Biochemistry, 4th Edition P71 pot

22.1 What Is Gluconeogenesis, and How Does It Operate? 663 esis are the liver and kidneys, which account for about 90% and 10% of the body’s gluconeogenic activity, respectively. Glucose produced by gluconeogenesis in the liver and kidneys is released into the blood and is subsequently absorbed by brain, heart, muscle, and red blood cells to meet their metabolic needs. In turn, pyruvate and lactate produced in these tissues are returned to the liver and kidneys to be used as gluconeogenic substrates. Gluconeogenesis Is Not Merely the Reverse of Glycolysis In some ways, gluconeogenesis is the reverse, or antithesis, of glycolysis. Glucose is synthesized, not catabolized; ATP is consumed, not produced; and NADH is oxidized to NAD ϩ , rather than the other way around. However, gluconeogenesis cannot be merely the reversal of glycolysis, for two reasons. First, glycolysis is exergonic, with a ⌬G°Ј of approximately Ϫ74 kJ/mol. If gluconeogenesis were merely the reverse, it would be a strongly endergonic process and could not occur spontaneously. Somehow the energetics of the process must be augmented so that gluconeogenesis can proceed spontaneously. Second, the processes of glycolysis and gluconeogenesis must be regulated in a reciprocal fashion so that when glycolysis is active, gluconeogenesis is inhibited, and when gluconeogenesis is proceeding, glycolysis is turned off. Both of these limitations are overcome by having unique reactions within the routes of glycolysis and gluconeogenesis, rather than a com- pletely shared pathway. Gluconeogenesis—Something Borrowed, Something New The complete route of gluconeogenesis is shown in Figure 22.1, side by side with the glycolytic pathway. Gluconeogenesis employs four different reactions, catalyzed by four different enzymes, for the three steps of glycolysis that are highly exergonic HUMAN BIOCHEMISTRY The Chemistry of Glucose Monitoring Devices Individuals with diabetes must measure their serum glucose concentration frequently, often several times a day. The advent of com- puterized, automated devices for glucose monitoring has made this necessary chore easier, far more accurate, and more convenient than it once was. These devices all use a simple chemical scheme for glucose measurement that involves oxidation of glucose to gluconic acid by glucose oxidase. This reaction produces two molecules of hydrogen peroxide per molecule of glucose oxidized. The H 2 O 2 is then used to oxidize a dye, such as o-dianisidine, to a colored product that can be measured: Glucose ϩ 2 H 2 O ϩ O 2 ⎯⎯→ gluconic acid ϩ 2 H 2 O 2 o-dianisidine (colorless) ϩ H 2 O 2 ⎯⎯→ oxidized o-anisidine (colored) ϩ H 2 O The amount of colored dye produced is directly proportional to the amount of glucose in the sample. The patient typically applies a drop of blood (from a finger- prick*) to a plastic test strip that is then inserted into the glucose monitor. Within half a minute, a digital readout indicates the blood glucose value. Modern glucose monitors store several days of glucose measurements, and the data can be easily transferred to a computer for analysis and graphing. *How does the monitor deal with getting just the right amount of blood? The blood flows up an absorbent “wick” by capillary action. It is impossible to overfill this device, but the monitor will give an error signal if not enough blood flows up the strip. 664 Chapter 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway (and highly regulated). In essence, seven of the ten steps of glycolysis are merely reversed in gluconeogenesis. The six reactions between fructose-1,6-bisphosphate and PEP are shared by the two pathways, as is the isomerization of glucose-6-P to fructose- 6-P. The three exergonic, regulated reactions—the hexokinase (glucokinase), phosphofructokinase, and pyruvate kinase reactions—are replaced by alternative reactions in the gluconeogenic pathway. The conversion of pyruvate to PEP that initiates gluconeogenesis is accom- plished by two unique reactions. Pyruvate carboxylase catalyzes the first, convert- ing pyruvate to oxaloacetate. Then, PEP carboxykinase catalyzes the conversion of oxaloacetate to PEP. Conversion of fructose-1,6-bisphosphate to fructose-6- phosphate is catalyzed by a specific phosphatase, fructose-1,6-bisphosphatase. The final step to produce glucose, hydrolysis of glucose-6-phosphate, is mediated ATP ATP Glucose + Glucose-6-P Fructose-6-P H 2 O H 2 O Fructose-1,6-bisP Glyceraldehyde-3-P Dihydroxyacetone-P Glycerol 1,3-Bisphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate PEP Pyruvate Lactate Oxaloacetate Amino acids This reaction occurs in the ER Gluconeogenesis Glycolysis Mitochondrial matrix ATP ATP ATP ATP ADP P i P i P i ADPADP ADP ADP ADP GTP GDP CO 2 NAD + NADH NAD + NADH Glucose-6-phosphatase Fructose-1,6-bisphosphatase Pyruvate carboxylase PEP carboxykinase FIGURE 22.1 The pathways of gluconeogenesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate). 22.1 What Is Gluconeogenesis, and How Does It Operate? 665 by glucose-6-phosphatase. Each of these steps is considered in detail in the following paragraphs. The overall conversion of pyruvate to PEP by pyruvate carboxylase and PEP carboxykinase has a ⌬G°Ј close to zero but is pulled along by sub- sequent reactions. The conversion of fructose-1,6-bisphosphate to glucose in the last three steps of gluconeogenesis is strongly exergonic, with a ⌬G°Ј of about Ϫ30.5 kJ/mol. This sequence of two phosphatase reactions separated by an isomerization accounts for most of the free energy release that makes the gluconeogenesis pathway spontaneous. Four Reactions Are Unique to Gluconeogenesis 1. Pyruvate Carboxylase—A Biotin-Dependent Enzyme Initiation of gluconeogenesis occurs in the pyruvate carboxylase reaction—the conversion of pyruvate to oxaloacetate. The reaction takes place in two discrete steps, involves ATP and bicarbonate as substrates, and utilizes biotin as a coenzyme and acetyl-CoA as an allosteric activator. Pyruvate carboxylase is a tetrameric enzyme (with a molecular mass of about 500 kD). Each monomer possesses a biotin covalently linked to the ⑀-amino group of a lysine residue at the active site (Figure 22.2). The first step of the reaction involves nucleophilic attack of a bicarbonate oxygen at the ␥-P of ATP to form carbonylphosphate, an activated form of CO 2 , and ADP (Figure 22.3). Reaction of carbonylphosphate with biotin occurs rapidly to form N-carboxybiotin, liberating inorganic phosphate. The third step involves abstraction of a proton from the C-3 of pyruvate, forming a carbanion that can attack the carbon of N-carboxybiotin to form oxaloacetate. Pyruvate Carboxylase Is Allosterically Activated by Acetyl-Coenzyme A Two particu- larly interesting aspects of the pyruvate carboxylase reaction are (1) allosteric activation of the enzyme by acyl-CoA derivatives and (2) compartmentation of the reaction CH 3 C O + HCO 3 – + CH 2 C – OOC O COO – COO – ATP Pyruvate Bicarbonate ++ Oxaloacetate ADP P i CH 2 CH 2 CH 2 CH 2 NH C CH 2 CH 2 CH 2 CH 2 S H N N H O Lysine Biotin O E FIGURE 22.2 Covalent linkage of biotin to an active-site lysine in pyruvate carboxylase. In most organisms, pyruvate carboxylase is a homo- tetramer of 130-kD subunits, with each subunit com- posed of three functional domains named biotin carboxylase, carboxyl transferase, and biotin carboxyl carrier protein. Shown here is the biotin carboxylase domain of pyruvate carboxylase from Bacillus thermodenitrificans. (pdb id ϭ 2DZD). – O C O HO P O O O – O P O O – O P O O – O – C O P O O O – O NNH S O HN NH S – O – O – O C O – O C O – CH 2 C COO – O CH 2 C COO – O Lys Lys H CH 2 C COO – O B E Biotin Adenosine ADP P i FIGURE 22.3 A mechanism for the pyruvate carboxylase reaction. Bicarbonate must be activated for attack by the pyruvate carbanion.This activation is driven by ATP and involves formation of a carbonylphosphate intermediate—a mixed anhydride of carbonic and phosphoric acids. (Carbonylphosphate and carboxyphos- phate are synonyms.) 666 Chapter 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway in the mitochondrial matrix. The carboxylation of biotin requires the presence (at an allosteric site) of acetyl-CoA or other acylated CoA derivatives. The second half of the carboxylase reaction—the attack by pyruvate to form oxaloacetate—is not af- fected by CoA derivatives. Activation of pyruvate carboxylase by acetyl-CoA provides an important physiological regulation. Acetyl-CoA is the primary substrate for the TCA cycle, and oxaloacetate (formed by pyruvate carboxylase) is an important intermediate in both the TCA cycle and the gluconeogenesis pathway. If levels of ATP and/or acetyl-CoA (or other acyl-CoAs) are low, pyruvate is directed primarily into the TCA cycle, which eventually promotes the synthesis of ATP. If ATP and acetyl-CoA levels are high, pyruvate is converted to oxaloacetate and consumed in gluconeogenesis. Clearly, high levels of ATP and CoA derivatives are signs that energy is abundant and that metabolites will be converted to glucose (and perhaps even glycogen). If the energy status of the cell is low (in terms of ATP and CoA derivatives), pyruvate is consumed in the TCA cycle. Also, as noted in Chapter 19, pyruvate carboxylase is an important anaplerotic enzyme. Its activation by acetyl-CoA leads to oxaloacetate formation, replenishing the level of TCA cycle intermediates. Compartmentalized Pyruvate Carboxylase Depends on Metabolite Conversion and Transport The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol, in the mitochondria, or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix (Figure 22.4), where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis; see Figure 24.1). Alternatively, it may be converted directly to OAA by pyruvate carboxylase and used in gluconeogenesis. In tissues where PEP carboxykinase is found only in the mitochondria, oxaloacetate is converted to PEP, which is then transported to the cytosol for gluconeogenesis. However, in tissues that must convert some oxaloacetate to PEP in the cytosol, a problem arises. Oxaloacetate cannot be transported directly across the mitochondrial membrane. Instead, it must first be transformed into malate or aspartate for transport across the mitochondrial inner membrane (Figure 22.4). Cytosolic malate and aspartate must be reconverted to oxaloacetate before continuing along the gluconeogenic route. 2. PEP Carboxykinase The second reaction in the gluconeogenic pyruvate–PEP bypass is the conversion of oxaloacetate to PEP. Production of a high-energy metabolite such as PEP requires energy. The energetic requirements are handled in two ways here. First, the CO 2 added to pyruvate in the pyruvate carboxylase step is removed in the PEP carboxykinase reaction. Decarboxy- lation is a favorable process and helps drive the formation of the very high-energy enol phosphate in PEP. This decarboxylation drives a reaction that would otherwise be highly endergonic. Note the inherent metabolic logic in this pair of reactions: Pyruvate carboxylase consumed an ATP to drive a carboxylation so that the PEP carboxykinase could use the decarboxylation to facilitate formation of PEP. Second, another high-energy phosphate is consumed by the carboxykinase. Mammals and several other species use GTP in this reaction, rather than ATP. The use of GTP here is equivalent to the consumption of an ATP, due to the activity of the nucleoside diphosphate kinase (see Figure 19.2). The substantial free energy of hydrolysis of GTP is crucial to the synthesis of PEP in this step. The overall ⌬G for the pyruvate carboxylase and PEP carboxykinase reactions under physiological conditions in the liver O PO 2– O 3 C CH 2 C H 2 C COO – COO – COO – Oxaloacetate + PEP carboxykinase PEP ++ CO 2 GTP GDP NAD + NADH Pyruvate Malate Pyruvate Oxaloacetate Malate Oxaloacetate Gluconeogenesis NADH NAD + FIGURE 22.4 Pyruvate carboxylase is a compartmentalized reaction. Pyruvate is converted to oxaloacetate in the mitochondria. Because oxaloacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, transported to the cytosol, and then oxidized back to oxaloacetate before gluconeogenesis can continue. PEP carboxykinase from Escherichia coli with ADP (blue), pyruvate (purple), and Mg 2ϩ (pdb id ϭ 1OS1). Go to CengageNOW and click CengageInteractive to learn more about the pyruvate carboxylase reaction. 22.1 What Is Gluconeogenesis, and How Does It Operate? 667 is Ϫ22.6 kJ/mol. Once PEP is formed in this way, the phosphoglycerate mutase, phosphoglycerate kinase, glyceraldehyde-3-P dehydrogenase, aldolase, and triose phosphate isomerase reactions act to eventually form fructose-1,6-bisphosphate, as shown in Figure 22.1. 3. Fructose-1,6-Bisphosphatase The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate, like all phosphate ester hydrolyses, is a thermodynamically favorable (exergonic) reaction under standard-state conditions (⌬G°ЈϭϪ16.7 kJ/mol). Under physiological conditions in the liver, the reaction is also exergonic (⌬G ϭ Ϫ8.6 kJ/mol). Fructose-1,6-bisphosphatase is an allosterically regulated enzyme. Citrate stimulates bisphosphatase activity, but fructose-2,6-bisphosphate is a potent allosteric inhibitor. AMP also inhibits the bisphosphatase; the inhibition by AMP is enhanced by fructose-2,6-bisphosphate. 4. Glucose-6-Phosphatase The final step in the gluconeogenesis pathway is the conversion of glucose-6-phosphate to glucose by the action of glucose-6- phosphatase (Figure 22.5). This enzyme is present in the membranes of the endoplasmic reticulum of liver and kidney cells but is absent in muscle and brain. For this reason, gluconeogenesis is not carried out in muscle and brain. The glucose- 6-phosphatase system includes the phosphatase itself and three transport proteins, T1, T2, and T3. The glucose-6-phosphate transporter (T1) takes glucose-6-phosphate into the endoplasmic reticulum, where it is hydrolyzed to glucose and P i . The T2 and T3 transporters export glucose and P i , respectively, to the cytosol, and glucose is then exported (to the circulation) by the GLUT2 transporter. The glucose-6-phosphatase reaction involves a phosphorylated enzyme intermediate, phosphohistidine (Figure 22.6). The ⌬G for the glucose-6-phosphatase reaction in liver is Ϫ5.1 kJ/mol. O HHO HO H HOH O 3 POH 2 CCH 2 OPO 3 + O HHO HO H HOH O 3 POH 2 C CH 2 OH + 2 – 2 – 2 – P H 2 O Fructose-6-phosphateFructose-1,6-bisphosphate ΔGЊЈ = –16.7 kJ/mol Fructose-1,6- bisphosphatase Fructose-1,6-bisphosphatase from pig with fructose-6- phosphate (orange), AMP (blue), and Mg 2ϩ (dark blue) (pdb id ϭ 1FBP). Glucose-6-P Glucose + P i P i transporter GLUT2 trans p orter Glucose transporter Glucose-6-phosphatase Glucose-6-phosphate G-6-P transporter Cytosol ER membrane ER lumen T1 T2 T3 Plasma membrane FIGURE 22.5 Glucose-6-phosphatase is localized in the endoplasmic reticulum. Conversion of glucose-6- phosphate to glucose occurs following transport into the endoplasmic reticulum. Glucose-6-phosphatase and the three transporters,T1,T2, and T3, are known collec- tively as the glucose-6-phosphatase system. 668 Chapter 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway Coupling with Hydrolysis of ATP and GTP Drives Gluconeogenesis The net reaction for the conversion of pyruvate to glucose in gluconeogenesis is 2 Pyruvate ϩ 4 ATP ϩ 2 GTP ϩ 2 NADH ϩ 2 H ϩ ϩ 6 H 2 O⎯⎯→ glucose ϩ 4 ADP ϩ 2 GDP ϩ 6 P i ϩ 2 NAD ϩ The net free energy change, ⌬G°Ј, for this conversion is Ϫ37.7 kJ/mol. The consumption of a total of six nucleoside triphosphates drives this process forward. If glycolysis were merely reversed to achieve the net synthesis of glucose from pyruvate, the net reaction would be 2 Pyruvate ϩ 2 ATP ϩ 2 NADH ϩ 2 H ϩ ϩ 2 H 2 O⎯⎯→ glucose ϩ 2 ADP ϩ 2 P i ϩ 2 NAD ϩ E O P O – O – O O OH OH HO H 2 C CHOH NNH E – O P O – O O OH OH HO CH 2 OH CHOH NNH + E NNHHOPO 3 2 – + HBH O FIGURE 22.6 The glucose-6-phosphatase reaction involves formation of a phosphohistidine intermediate. HUMAN BIOCHEMISTRY Gluconeogenesis Inhibitors and Other Diabetes Therapy Strategies Diabetes, the inability to assimilate and metabolize blood glucose, afflicts millions of people. People with type 1 diabetes are unable to synthesize and secrete insulin. On the other hand, people with type 2 diabetes make sufficient insulin, but the molecular pathways that respond to insulin are defective. Many type 2 diabetic people exhibit a condition termed insulin resistance even before the onset of diabetes. Metformin (see accompanying figure) is a drug that improves sensitivity to insulin, primarily by stimulating glucose uptake by glucose transporters in peripheral tissues. It also increases binding of insulin to insulin receptors, stimulates tyro- sine kinase activity (see Chapter 32) of the insulin receptor, and inhibits gluconeogenesis in the liver. Gluconeogenesis inhibitors may be the next wave in diabetes therapy. Drugs that block gluconeogenesis without affecting glycolysis would need to target one of the enzymes unique to gluconeogenesis. 3-Mercaptopicolinate and hydrazine inhibit PEP carboxykinase, and chlorogenic acid, a natural product found in the skin of peaches, inhibits the transport activity of the glucose-6-phosphatase system (but not the glucose-6-phosphatase enzyme activity). The drug S-3483, a derivative of chlorogenic acid, also inhibits the glucose-6-phosphatase transport activity and binds a thousand times more tightly to the transporter than chlorogenic acid. Drugs of this type may be useful in the treatment of type 2 diabetes. CH 3 H 3 C H CC H 2 NNH 2 NH 2 NN NH NH HH HO HO OH O O O O C OH Cl HS N COO – HO HO HO OH O O O C OH OH Metformin 3-Mercaptopicolinate Hydrazine S-3483 Chlorogenic acid 22.2 How Is Gluconeogenesis Regulated? 669 and the overall ⌬G°Ј would be about ϩ74 kJ/mol. Such a process would be highly endergonic and therefore thermodynamically unfeasible. Hydrolysis of four addi- tional high-energy phosphate bonds makes gluconeogenesis thermodynamically favorable. Under physiological conditions, however, gluconeogenesis is somewhat less favorable than at standard state, with an overall ⌬G of Ϫ15.6 kJ/mol for the conversion of pyruvate to glucose. Lactate Formed in Muscles Is Recycled to Glucose in the Liver A final point on the re- distribution of lactate and glucose in the body serves to emphasize the metabolic in- teractions between organs. Vigorous exercise can lead to oxygen shortage (anaero- bic conditions), and energy requirements must be met by increased levels of glycolysis. Under such conditions, glycolysis converts NAD ϩ to NADH, yet O 2 is un- available for regeneration of NAD ϩ via cellular respiration. Instead, large amounts of NADH are reoxidized by the reduction of pyruvate to lactate. The lactate thus produced can be transported from muscle to the liver, where it is reoxidized by liver lactate dehydrogenase to yield pyruvate, which is converted eventually to glucose. In this way, the liver shares in the metabolic stress created by vigorous exercise. It ex- ports glucose to muscle, which produces lactate, and lactate from muscle can be processed by the liver into new glucose. This is referred to as the Cori cycle (Figure 22.7). Liver, with a typically high NAD ϩ /NADH ratio (about 700), readily produces more glucose than it can use. Muscle that is vigorously exercising will enter anaer- obiosis and show a decreasing NAD ϩ /NADH ratio, which favors reduction of pyruvate to lactate. 22.2 How Is Gluconeogenesis Regulated? Nearly all of the reactions of glycolysis and gluconeogenesis take place in the cytosol. If metabolic control were not exerted over these reactions, glycolytic degradation of glucose and gluconeogenic synthesis of glucose could operate simultaneously, with no net benefit to the cell and with considerable consumption of ATP. This is prevented by a sophisticated system of reciprocal control, which inhibits glycolysis when gluconeogenesis is active, and vice versa. Reciprocal regulation of 2 NTP Glucose Pyruvate LDH Lactate Muscle Liver (low ) [NAD + ] [NADH] 6 NTP Gluconeogenesis Glycolysis Glucose Pyruvate LDH Lactate (high ) [NAD + ] [NADH] Blood NADH NAD + NADH NAD + FIGURE 22.7 The Cori cycle. 670 Chapter 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway these two pathways depends largely on the energy status of the cell. When the energy status of the cell is low, glucose is rapidly degraded to produce needed energy. When the energy status is high, pyruvate and other metabolites are utilized for synthesis (and storage) of glucose. Moreover, when blood glucose levels are low, gluconeogenesis is active. In glycolysis, the three regulated enzymes are those catalyzing the strongly exergonic reactions: hexokinase (glucokinase), phosphofructokinase, and pyruvate kinase. As noted, the gluconeogenic pathway replaces these three reactions with corresponding reactions that are exergonic in the direction of glucose synthesis: glucose-6-phosphatase, fructose-1,6-bisphosphatase, and the pyruvate carboxylase– PEP carboxykinase pair, respectively. These are the three most appropriate sites of regulation in gluconeogenesis. Gluconeogenesis Is Regulated by Allosteric and Substrate-Level Control Mechanisms The mechanisms of regulation of gluconeogenesis are shown in Figure 22.8. Control is exerted at all of the predicted sites, but in different ways. Glucose-6-phosphatase is not under allosteric control. However, the K m for the substrate, glucose-6-phosphate, is considerably higher than the normal range of substrate concentrations. As a result, glucose-6-phosphatase displays a near-linear dependence of activity on substrate concentrations and is thus said to be under substrate-level control by glucose- 6-phosphate. Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis. It allosterically inhibits pyruvate kinase (as noted in Chapter 18) and activates pyruvate carboxylase. Because it also allosterically inhibits pyruvate dehydrogenase (the enzymatic link between glycolysis and the TCA cycle), the cellular fate of pyruvate is strongly dependent on acetyl-CoA levels. A rise in [acetyl-CoA] indicates that cellular energy levels are high and that carbon metabolites can be directed to glucose synthesis and storage. When acetyl-CoA levels drop, the activities of pyruvate kinase and pyruvate dehydrogenase increase and flux through the TCA cycle increases, providing needed energy for the cell. Fructose-1,6-bisphosphatase is another important site of gluconeogenic regulation. This enzyme is inhibited by AMP and activated by citrate. These effects by AMP and citrate are the opposites of those exerted on phosphofructokinase in glycolysis, providing another example of reciprocal regulatory effects. When AMP levels increase, gluconeogenic activity is diminished and glycolysis is stimulated. An increase in citrate concentration signals that TCA cycle activity can be curtailed and that pyruvate should be directed to sugar synthesis instead. Fructose-2,6-Bisphosphate—Allosteric Regulator of Gluconeogenesis Emile Van Schaftingen and Henri-Géry Hers demonstrated in 1980 that fructose-2,6- CRITICAL DEVELOPMENTS IN BIOCHEMISTRY The Pioneering Studies of Carl and Gerty Cori The Cori cycle is named for Carl and Gerty Cori, who received the Nobel Prize in Physiology or Medicine in 1947 for their studies of glycogen metabolism and blood glucose regulation. Carl Ferdi- nand Cori and Gerty Theresa Radnitz were both born in Prague (then in Austria). They earned medical degrees from the German University of Prague in 1920 and were married later that year. They joined the faculty of the Washington University School of Medicine in St. Louis in 1931. Their remarkable collaboration re- sulted in many fundamental advances in carbohydrate and glycogen metabolism. They were credited with the discovery of glucose- 1-phosphate, also known at the time as the “Cori ester.” They also showed that glucose-6-phosphate was produced from glucose-1-P by the action of phosphoglucomutase. They isolated and crystal- lized glycogen phosphorylase and elucidated the pathway of glycogen breakdown. In 1952, they showed that absence of glucose-6- phosphatase in the liver was the enzymatic defect in von Gierke’s disease, an inherited glycogen-storage disease. Six eventual Nobel laureates received training in their laboratory. Gerty Cori was the first American woman to receive a Nobel Prize. Carl Cori said of their remarkable collaboration: “Our efforts have been largely complementary and one without the other would not have gone so far…” CH 2 HHO CH 2 OHH O OPO 3 H OH 2– O 3 PO 2– Fructose-2,6-bisphosphate 22.2 How Is Gluconeogenesis Regulated? 671 bisphosphate is a potent stimulator of phosphofructokinase (see Chapter 18). Cog- nizant of the reciprocal nature of regulation in glycolysis and gluconeogenesis, Van Schaftingen and Hers also considered the possibility of an opposite effect— inhibition—for fructose-1,6-bisphosphatase. In 1981, they reported that fructose- 2,6-bisphosphate was indeed a powerful inhibitor of fructose-1,6-bisphosphatase (Figure 22.9). Inhibition occurs in either the presence or absence of AMP, and the effects of AMP and fructose-2,6-bisphosphate are synergistic. Cellular levels of fructose-2,6-bisphosphate are controlled by phosphofructokinase-2 (PFK-2), an enzyme distinct from the phosphofructokinase of the glycolytic pathway, and by fructose-2,6-bisphosphatase (F-2,6-BPase). Remarkably, these two enzymatic activities are both found in the same protein molecule, which is an example of a bifunctional, or tandem, enzyme (Figure 22.10). The opposing activities of this bifunctional enzyme are themselves regulated in two ways. First, fructose-6- phosphate, the substrate of phosphofructokinase and the product of fructose-1,6- bisphosphatase, allosterically activates PFK-2 and inhibits F-2,6-BPase. Second, the phosphorylation by cAMP-dependent protein kinase of a single Ser (Ser 32 ) residue on the liver enzyme exerts reciprocal control of the PFK-2 and F-2,6-BPase activities. Phos- phorylation inhibits PFK-2 activity (by increasing the K m for fructose-6-phosphate) and stimulates F-2,6-BPase activity. To bloodstream Glucose Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-bisphosphate Phosphoenolpyruvate Oxaloacetate Pyruvate Glucose-6-phosphataseHexokinase Fructose-1,6-bisphosphatasePhosphofructokinase Phosphoenolpyruvate carboxykinase Pyruvate kinase Pyruvate carboxylase Glucose-6-phosphate Fructose-2,6-bisphosphate AMP ATP Citrate F-1,6-BP Acetyl-CoA ATP Alanine cAMP-dependent phosphorylation [Glucose-6-phosphate] (substrate level control) F-2,6-BP AMP Acetyl-CoA Regulation of glycolysis Regulation of gluconeogenesis – + + – – + – – – – – – + FIGURE 22.8 The principal regulatory mechanisms in glycolysis and gluconeogenesis. Activators are indicated by plus signs and inhibitors by minus signs. 672 Chapter 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway Substrate Cycles Provide Metabolic Control Mechanisms If fructose-1,6-bisphosphatase and phosphofructokinase acted simultaneously, they would constitute a substrate cycle in which fructose-1,6-bisphosphate and fructose- 6-phosphate became interconverted with net consumption of ATP: Fructose-1,6-BP ϩ H 2 O ⎯⎯→ fructose-6-P ϩ P i Fructose-6-P ϩ ATP ⎯⎯→ fructose-1,6-BP ϩ ADP Net: ATP ϩ H 2 O ⎯⎯→ ADP ϩ P i Because substrate cycles such as this appear to operate with no net benefit to the cell, they were once regarded as metabolic quirks and were referred to as futile cycles. More recently, substrate cycles have been recognized as important devices for controlling metabolite concentrations. The three steps in glycolysis and gluconeogenesis that differ constitute three such substrate cycles, each with its own particular metabolic raison d’être. Consider, for example, the regulation of the fructose-1,6-BP–fructose-6-P cycle by fructose-2,6- bisphosphate. As already noted, fructose-1,6-bisphosphatase is subject to allosteric in- 20 Fructose-1,6-bisphosphatase activity, units/mg protein (a) 15 10 5 0 0 Fructose-1,6-bisphosphate, ␮M 50 100 200 Fructose-1,6-bisphosphatase activity, units/mg protein (b) 15 10 5 0 0 Fructose-1,6-bisphosphate, ␮M 50 100 200 0 1 5 25 0 0.2 1 2.5 5 100 Relative activity (c) 75 50 25 0 0 Fructose-2,6-bisphosphate, ␮M 12345 0 10 25 FIGURE 22.9 Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and (b) presence of 25 mM AMP. In (a) and (b),enzyme activity is plotted against substrate (fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in mM) are indicated above each curve. (c) The effect of AMP (0, 10, and 25 mM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Activity was measured in the presence of 10 mM fructose-1,6-bisphosphate. (Adapted from Van Schaftingen, E.,and Hers, H-G., 1981. Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Proceedings of the National Academy of Science,U.S.A. 78:2861–2863.) P i H 2 O F2,6-BP F2,6-BPase F-6-P F-6-P ADPF2,6-BP (a) (b) Glucagon cAMP-dep. PK ADP PFK-1 PFK-2 F2,6-BPase PFK-2 F1,6-BPase ATP ATP + – – FIGURE 22.10 (a) Synthesis and degradation of fructose-2,6-bisphosphate are catalyzed by the same bifunctional enzyme. (b) The structure of PFK-2/F-2,6-BPase from rat liver. PFK-2 activity resides in the N-terminal portion of the protein (left), and the C-terminal domain (right) contains F-2,6-BPase activity.The PFK-2 domain has a bound ATP analog; the F2,6-Pase has two phosphates bound (pdb id ϭ 1BIF). . regulated enzyme. Citrate stimulates bisphosphatase activity, but fructose-2,6-bisphosphate is a potent allosteric inhibitor. AMP also inhibits the bisphosphatase; the inhibition by AMP is enhanced by. concentrations and is thus said to be under substrate-level control by glucose- 6-phosphate. Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis. It allosterically inhibits pyruvate. far…” CH 2 HHO CH 2 OHH O OPO 3 H OH 2– O 3 PO 2– Fructose-2,6-bisphosphate 22.2 How Is Gluconeogenesis Regulated? 671 bisphosphate is a potent stimulator of phosphofructokinase (see Chapter 18). Cog- nizant of the reciprocal nature