Principles of BIOCHEMISTRY

Principles of BIOCHEMISTRY
Fifth Edition Gluconeogenesis, The Pentose Phosphate Pathway, and Glycogen Metabolism

OUTLINE 12.1 Gluconeogenesis 12.2 Precursors for Gluconeogenesis
A. Lactate B. Amino Acids C. Glycerol D. Propionate and Lactate E. Acetate 12.3 Regulation of Gluconeogenesis 12.4 The Pentose Phosphate Pathway A. Oxidative Stage B. Nonoxidative Stage C. Interconversions Catalyzed by Transketolase and Transaldolase

12.5 Glycogen Metabolism A. Glycogen Synthesis B. Glycogen Degradation 12.6 Regulation of Glycogen Metabolism in Mammals A. Regulation of Glycogen Phosphorylase B. Hormones Regulate Glycogen Metabolism C. Hormones Regulate Gluconeogenesis and Glycolysis 12.7 Maintenance of Glucose Levels in Mammals 12.8 Glycogen Storage Diseases

Carbohydrate metabolism
All species can synthesize glucose from simple two carbon and three-carbon precursors by gluconeogenesis (糖質新生作用). Some species, notably photosynthetic organisms (光合作用生物), can make these precursors by fixing (固定) CO2 leading to the net synthesis of glucose from inorganic compounds. Every glucose molecule used in glycolysis had to be synthesized in some species. The pathway for gluconeogenesis shares some steps with glycolysis, the pathway for glucose degradation, but four reactions specific to the gluconeogenic pathway are not found in the degradation pathway. These reactions replace the metabolically irreversible reactions of glycolysis. Carbohydrate metabolism

Carbohydrate metabolism
In addition to fueling the production of ATP (via glycolysis and the citric acid cycle), glucose is also a precursor of the ribose (核糖) and deoxyribose (去氧核糖) moieties of nucleotides (核苷酸) and deoxynucleotides (去氧核苷酸) . The pentose phosphate pathway (戊糖磷酸途徑) is responsible for the synthesis of ribose as well as the production of reducing equivalents in the form of NADPH. Glucose availability is controlled by regulating the uptake and synthesis of glucose and related molecules and by regulating the synthesis and degradation of storage polysaccharides composed of glucose residues. Glucose is stored as glycogen (肝醣) in bacteria and animals and as starch in plants. Glycogen and starch can be degraded to release glucose monomers that can fuel energy production via glycolysis or serve as precursors in biosynthesis reactions. Carbohydrate metabolism

12.1 Gluconeogenesis

Gluconeogenesis All organisms have a pathway for glucose biosynthesis, or gluconeogenesis. This is true even for animals that use exogenous glucose (外源性葡萄糖) as an important energy source because glucose may not always be available from external sources or intracellular stores. Some mammalian tissues, primarily liver and kidney can synthesize glucose from noncarbohydrate precursors such as lactate and alanine. Under fasting conditions, gluconeogenesis supplies almost all of the body’s glucose. 2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O + 2 H+ Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi 12.1 Gluconeogenesis

It is convenient to consider pyruvate as the starting point for the synthesis of glucose. The pathway for gluconeogenesis from pyruvate is compared to the glycolytic pathway in Figure Note that many of the intermediates and enzymes are identical. All seven of the near-equilibrium reactions of glycolysis proceed in the reverse direction during gluconeogenesis. Enzymatic reactions unique to gluconeogenesis are required for the three metabolically irreversible reactions of glycolysis. These irreversible glycolytic reactions are catalyzed by PK, PFK-1, and hexokinase. In the biosynthesis direction these reactions are catalyzed by different enzymes. 12.1 Gluconeogenesis

Figure 12.1 Comparison of gluconeogenesis and glycolysis

The synthesis of one molecule of glucose from two molecules of pyruvate requires four ATP and two GTP molecules as well as two molecules of NADH. The net equation for gluconeogenesis is: Four ATP equivalents are needed to overcome the thermodynamic barrier to the formation of two molecules of the energy-rich compound PEP from two molecules of pyruvate. In glycolysis the conversion of PEP to pyruvate is a metabolically irreversible reaction catalyzed by PK. 12.1 Gluconeogenesis

A. Pyruvate carboxylase (PC)
(丙酮酸羧化酶) In the conversion of pyruvate to glucose with the two enzymes required for synthesis of PEP. The two steps involve a carboxylation followed by decarboxylation. In the first step, pyruvate carboxylase catalyzes the conversion of pyruvate to OAA. Catalyzes a metabolically irreversible reaction. The reaction is coupled to the hydrolysis of one molecule of ATP (Figure 12.2). Allosterically activated by acetyl CoA. Accumulation of acetyl CoA from fatty acid oxidation signals abundant energy, and directs pyruvate to OAA for gluconeogenesis. 12.1 Gluconeogenesis

Figure 12.2 Pyruvate carboxylase reaction.

Bicarbonate (HCO3-) is one of the substrates in the reaction shown in Figure Bicarbonate is formed when CO2 dissolves in water so the reaction is sometimes written with CO2 as a substrate. The pyruvate carboxylase reaction plays an important role in fixing CO2 in bacteria and some eukaryotes. This role may not be so obvious when we examine gluconeogenesis since the CO2 is released in the very next reaction; however, much of the OAA that is made is not used for gluconeogenesis. Instead, it replenishes (回補) the pool of citric acid cycle intermediates that serve as precursors to the biosynthesis of amino acids and lipids. 12.1 Gluconeogenesis

B. Phosphoenolpyruvate Carboxykinase (PEPCK)
(磷酸烯醇式丙酮酸羧激酶) Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of OAA to PEP (Figure 12.3). This is a well-studied enzyme with an induced-fit binding mechanism similar to that described for yeast hexokinase and citrate synthase (CS；檸檬酸合成酶). In most species, the enzyme displays no allosteric kinetic properties and has no known physiological modulators. The level of PEPCK activity in cells influences the rate of gluconeogenesis. This is especially true in mammals where gluconeogenesis is mostly confined to cells in the liver, kidneys, and small intestine. 12.1 Gluconeogenesis

Figure 12.3 Phosphoenolpyruvate carboxykinase reaction.

The two-step synthesis of PEP from pyruvate is common in most eukaryotes, including humans. This is the main reason why it’s usually shown when the gluconeogenesis pathway is described (Figure 12.1). However, many species of bacteria can convert pyruvate directly to PEP in an ATP-dependent reaction catalyzed by phosphoenolpyruvate synthetase (磷酸烯醇式丙酮酸合成酶) (Figure 12.4). The products of this reaction include AMP and Pi. The second phosphoryl from ATP is transferred to pyruvate. Thus, two ATP equivalents are used in the conversion of pyruvate to PEP. This is a much more efficient route than the eukaryotic two-step pathway catalyzed by PC and PEPCK. The presence of phosphoenolpyruvate synthetase in bacterial cells is due to the fact that efficient gluconeogenesis is much more important in bacteria than in eukaryotes. 12.1 Gluconeogenesis

Figure 12.4 Phosphoenolpyruvate synthetase reaction.

C. Fructose 1,6-bisphosphatase (F1,6BPase)
(果糖-1,6-二磷酸酶) The reactions of gluconeogenesis between PEP and F1,6BP are simply the reverse of the near-equilibrium reactions of glycolysis. The next reaction in the glycolysis pathway, catalyzed by PFK-1, is metabolically irreversible. In the biosynthesis direction, this reaction is catalyzed by the third enzyme specific to gluconeogenesis, fructose 1,6-bisphosphatase (F1,6BPase). This enzyme catalyzes the conversion of F1,6BP to F6P. F1,6BPase is allosterically inhibited by AMP and fructose 2,6-bisphosphate (F2,6BP). PFK-1 and F1,6BPase that catalyze the interconversion of F6P and F1,6BP are reciprocally controlled by the concentration of F2,6BP. 12.1 Gluconeogenesis

The fructose 1,6-bisphosphatase reaction

D. Glucose 6-phosphatase
(葡萄糖-6-磷酸酶) The final step of gluconeogenesis is the hydrolysis of G6P to form glucose. The enzyme is glucose 6-phosphatase. In mammals, glucose is an important end product of gluconeogenesis since it serves as an energy source for glycolysis in many tissues. Glucose is made in the cells of the liver, kidneys, and small intestine (小腸) and exported to the bloodstream. 12.1 Gluconeogenesis

In these cells, glucose 6-phosphatase is bound to the endoplasmic reticulum (ER; 內質網) with its active site in the lumen. The enzyme is part of a complex that includes a glucose 6-phosphate transporter (G6PT) and a phosphate transporter. G6PT moves G6P from the cytosol to the interior of the ER where it is hydrolyzed to glucose and inorganic phosphate. Phosphate is returned to the cytosol and glucose is transported to the cell surface (and the bloodstream) via the secretary pathway. Glucose 6-phosphatase is found only in cells from the liver, kidneys, and small intestine, so only these tissues can synthesize free glucose. 12.1 Gluconeogenesis

Precursors for Gluconeogenesis
12.2 Precursors for Gluconeogenesis

Precursors for Gluconeogenesis
The main substrates for G6P synthesis are pyruvate, citric acid cycle intermediates, three-carbon intermediates in the pathway (e.g. G3P), and two-carbon compounds such as acetyl CoA. Acetyl CoA is converted to OAA in the glyoxylate cycle (乙醛酸循環), that operates in bacteria, protists, fungi, plants, and some animals. Some organisms can fix inorganic carbon by incorporating it into two carbon and three-carbon organic compounds (e.g., Calvin cycle；卡爾文循環). These compounds enter the gluconeogenesis pathway resulting in net synthesis of glucose from CO2. Major gluconeogenic precursors in mammals: (1) Lactate (2) Most amino acids (especially alanine), (3) Glycerol (from triacylglycerol hydrolysis) 12.2 Precursors for Gluconeogenesis

12.2 Precursors for Gluconeogenesis
A. Lactate Glycolysis generates large amounts of lactate in active muscle and red blood cells. Lactate from these and other sources enters the bloodstream and travels to the liver where it is converted to pyruvate by the action of lactate dehydrogenase (LDH；乳酸脫氫酶). Pyruvate can then be a substrate for gluconeogenesis. Glucose produced by the liver enters the bloodstream for delivery to peripheral tissues, including muscle and red blood cells. This sequence is known as the Cori cycle (Figure 12.5). The conversion of lactate to glucose requires energy, most of which is derived from the oxidation of fatty acids in the liver. Thus, the Cori cycle transfers chemical potential energy in the form of glucose from the liver to the peripheral tissues (週邊組織). 12.2 Precursors for Gluconeogenesis

Figure Cori cycle. Glucose is converted to L-lactate in muscle cells. Some of this lactate is secreted and passes via the bloodstream to the liver. Lactate is converted to glucose in the liver and the glucose is secreted into the bloodstream where it is taken up by muscle cells. Both tissues are capable of synthesizing glycogen and mobilizing it. 12.2 Precursors for Gluconeogenesis

B. Amino Acids Carbon skeletons of most amino acids are catabolized to pyruvate or citric acid cycle intermediates. The end products of these catabolic pathways can serve directly as precursors for synthesis of G6P in cells that are capable of gluconeogenesis. In peripheral mammalian tissues, pyruvate formed from glycolysis or amino acid catabolism must be transported to the liver before it can be used in glucose synthesis. The Cori cycle is one way of accomplishing this transfer by converting pyruvate to lactate in muscle and reconverting it to pyruvate in liver cells. The glucose-alanine cycle (葡萄糖-丙氨酸循環): (1) Transamination of pyruvate yields alanine which travels to the liver. (2) Transamination of alanine in the liver yields pyruvate for gluconeogenesis. (3) Glucose is released to the bloodstream. 12.2 Precursors for Gluconeogenesis

Amino acids become a major source of carbon for gluconeogenesis during fasting when glycogen supplies are depleted. The carbon skeleton of aspartate is also a precursor of glucose. Aspartate is the amino group donor in the urea cycle, a pathway that eliminates excess nitrogen from the cell (Section 17.9B). Aspartate is converted to fumarate in the urea cycle and then fumarate is hydrated to malate that is oxidized to OAA. In addition, the transamination of aspartate with a-ketoglutarate directly generates OAA. 12.2 Precursors for Gluconeogenesis

Figure 12.6 Conversion of pyruvate to alanine.

C. Glycerol The catabolism of triacylglycerols produces glycerol and acetyl CoA. As mentioned earlier, acetyl CoA contributes to the net formation of glucose through reactions of the glyoxylate cycle (Section 13.8). Glycerol can be converted to glucose by a route that begins with phosphorylation to glycerol 3-phosphate, catalyzed by glycerol kinase (Figure 12.7). Glycerol 3-phosphate enters gluconeogenesis after conversion to DHAP. This oxidation can be catalyzed by a flavin containing glycerol 3-phosphate dehydrogenase complex embedded in the inner mitochondrial membrane. The outer face of this enzyme binds glycerol 3-phosphate and electrons are passed to ubiquinone (Q) and subsequently to the rest of the membrane-associated electron transport chain. 12.2 Precursors for Gluconeogenesis

Figure 12.7 Gluconeogenesis from glycerol.

D. Propionate and Lactate
In ruminants (反芻動物)- cattle, sheep, giraffes, deer, and camels- the propionate (丙酸鹽) and lactate produced by the microorganisms in the rumen (瘤胃；反芻動物的第一胃) are absorbed and enter the gluconeogenesis pathway. Propionate is converted to propionyl CoA (丙醯基輔酶A) and then to succinyl CoA (琥珀醯輔酶A). Succinyl CoA is an intermediate of the citric acid cycle that can be metabolized to OAA. Lactate from the rumen is oxidized to pyruvate. Pyruvate and OAA are substrates for gluconeogenesis 12.2 Precursors for Gluconeogenesis

Precursors for gluconeogenesis

Regulation for Gluconeogenesis
12.3 Regulation for Gluconeogenesis

Regulation of Gluconeogenesis
Gluconeogenesis is carefully regulated in vivo. Glycolysis and gluconeogenesis are opposing catabolic and anabolic pathways that share some enzymatic steps but certain reactions are unique to each pathway. Short-term regulation of gluconeogenesis is exerted at two sites—the reactions involving pyruvate and PEP and those that interconvert F1,6BP and F6P (Figure 12.8). When there are two enzymes catalyzing the same reaction (in different directions), modulating the activity of either enzyme can alter the flux through the two opposing pathways. For example, inhibiting PFK-1 stimulates gluconeogenesis since more F6P enters the pathway leading to glucose rather than being converted to F1,6BP. Simultaneous control of F1,6BPase also regulates the flux of F1,6BP toward either glycolysis or gluconeogenesis. 12.3 Regulation for Gluconeogenesis

12.3 Regulation for Gluconeogenesis
Figure12.8 Regulation of liver glycolysis and gluconeogenesis by metabolites 12.3 Regulation for Gluconeogenesis

F2,6BP is formed from F6P by the action of the enzyme phosphofructokinase-2 (PFK-2) (Figure 12.9). In mammalian liver, a different active site on the same protein catalyzes the hydrolytic dephosphorylation of F2,6BP, re-forming F6P. This activity of the enzyme is called F2,6BPase. The dual activities of this bifunctional enzyme control the steady state concentration of F2,6BP and, ultimately, the switch between glycolysis and gluconeogenesis. The allosteric effector F2,6BP activates PFK-1 and inhibits F1,6BPase. Note that an increase in F2,6BP has reciprocal effects: it stimulates glycolysis and inhibits gluconeogenesis. Similarly, AMP affects the two enzymes in a reciprocal manner; inhibiting F1,6BPase and activating PFK-1. The regulation of the bifunctional enzyme PFK-2/F2,6BPase will be described after we cover glycogen metabolism. 12.3 Regulation for Gluconeogenesis

Figure12.9 Interconversion of b-D-F6P and b-D-F2,6BP. 12.3 Regulation for Gluconeogenesis

The Pentose Phosphate Pathway
12.4 The Pentose Phosphate Pathway

The Pentose Phosphate Pathway
(五碳糖磷酸途徑) 五碳糖磷酸途徑意義： (1) 合成許多反應所需之NADPH + H+，如：脂肪酸及膽固醇之合成。 G6P + 12 NADP+ + 7H2O → 6 CO NADPH + 12 H+ + H3PO4 (2) 生成核酸(嘌呤或嘧啶)合成所需之ribose 5-phosphate G6P + 2 NADP+ +H2O → R5P + CO2 + 2 NADPH + 2 H+ The pentose phosphate pathway is a pathway for the synthesis of three pentose phosphates: ribulose 5-phosphate (Ru5P；核酮糖-5-磷酸), ribose 5-phosphate (R5P；核糖-5-磷酸), and xylulose 5-phosphate (Xu5P；木酮糖-5-磷酸). R5P is required for the synthesis of RNA and DNA. The complete pathway has two stages: an oxidative stage and a nonoxidative stage (Figure 12.10). In the oxidative stage, NADPH is produced when glucose 6-phosphate is converted to the five-carbon compound Ru5P. 12.4 The Pentose Phosphate Pathway

12.4 The Pentose Phosphate Pathway
The nonoxidative stage of the pentose phosphate pathway disposes of the pentose phosphate formed in the oxidative stage by providing a route to gluconeogenesis or glycolysis. In this stage, Ru5P is converted to the intermediates F6P and G3P. If all the pentose phosphate were converted to these intermediates, the sum of the nonoxidative reactions would be the conversion of three pentose molecules to two hexose molecules plus one triose molecule. (Ru5P) (F6P) (G3P) 12.4 The Pentose Phosphate Pathway

Figure 12.10 Pentose phosphate Pathway (2 slides)

Occurrence of the Pentose Phosphate Pathway
Pathway is active in tissues that synthesize fatty acids or steroids (liver, mammary and adrenal glands, and adipose tissue). Red blood cells use this pathway to produce needed NADPH to maintain reduced iron. All enzymes in the cycle occur in the cytosol. 12.4 The Pentose Phosphate Pathway

A. Oxidative Stage The three reactions of the oxidative stage of the pentose phosphate pathway are shown in Figure The first two steps are the same as those in the bacterial Entner-Doudoroff pathway (Section 11.7). The first reaction, catalyzed by glucose 6-phosphate dehydrogenase (G6PDH), is the oxidation of G6P to 6-phosphogluconolactone. This step is the major regulatory site for the entire pentose phosphate pathway. G6PDH is allosterically inhibited by NADPH (feedback inhibition). This simple regulatory feature ensures that the production of NADPH by the pentose phosphate pathway is self-limiting. 12.4 The Pentose Phosphate Pathway

The next enzyme of the oxidative phase is 6-phosphogluconolactonase (6GPL；6-磷酸葡糖酸内酯酶) that catalyzes the hydrolysis of 6-phosphogluconolactone to the sugar acid 6-phosphogluconate. Finally, 6-phosphogluconate dehydrogenase (6-磷酸葡萄糖脫氫酶) catalyzes the oxidative decarboxylation of 6-phosphogluconate. This reaction produces a second molecule of NADPH, Ru5P, and CO2. In the oxidative stage, therefore, a six-carbon sugar is oxidized to a five-carbon sugar plus CO2 and two molecules of NADP are reduced to two molecules of NADPH. 12.4 The Pentose Phosphate Pathway

(6-磷酸葡萄糖脫氫酶) (6-磷酸葡萄糖酸) Figure Oxidative stage of the pentose phosphate pathway 12.4 The Pentose Phosphate Pathway

B. Nonoxidative Stage The nonoxidative stage of the pentose phosphate pathway consists entirely of near equilibrium reactions. This stage of the pathway provides five-carbon sugars for biosynthesis and introduces sugar phosphates into glycolysis or gluconeogenesis. Ru5P has two fates: an epimerase can catalyze the formation of Xu5P, or an isomerase can catalyze the formation of R5P (Figure 12.12). R5P is the precursor of the ribose (or deoxyribose) portion of nucleotides. The remaining steps of the pathway convert the five-carbon sugars into glycolytic intermediates. Rapidly dividing cells that require both R5P and NADPH (for the reduction of ribonucleotides to deoxyribonucleotides) generally have high pentose phosphate pathway activity. 12.4 The Pentose Phosphate Pathway

Figure 12.12 Conversions of ribulose 5-phosphate.

In most cells, the G3P and F6P produced by the pentose phosphate pathway are used to resynthesize G6P. This G6P molecule can reenter the pentose phosphate pathway. In that case, the equivalent of one molecule of glucose is completely oxidized to CO2 by six passages through the pathway. After six molecules of G6P are oxidized, the six Xu5P produced can be rearranged by the reactions of the pentose phosphate pathway and part of the gluconeogenic pathway to form five G6P molecules. (Recall that two G3P molecules are equivalent to one F1,6BP molecule.) If we disregard H2O and H+, the overall stoichiometry for this process is 12.4 The Pentose Phosphate Pathway

C. Interconversions Catalyzed by Transketolase and Transaldolase
The interconversions of the nonoxidative stage of the pentose phosphate pathway are catalyzed by two enymes called transketolase (轉酮醇酶) and transaldolase (轉醛醇酶). Both enzymes have broad substrate specificities (基質專一性). They catalyze the exchange of two- and three-carbon fragments between sugar phosphates For both enzymes, one substrate is an aldose, one substrate is a ketose. 12.4 The Pentose Phosphate Pathway

Figure 12.13 Reaction catalyzed by transketolase.

Figure 12.14 Reaction catalyzed by Transaldolase.

12.5 Glycogen Metabolism

Glycogen Metabolism A. Glycogen Synthesis
De novo (重新) glycogen synthesis requires a preexisting primer of four to eight a-1-4 linked glucose residues. This primer is attached to a specific tyrosine residue of a protein called glycogenin (肝醣原) (Figure 12.15) via the 1-hydroxyl group of the reducing end of the short polysaccharide. The primer is formed in two steps. The first glucose residue is attached to glycogenin by the action of a glucosyltransferase (葡萄糖基轉移酶) activity that requires UDP-glucose. Glycogenin itself catalyzes this reaction as well as the extension of the primer by up to seven more glucose residues. Thus, glycogenin is both a protein scaffold for glycogen and an enzyme. 12.5 Glycogen Metabolism

Figure 12.15 Glycogenin from rabbit.

Cellular glucose converted to G6P by hexokinase.
Each glycogen molecule (which can contain thousands of glucose residues) contains a single glycogenin protein at its center. Synthesis and degradation of glycogen require separate enzymatic steps. Further glycogen addition reactions begin with G6P that can be converted to G1P by the action of phosphoglucomutase (葡萄糖磷酸變位酶). Glycogen synthesis and degradation is mostly a way of storing G6P until it is needed by the cell. Cellular glucose converted to G6P by hexokinase. Three separate enzymatic steps are required to incorporate one G6P into glycogen. Glycogen synthase (肝醣合成酶) is the major regulatory step. 12.5 Glycogen Metabolism

Figure 12.16 Synthesis of glycogen in eukaryotes.
(UDP-葡萄糖焦磷酸化酶) Figure Synthesis of glycogen in eukaryotes. 12.5 Glycogen Metabolism

Figure 12.17 The glycogen synthase reaction.

Another enzyme, amylo-(1,4→1,6)-transglycosylase (轉糖苷酶), catalyzes branch formation in glycogen. This enzyme, also known as the branching enzyme, removes an oligosaccharide of at least six residues from the nonreducing end of an elongated chain and attaches it by an linkage to a position at least four glucose residues from the nearest branch point. These branches provide many sites for adding or removing glucose residues, thereby contributing to the speed with which glycogen can be synthesized or degraded. The complete glycogen molecule has many layers of polysaccharide chains extending out from the glycogenin core (Figure 12.18). 12.5 Glycogen Metabolism

Figure 12.18 A glycogen molecule.
Two polysaccharides (blue) are attached to each core glycogenin molecule. Each chain core has 8~14 residues and two branches. Not all branches are shown. Seven layers are numbered but typical glycogen molecules have 8~12 layers, depending on the species. 12.5 Glycogen Metabolism

B. Glycogen Degradation
The glucose residues of starch and glycogen are released from storage polymers through the action of enzymes called polysaccharide phosphorylases: starch phosphorylase (澱粉磷解酶；in plants) and glycogen phosphorylase (in other organisms). These enzymes catalyze the removal of glucose residues from the nonreducing ends of starch or glycogen, provided the monomers are attached by a 1→4 linkages. In contrast to hydrolysis (group transfer to water), phosphorolysis (磷酸解作用) produces phosphate esters. Thus, the first product of polysaccharide breakdown is a-D-glucose 1-phosphate (the Cori ester) (柯里酯、葡萄糖-1-磷酸酯，葡萄釀酒的中間產物), not free glucose. 12.5 Glycogen Metabolism

The phosphorolysis reaction catalyzed by glycogen phosphorylase is shown in Figure 12.19.
Pyridoxal phosphate (PLP；磷酸吡哆醛) is a prosthetic group (輔基) in the active site of the enzyme. The phosphate group of PLP appears to relay a proton to the substrate phosphate to help cleave the scissile (易切斷的) bond of glycogen. Note that glycogen phosphorylase catalyzes a remarkable reaction since it only uses glycogen and inorganic phosphates as substrates in a reaction that produces a relatively “high energy” compound, G1P (Table 10.1). 12.5 Glycogen Metabolism

Figure Cleavage of a glucose residue from the nonreducing end of a glycogen chain, catalyzed by glycogen phosphorylase. 12.5 Glycogen Metabolism

Glycogen phosphorylase is a dimer of identical subunits
Glycogen phosphorylase is a dimer of identical subunits. The catalytic sites lie in the middle of each subunit. It binds phosphate and the end of a glycogen chain (Figure 12.20). The large glycogen particle binds to a nearby site and the chain being degraded passes along a groove on the surface of the enzyme. Four or five glucose residues can be cleaved sequentially before the enzyme has to release a glycogen particle and re-bind. Thus, in contrast to glycogen synthase, glycogen phosphorylase is partially processive (the enzyme remains bound to the end of the growing chain and addition reactions are very rapid)(酵素在重複的催化過程中不分離). 12.5 Glycogen Metabolism

Figure 12.20 Binding and catalytic sites on glycogen phosphorylase.

The enzyme stops four glucose residues from a branch point (an a (1→6) glucosidic bond) leaving a limit dextrin (限制糊精). The limit dextrin can be further degraded by the action of the bifunctional glycogen debranching enzyme (Figure 12.21). A glucanotransferase (葡聚醣基轉移酶) activity of the debranching enzyme catalyzes the relocation of a chain of three glucose residues from a branch to a free 4-hydroxyl end of the glycogen molecule. Both the original linkage and the new one are a (1→4). The other activity of glycogen debranching enzyme, amylo-1,6-glucosidase, catalyzes hydrolytic (not phosphorolytic) removal of the remaining a (1→6) linked glucose residue. 12.5 Glycogen Metabolism

Figure 12.21 Degradation of glycogen.

Regulation of Glycogen Metabolism in Mammals
12.6 Regulation of Glycogen Metabolism in Mammals

12.6 Regulation of Glycogen Metabolism in Mammals
Muscle glycogen is fuel for muscle contraction. Liver glycogen is mostly converted to glucose for bloodstream transport to other tissues. Both mobilization and synthesis of glycogen are regulated by hormones. Insulin (胰島素), glucagon (升糖激素、血糖激素) and epinephrine (腎上腺素) regulate mammalian glycogen metabolism. 12.6 Regulation of Glycogen Metabolism in Mammals

A. Regulation of Glycogen Phosphorylase
Glycogen phosphorylase (GP) and glycogen synthase (GS) control glycogen metabolism in liver and muscle cells. GP is responsible for the breakdown of glycogen to produce G1P. In muscle cells, G1P is converted to G6P that is used in glycolysis to produce ATP. In liver cells, G6P is hydrolyzed to free glucose that is secreted into the bloodstream where it can be taken up by other tissues. GP and GS are reciprocally regulated both covalently and allosterically (when one is active the other is inactive). Covalent regulation by phosphorylation (-P) and dephosphorylation (-OH). 12.6 Regulation of Glycogen Metabolism in Mammals

The activity of GP is regulated by several allosteric effectors and by covalent modification (phosphorylation). Let’s take a few minutes to study the regulation of GP because not only is it important in glycogen metabolism, it’s also historically important. The enzyme exists in four different forms as shown in Figure The unphosphorylated form is called glycogen phosphorylase b (GPb) and the phosphorylated form is called glycogen phosphorylase a (GPa). The enzyme is phosphorylated by a kinase enzyme and dephosphorylated by a phosphatase. 12.6 Regulation of Glycogen Metabolism in Mammals

Like other allosterically regulated enzymes, GP adopts two conformations; the R conformation is the active conformation and the T conformation is much less active. This is depicted in Figure as a change in the shape of the catalytic site: In the R conformation, inorganic phosphate (a substrate of the reaction) can bind and in the T conformation binding of inorganic phosphate is inhibited. Unphosphorylated GPb can exist in both inactive T conformations and active R conformations. The allosteric site of the enzyme binds several effectors that cause a shift in conformation. 12.6 Regulation of Glycogen Metabolism in Mammals

Figure 12.22 Regulation of glycogen Phosphorylase.
12.6 Regulation of Glycogen Metabolism in Mammals

When ATP is bound, the activity of the enzyme is inhibited (T state). This is the normal state of activity since physiological concentrations of ATP are high and relatively constant. When the AMP concentration rises, it displaces ATP from the allosteric site causing a shift to the active R conformation and activation of glycogen breakdown. In muscle cells, increasing AMP concentration results from strenuous muscle activity and signals the need for more G1P to stimulate ATP production by glycolysis. The enzyme is inhibited by G6P (feedback inhibition). There’s no need to continue glycogen breakdown if G6P concentration is sufficient to fuel glycolysis. 12.6 Regulation of Glycogen Metabolism in Mammals

The structures of GPa and GPb are shown in Figure in order to illustrate the structural changes that take place when the enzyme is phosphorylated and dephosphorylated. The phosphoryl group is covalently attached to serine residue 14 (Ser-14) near the N-terminal end of the protein. In the unphosphorylated state (GPb), the N-terminal residues, including Ser-14, associate with the surface near the catalytic site. In the phosphorylated state (GPa), phosphoserine-14 interacts with two positively charged arginine residues near the allosteric site. The remarkable shift in the location of the N-terminal end of the chain cause other conformation changes in the enzyme; notably, a reorientation of two a helices, the tower helices, on the other side of the dimer interface. This, in turn, affects the position of the 280s loop controlling the transition between the active R conformation and the inactive T conformation. 12.6 Regulation of Glycogen Metabolism in Mammals

Figure Phosphorylated and unphosphoylated forms of glycogen phosphorylase. 12.6 Regulation of Glycogen Metabolism in Mammals

GPa is relatively insensitive to ATP, AMP, and G6P. In muscle cells, GPa will be formed in response to hormones that signal the need for glucose and strenuous (激烈的) muscle activity. This promotes rapid mobilization of glycogen. In liver cells, the liver version of GP responds to the same hormones but in this case glycogen breakdown leads to excretion of glucose that can be taken up by muscle cells. Liver GPa is inhibited by glucose by shifting GPa to the T conformation. This makes sense since the presence of a high concentration of free glucose means that it’s not necessary to continue producing glucose from glycogen. The muscle version of GP is not inhibited by glucose since muscle cells rarely see significant concentrations of free glucose. Muscle cells don’t convert G6P to glucose and any glucose taken up from the bloodstream is quickly phosphoryated by hexokinase to G6P. 12.6 Regulation of Glycogen Metabolism in Mammals

B. Hormones Regulate Glycogen Metabolism
Insulin, glucagon, and epinephrine are the principal hormones that control glycogen metabolism in mammals. Insulin is produced by b-cells of the pancreas, is secreted when the concentration of glucose in the blood increases. High levels are associated with the fed state. Insulin increases rate of glucose transport into muscle, adipose tissue via GLUT 4 transporter. Insulin stimulates glycogen synthesis in the liver via the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). 12.6 Regulation of Glycogen Metabolism in Mammals

Glucagon (29 AAs) is secreted by the a cells of the pancreas in response to low blood glucose (elevated glucagon is associated with the fasted state). Stimulates glycogen degradation to restore blood glucose to steady-state levels Glucagon is extremely selective in its target because only liver cells are rich in glucagon receptors. The effect of glucagon is opposite that of insulin and an elevated glucagon concentration is associated with the fasted state. 12.6 Regulation of Glycogen Metabolism in Mammals

The adrenal glands release the catecholamine epinephrine (兒茶酚胺腎上腺素) (aka adrenaline), in response to neural signals that trigger the fight or flight response (Figure 3.5c). The epinephrine precursor, norepinephrine (正腎上腺素), also has hormone activity. Epinephrine stimulates the breakdown of glycogen. It triggers a response to a sudden energy requirement whereas glucagon and insulin act over longer periods to maintain a relatively constant concentration of glucose in the blood. Epinephrine binds to b-adrenergic receptors of liver and muscle cells and to a1-adrenergic receptors of liver cells. The binding of epinephrine to b-adrenergic receptors or of glucagon to its receptors activates the adenylyl cyclase signaling pathway. The second messenger, cyclic AMP (cAMP), then activates protein kinase A (PKA). 12.6 Regulation of Glycogen Metabolism in Mammals

PKA phosphorylates a number of other proteins causing significant changes in metabolism. When glucagon binds to its receptor it stimulates adenylate cyclase causing an increase in cAMP that leads to activation of PKA. PKA phosphorylates GS converting the “a” form to the inactive “b” form. This blocks glycogen synthesis. PKA also phosphorylates another kinase called phosphorylase kinase. As the name implies, this is the kinase that phosphorylates GP. PKA activates phosphorylase kinase leading to conversion of GPb to the active form, GPa. The result is an increase in the rate of degradation of glycogen (Figure 12.24). 12.6 Regulation of Glycogen Metabolism in Mammals

Figure 12.24 Effects of glycogen metabolism.
PP1 PP1 Figure Effects of glycogen metabolism. 12.6 Regulation of Glycogen Metabolism in Mammals

The net effect of glucagon (or epinephrine) is to block synthesis of glycogen and stimulate its breakdown. The reciprocal regulation of these two enzymes is an important feature of regulation in this pathway. GS and GP are dephosphorylated by phosphoprotein phosphatase-1 (PP1；第一型磷蛋白磷酸水解酶), an enzyme that acts on many other substrates. As shown in Figure 12.25, dephosphorylation leads to reciprocal inactivation of GP and activation of GS. This results in synthesis of glycogen from UDP-glucose and inhibition of glycogen breakdown. Insulin stimulates the activity of PP1, thus causing the uptake of glucose into glycogen and its depletion in the bloodstream. 12.6 Regulation of Glycogen Metabolism in Mammals

Figure Effects of glycogen metabolism. 12.6 Regulation of Glycogen Metabolism in Mammals

Reciprocal Regulation of GP and GS Covalent Regulation Active form “a” Inactive form “b” Glycogen phosphorylase -P OH Glycogen synthase OH P Allosteric Regulation by G6P GP a (active form) - inhibited by G6P GS b (inactive form) - activated by G6P 12.6 Regulation of Glycogen Metabolism in Mammals

C. Hormones Regulate Gluconeogenesis and Glycolysis
Fructose 1,6-bisphosphatase (F1,6BPase) and PFK-1 are the key enzymes involved in the decision to either degrade glucose or synthesize it. These two enzymes are reciprocally regulated by the effector F2,6BP (Figure 12.8). This effector molecule is synthesized from F6P by PFK-2 and it is dephosphorylated back to F6P by fructose 2,6-bisphosphatase (F2,6BPase) (Figure 12.9). These two enzymatic activities are located on the same bifunctional protein. The relationship among the four enzymes and their products is summarized in Figure The F2,6BPase and PFK-2 activities in the bifunctional enzyme (雙功酵素) are regulated by phosphorylation in a reciprocal manner. When the protein is phosphorylated, the enzyme acts as a F2,6BPase and the phosphofructokinase activity is inhibited. Conversely, when the enzyme is unphosphorylated it acts as a phosphofructokinase and the F2,6BPase activity is inhibited. 12.6 Regulation of Glycogen Metabolism in Mammals

Figure The role of fructose 2,6-bisphosphate in regulating glycolysis and gluconeogenesis. 12.6 Regulation of Glycogen Metabolism in Mammals

This is the same mode of reciprocal regulation we encountered with GP and GS, except this time the two enzyme activities are on the same molecule. In the presence of glucagon, PKA is active and it phosphorylates the bifunctional enzyme (Figure 12.27). Thus, glucagon stimulates gluconeogenesis and inhibits glycolysis in liver cells causing glucose levels in the bloodstream to rise. At the same time, epinephrine (腎上腺素) can stimulate glycogen degradation and inhibit glycogen synthesis in muscle cells. The result is more glucose for muscle cells and more ATP from glycolysis. 12.6 Regulation of Glycogen Metabolism in Mammals

Figure Effect of glucagon on gluconeogenesis. 12.6 Regulation of Glycogen Metabolism in Mammals

Maintenance of Glucose Levels in Mammals
12.7 Maintenance of Glucose Levels in Mammals

Maintenance of Glucose Levels in Mammals
Mammals maintain blood glucose levels within strict limits by regulating both the synthesis and degradation of glucose. Glucose is the major metabolic fuel in the body. Some tissues, such as brain, rely almost entirely on glucose for their energy needs. The concentration of glucose in the blood seldom drops below 3 mM or exceeds 10 mM. When the concentration of glucose in the blood falls below 2.5 mM, glucose uptake into the brain is compromised, with severe consequences. Conversely, when blood glucose levels are very high, glucose is filtered out of the blood by the kidneys accompanied by osmotic loss of water and electrolytes. 12.7 Maintenance of Glucose Levels in Mammals

Role of the Liver in Metabolism
The liver participates in the interconversions of all types of metabolic fuels: carbohydrates, amino acids and fatty acids. Anatomically (在解剖學上), the liver is centrally located in the circulatory system (Figure 12.28). Most tissues are perfused (佈滿) in parallel with arterial system (動脈系統) supplying oxygenated blood and the venous circulation (靜脈循環) returning blood to the lungs for oxygenation. The liver, however, is perfused in series with the visceral tissue (內臟組織) (gastrointestinal tract [胃腸道], pancreas [胰臟], spleen [脾臟], and adipose tissue [脂肪組織]); blood from these tissues drains into the portal vein (門靜脈) and then flows to the liver. 12.7 Maintenance of Glucose Levels in Mammals

Figure 12.28 Placement of the liver in circulation system
12.7 Maintenance of Glucose Levels in Mammals

12.7 Maintenance of Glucose Levels in Mammals
The consumption of glucose by tissues removes dietary glucose from the blood. When glucose levels fall, liver glycogen and gluconeogenesis become the sources of glucose. However, since these sources are limited, hormones act to restrict the use of glucose to those cells and tissues that absolutely depend on glycolysis for generating ATP (kidney medulla [腎髓質], retina [視網膜], red blood cells, and part of brain). Other tissues can generate ATP by oxidizing fatty acids mobilized from adipose tissue. In the a960’s, George Cahill examined the glucose utilization of obese patients (肥胖的患者) as they underwent therapeutic starvation (Figure 12.29). 12.7 Maintenance of Glucose Levels in Mammals

Figure 12.29 Five phases of glucose homeostasis.
Graph illustrates glucose utilization after 100 g glucose consumption then 40 day fast. 12.7 Maintenance of Glucose Levels in Mammals

Glycogen Storage Diseases
12.8 Glycogen Storage Diseases

Glycogen Storage Diseases
Many metabolic enzymes in humans are encoded by gene families. Different versions are expressed in different tissues. In the case of enzymes involved in glycogen metabolism, the most common versions are found in liver and muscle. A deficiency in one of these enzymes will produce severe symptoms but may not be lethal. There are nine types of glycogen storage diseases resulting from defects in glycogen metabolism. 12.8 Glycogen Storage Diseases

12.8 Glycogen Storage Diseases
Type I: The most common glycogen storage disease is called von Gierke disease (肝醣儲積症、馮吉爾克氏症). It is caused by a deficiency in glucose 6-phosphatase. Patients are unable to secrete glucose leading to accumulation of glycogen in the liver and kidneys. Type II: Patients suffering from type II disease, known as Pompe’s disease (龐貝氏症), suffer from reduced activity of a-1,4-glucosidase, or acid maltase, an enzyme required for glycogen breakdown in lysozomes. The defect causes glycogen to accumulate in lysosomes leading to problems with muscle tissue, especially in the heart. In the most severe forms, children die within the first few years of life. 12.8 Glycogen Storage Diseases

Type III: Cori disease (肝醣儲積症第三型、科里氏症), characterized by defects in the gene encoding the glycogen debranching enzyme in liver and muscle. People suffering from this disease have weakened muscles because they are unable to mobilize all of the stored glycogen. Some defects have very mild symptoms. Type IV: Anderson’s disease (安德森氏症), the mutations occur in the gene for liver branching enzyme found on chromosome 3. Long-chain polysaccharides accumulate in patients with these mutations, resulting in death within a few years from heart failure or liver failure. Type V: McArdle’s disease (麥克阿德爾氏症) is caused by a deficiency of muscle glycogen phosphorylase. Individuals having this genetic disease cannot perform strenuous exercise and suffer painful muscle cramps (痙攣). 12.8 Glycogen Storage Diseases

Type VI: Hers’ disease (赫斯氏症) is a mild form of glycogen storage disease due to a deficiency in liver glycogen phosphorylase. Type VII: Mutations in the gene for muscle PFK-1 cause Tarui’s disease (肌肉磷酸果糖激酶缺乏症), characterized by inability to exercise and muscle cramps. Type VIII: Now recognized as a subtype of type IX. Type IX: This form of glycogen storage disease manifests as muscle weakness and/or muscle cramps. The symptoms are usually mild. All subtypes are due to mutations in the genes for the various subunits of glycogen phosphorylase kinase. 12.8 Glycogen Storage Diseases

Summary 1. Gluconeognesis is the pathway for glucose synthesis from noncarbohydrate precursors. The seven near-equilibrium reactions of glycolysis proceed in the reverse direction in gluconeogenesis. Four enzymes specific to gluconeogenesis catalyze reactions that bypass the three metabolically irreversible reactions of glycolysis. 2. Noncarbohydrate precursors of glucose include pyruvate, lactate, alanine, and glycerol. 3. Gluconeogenesis is regulated by glucagon, allosteric modulators, and the concentrations of its substrates. Summary

4. The pentose phosphate pathway metabolizes glucose 6-phosphate to generate NADPH and ribose 5-phosphate. The oxidative stage of the pathway generates two molecules of NADPH per molecule of glucose 6-phosphate converted to ribulose 5-phosphate and CO2. 5. The nonoxidative stage includes isomerization of ribulose 5-phosphate to ribose 5-phosphate. Further metabolism of pentose phosphate molecules can covert them to glycolytic intermediates. The combined activities of transketolase and transaldolase convert pentose phosphates to triose phosphates and hexose phosphates. 6. Glycogen synthesis is catalyzed by glycogen synthase, using a glycogen primer and UDP-glucose. Summary

7. Glucose residues are mobilized from glycogen by the action of glycogen phosphorylase. Glucose 1-phosphate is then converted to glucose 6-phosphate. 8. Glycogen degradation and glycogen synthesis are reciprocally regulated by hormones. Kinases and phosphatases control the activities of the interconvertible enzymes glycogen phosphorylase and glycogen synthase. 9. Mammals maintain a nearly constant concentration of glucose blood. The liver regulates the amount of glucose supplied by the diet, glycogenolysis, and other fuels. 10. Glycogen storage diseases result from defects in genes required for glycogen metabolism. Summary

Cori Cycle 就是能量於骨骼肌組織、肝臟之間的循環。在新陳代謝過程中，乳酸(lactate)可經由無氧糖解反應(anaerobic glycolysis)於肌肉組織中產生，而部分的乳酸會進入肝臟中，並進一步利用gluconeogenesis產生葡萄糖。而葡萄糖又可回到肌肉組織中，再次利用後產生乳酸，以此循環。肌肉組織的活動需要能量，而可藉由分解在肌肉中的肝醣得之，分解肝醣產生的ATP可為被肌肉組織利用的能量來源。儘管遍佈肌肉組織活動的ATP用於提供能量，但其必須持續的補充。當氧氣供應充足時，經分解肝糖所產生的G6P可進行有氧氧化作用，使pyruvate進入citric acid cycle中，完成有氧呼吸作用，並產生大量ATP。然而當氧氣短缺時，例如肌肉進行劇烈運動時，只單靠一般呼吸作用不能將足夠的氧氣供給到組織進行citric acid cycle，導致無法完全氧化，所以能量經由無氧呼吸過程的G6P釋放。分解葡萄糖經G6P至pyruvate，此過程為glycolysis。而糖解作用單經由無氧呼吸可產生丙酮酸酯，又可被轉換成乳糖。在柯氏循環中，當pyruvate被轉換成乳酸可使糖解作用繼續進行。在肌肉組織中pyruvate被還原成乳酸的機制為乳酸脫氫酶(lactate dehydrogenase)。作用產生機制是利用在糖解作用中氧化NADH產生NAD+，利用NADH氧化後的兩個電子傳遞給pyruvate以產生乳酸。此可維持NAD+的濃度並可使糖解作用的過程繼續。於是乳酸被釋放至血液中，同時可循環至心肌並氧化，而乳酸在長時間激烈運動下聚集會限制運動員的表現。部分的乳酸將進入肝臟中，並再次氧化成pyruvate。 Cori Cycle

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