulated step of the pathway. NADPH is a potent competitive inhibitor of G6PD, and the ratio of NADPH/NADP+ is sufficiently high to substantially inhibit the enzyme under most metabolic conditions. However, with increased demand for NADPH, the ratio of NADPH/NADP+ decreases, and flux through the pathway increases in response to the enhanced activity of G6PD. [Note: Insulin upregulates expression of the gene for G6PD, and flux through the pathway increases in the absorptive state (see p. 323).]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Ribulose 5-phosphate formation 6-Phosphogluconolactone is hydrolyzed by 6-phosphogluconolactone hydrolase in the second step. The oxidative decarboxylation of the product, 6-phosphogluconate, is catalyzed by 6-phosphogluconate dehydrogenase. This third irreversible step produces ribulose 5-phosphate (a pentose sugar– phosphate), CO2 (from carbon 1 of glucose), and a second molecule of NADPH (see Fig. 13.2). III. REVERSIBLE NONOXIDATIVE REACTIONS