Glycolysis and Pyruvate Oxidation PDF

Glycolysis and Pyruvate 6 Oxidation 4. Interface with other pathways: Many metabolic intermedi- CONTENTS...

Glycolysis and Pyruvate 6 Oxidation 4. Interface with other pathways: Many metabolic intermedi- CONTENTS ates are substrates for alternative pathways, providing a FIVE PERSPECTIVES FOR LEARNING METABOLISM means for interfacing one pathway with another. PATHWAY REACTION STEPS 5. Related diseases: Reduced or absent activity of enzymes Glycolysis—Glucose to Pyruvate creates a buildup or reduced availability of metabolites, Pyruvate Oxidation—Pyruvate to Acetyl–Coenzyme A leading to imbalances in homeostasis. REGULATED REACTIONS Regulation of Glycolysis Regulation of Pyruvate Oxidation lll PATHWAY REACTION STEPS UNIQUE CHARACTERISTICS Anaerobic Glycolysis Glycolysis—Glucose to Pyruvate Glucokinase Versus Hexokinase The glycolytic pathway is composed of two smaller pathways: Multienzyme Complexes (1) five reactions that require energy by converting glucose to Energy Production triose phosphates, and (2) five reactions that produce energy INTERFACE WITH OTHER PATHWAYS Glucose 6-Phosphate by converting triose phosphates to pyruvate (Fig. 6-1). Fructose 6-Phosphate Dihydroxyacetone Phosphate Conversion of Glucose to Glyceraldehyde Pyruvate 3-Phosphate Acetyl–Coenzyme A Hexokinase (or glucokinase in liver) RELATED DISEASES Glucose is first phosphorylated with adenosine triphosphate Lactic Acidosis (ATP), trapping glucose inside the cell. This is an irreversible Pyruvate Kinase Deficiency step. Pyruvate Dehydrogenase Deficiency Arsenate and Arsenite Poisoning Phosphoglucose isomerase Glucose 6-phosphate (G6P) is converted to its isomer, fruc- tose 6-phosphate (F6P). This moves the carbonyl nearer to the middle of the molecule, preparing it to be divided into two triose (3-carbon) molecules. lll FIVE PERSPECTIVES FOR Phosphofructokinase LEARNING METABOLISM Before F6P is cleaved, it acquires another phosphate from ATP, Intermediary metabolism is composed of interacting metabolic producing fructose 1,6-bisphosphate (F1,6-BP). Now the mol- pathways involved in the extraction and/or storage of energy ecule can be split into two phosphorylated products, the triose from fuel molecules. There are five perspectives that provide phosphates. a consistent organization for reviewing these pathways: 1. Pathway reaction steps: Each reaction has unique charac- Aldolase teristics with respect to substrates, products, enzymes, co- Cleavage of F1,6-BP produces the triose phosphates: factors, and inhibitors. dihydroxyacetone phosphate (DHAP) and glyceraldehyde 2. Regulated reactions: Some steps in metabolism are regu- 3-phosphate (G3P). lated by hormones, metabolites, or both, so as to restrict or accelerate the flow of metabolites through a pathway. Triose phosphate isomerase 3. Unique characteristics: Each pathway has features that de- DHAP is brought back into the glycolytic pathway by isomer- scribe unique aspects of its function and identify its general ization to G3P. In effect, this reaction allows two G3P mole- contribution to metabolism. cules to be formed from one F1,6-BP.  50 Glycolysis and Pyruvate Oxidation to prevent metabolism of glucose during blood transport and storage in the clinical laboratory. Glucose ATP Pyruvate kinase Kinase ADP Substrate-level phosphorylation of ADP with PEP produces G6P ATP and pyruvate. (The third substrate level phosphorylation Isomerase Energy reaction occurs in the citric acid cycle.) utilization F6P phase PFK-1 Pyruvate Oxidation—Pyruvate F1,6-BP to Acetyl–Coenzyme A Aldolase After transport of pyruvate into the mitochondrial matrix, it is oxidized by a multienzyme complex, the pyruvate dehydro- Isomerase Pi G3P DHAP genase complex (PDC). Pyruvate oxidation links glycolysis NAD ; with the citric acid cycle. Three steps produce acetyl- Dehydrogenase coenzyme A (CoA) and NADH as the final products (Fig. 6-2). NADH 1,3-BPG ADP Pyruvate Dehydrogenase Kinase Pyruvate binds to thiamine pyrophosphate (TPP) on the pyru- ATP Energy vate dehydrogenase (PDH) enzyme and undergoes decarbox- 3PG production Mutase ylation. CO2 is released, and two of the original carbons of phase pyruvate, in the form of a hydroxyethyl group, remain bound 2PG to TPP on the enzyme. Enolase PEP Dihydrolipoyl Transacetylase ADP The hydroxyethyl group is transferred from TPP to lipoic acid. Kinase ATP During this transfer, the lipoic acid is reduced and the hydro- Pyruvate xyethyl group is oxidized to an acetyl group, now attached to lipoic acid. The lipoyl group is attached to the dihydrolipoyl Figure 6-1. Glycolytic pathway reactions. See text for complete transacetylase, preventing the 2-carbon acetate intermedi- enzyme names and abbreviations. ate from diffusing away. The acetate is subsequently trans- ferred to CoA to produce acetyl-CoA. This leaves the lipoyl Conversion of Glyceraldehyde 3-Phosphate coenzyme in the reduced form, requiring reoxidation to its to Pyruvate active form. Glyceraldehyde 3-phosphate dehydrogenase Simultaneous oxidation and phosphorylation of G3P produces Dihydrolipoyl Dehydrogenase (Lipoamide 1,3-bisphosphoglycerate (1,3-BPG) and nicotine adenine dinu- Dehydrogenase) cleotide (NADH). Inorganic phosphate, rather than ATP, is The reduced lipoyl coenzyme is oxidized using flavin adenide used in this phosphorylation step. dinucleotide (FAD) as a coenzyme. The electrons from the re- duced form of FAD (FADH2) are used to reduce NADþ to pro- duce NADH as a reaction product. Phosphoglycerate kinase Transfer of a phosphate from 1,3-BPG to adenosine diphos- phate (ADP) produces ATP and leaves 3-phosphoglycerate Pyruvate (3PG) to be metabolized further. This is one of three reactions Dehydrogenase that create ADP outside the oxidative phosphorylation CO2 process; it is known as substrate-level phosphorylation of HE-TPP ADP because an identifiable high-energy substrate, 1,3-BPG, NAD; donates a phosphate to ADP to make ATP. Dehydrogenase Lipoate (red) Transacetylase Phosphoglyceromutase The phosphate group is shifted to carbon 2 to produce NADH 2-phosphoglycerate (2PG). Acetyl-CoA Figure 6-2. Sequence of reactions for the pyruvate dehydroge- Enolase nase multienzyme complex. Lipoate alternates between a Removal of a molecule of water produces phosphoenolpyr- reduced form (red) and an oxidized form. The oxidized form uvate (PEP); fluoride inhibits enolase by combining with has a disulfide bond. HE-TPP, hydroxyethyl thiamine pyrophos- Mgþþ. Fluoride is often included in blood collection tubes phate; NADH, nicotine adenine dinucleotide; CoA, coenzyme A.  Regulated reactions 51 Pi Glucagon + KEY POINTS ABOUT PATHWAY REACTION STEPS PFK-2 n The first half of the glycolytic pathway uses energy, and the last half produces energy. F6P F2,6-BP PFK-2 PO4ase n The enzymes in the PDH pathway are coordinated in a multien- is a PFK-2 zyme complex. bifunctional Kinase ATP Insulin + ADP enzyme + lll REGULATED REACTIONS PFK-1 Regulation of Glycolysis F1,6-BP Glycolysis is regulated at three points, each serving a different Figure 6-4. Regulation of phosphofructokinase (PFK-1) by function (Fig. 6-3). Hexokinase, present in all tissues except fructose 2,6-bisphosphate (F2,6-BP). PFK-2 can act as either a the liver, is allosterically inhibited by G6P. Hexokinase regu- kinase or a phosphatase. Glucagon increases the phosphatase lation ensures that cells do not take more glucose out of the activity by increasing the phosphorylated form of PFK-2. Insulin increases the kinase activity by increasing the dephosphorylated blood, and away from the brain, than they really need. Liver form. PO4ase, phosphatase. See text for expansion of all contains glucokinase, an isoform of hexokinase that is not abbreviations. inhibited by G6P. Phosphofructokinase (PFK-1) controls entry of G6P into glycolysis. When the rate of PFK-1 is slowed, G6P accumu- l Adenosine monophosphate (AMP): This effector is pro- lates and is routed toward glycogen synthesis or the pentose duced in increasing amounts from ATP during exercise. It phosphate pathway. PFK-1 is allosterically regulated by sev- allosterically stimulates PFK-1 in muscle, increasing glycol- eral effectors: ysis to restore the ATP concentrations to normal. l Fructose 2,6-bisphosphate (F2,6-BP): This effector is a l ATP and citrate: These negative effectors slow glycolysis “well-fed” signal that allosterically stimulates PFK-1 in when energy is abundant. the liver (Fig. 6-4). It is synthesized from F6P by PFK-2 Pyruvate kinase regulation controls the flow of PEP to py- when insulin (and glucose) levels are high. Elevated gluca- ruvate or to gluconeogenesis. gon, a fasting hormone, inhibits PFK-2 and lowers F2,6-BP In well-fed conditions, pyruvate kinase is allosterically stim- concentration. ulated by F1,6-BP; this prevents a metabolic roadblock when PFK is active. In fasting conditions, pyruvate kinase is allosterically inhib- Glucose ited by ATP and alanine (mobilized from muscle). This pre- − G6P Hexokinase vents PEP that is needed for gluconeogenesis from being G6P converted directly back to pyruvate. F2,6-BP + F6P AMP − PFK-1 Regulation of Pyruvate Oxidation ATP citrate F1,6-BP The PDC is regulated by covalent modification of the first en- zyme, pyruvate dehydrogenase (PDH). PDH kinase inactivates PDH by phosphorylation with ATP (Fig. 6-5). Reactivation is G3P DHAP achieved by the action of PDH phosphatase. Both of these regulatory enzymes are regulated. 1,3-BPG l PDH kinase is stimulated by NADH and acetyl-CoA. It is inhibited by pyruvate. þþ l PDH phosphatase is stimulated by Ca and insulin. 3-PG 2-PG KEY POINTS ABOUT REGULATED REACTIONS n Glycolysis is regulated at the steps catalyzed by hexokinase, F1,6-BP + PEP − Protein ATP PFK-1, and pyruvate kinase. − kinase A n The PDC is regulated by covalent modification through the action ATP ADP alanine Pyruvate of a specific kinase and phosphatase; the kinase and phospha- tase are regulated by changes in NADH, acetyl-CoA, pyruvate, Figure 6-3. Regulated reactions in glycolysis. Each regulated and insulin. step is irreversible. See text for expansion of all abbreviations.  52 Glycolysis and Pyruvate Oxidation − + Glucose Pyruvate NADH acetyl-CoA Kinase G6P PDH active PDH inactive F6P HE-TPP Phosphatase F1,6-BP NAD; + NAD; is regenerated Ca;; Lipoate (red) by lactate formation insulin G3P DHAP NADH NAD; NADH Acetyl-CoA G1,3-BP Figure 6-5. Regulation of the pyruvate dehydrogenase (PDH) complex. Only the first enzyme component, pyruvate dehydrogenase, is regulated. Both insulin and pyruvate 2PG stimulate production of the unphosphorylated, active form. The products of the reaction, nicotine adenine dinucleotide (NAD) 3PG and acetyl-coenzyme A (CoA), promote a lower percentage of the pyruvate dehydrogenase in the active form. HE-TPP, hydroxyethyl thiamine triphosphate. PEP lll UNIQUE CHARACTERISTICS Lactate Pyruvate Anaerobic Glycolysis Figure 6-6. Formation of lactic acid from nicotine adenine dinucleotide (NADþ) produced in glycolysis. See text for The capacity to recycle NADH back to NADþ anaerobically expansion of all abbreviations. (i.e., without mitochondrial involvement) serves important functions in several tissues. anaerobic metabolism (the slow-twitch fibers are adapted to aerobic metabolism), they contain few mitochondria. Liver Conversion of pyruvate to lactate to recycle NADH (Fig. 6-6) Red Blood Cells allows the liver to dispose of excess of either NADH (hypoxia, The lack of mitochondria in red blood cells (RBCs) prevents excessive alcohol consumption) or pyruvate (PDH deficiency) oxidative phosphorylation as a source of ATP. Thus there is produced under conditions that alter normal physiology. total reliance on anaerobic metabolism to provide energy The lactate can be either converted back to pyruvate when for RBC functions. conditions return to normal or excreted in the urine. The net production of energy from anaerobic glycolysis is Glucokinase Versus Hexokinase 2 ATP per glucose molecule; no CO2 is produced. Hexokinase exists in two different isoforms that have differ- ent kinetic and regulatory properties (Table 6-1). Muscle Glucokinase, the isoform in liver, has kinetic properties that Fast-twitch muscle fibers possess a substantial capacity for allow it to capture much of the dietary glucose that enters the glycolysis to supply energy rapidly. These muscle fibers con- liver from the intestines via the portal circulation. This high- tain high concentrations of lactate dehydrogenase to sustain capacity uptake provides glucose for conversion to glycogen high rates of glycolysis. Since these fibers are adapted to or fatty acids. The high Km also minimizes the uptake of TABLE 6-1. Comparison Between Glucokinase and Hexokinase CHARACTERISTIC GLUCOKINASE HEXOKINASE Kinetic properties Km ¼ 5 mmol/L Km ¼ 0.1 mol/L Substrate specificity Glucose only Glucose, fructose, and galactose Inhibition by glucose 6-phosphate Not inhibited Inhibited Insulin response Induced by insulin Constitutive  Interface with other pathways 53 glucose by the liver during fasting, thereby preventing unnec- essary synthesis of glycogen and the development of hypogly- KEY POINTS ABOUT UNIQUE CHARACTERISTICS cemia. Glucokinase also is present in the pancreatic B cells OF GLYCOLYSIS AND PYRUVATE OXIDATION that produce insulin so that the intracellular G6P increases n When anaerobic conditions prevent the use of NADH produced in only when the blood sugar is elevated following a meal. Insu- glycolysis, it is used by lactate dehydrogenase to form lactate; lin induces synthesis of glucokinase to help the liver adapt to conditions that accelerate this reaction lead to lactic acidosis. repeated high-carbohydrate meals. n Glucokinase is a specialized hexokinase in liver that allows for the Hexokinase is the most widely distributed isoform. Its low rapid uptake of dietary glucose from the hepatic portal vein. Km allows glucose to enter cells, especially brain cells and RBCs, under fasting conditions. Excess removal of glucose from the blood into tissues is prevented by the allosteric inhi- lll INTERFACE WITH OTHER bition of hexokinase by its product, G6P. PATHWAYS The metabolic pathway from glucose to acetyl-CoA has sev- HISTOLOGY eral branch points that connect with other metabolic path- ways (Fig. 6-7). Cellular Compartmentation The glycolytic pathway is compartmented in the cytoplasm, Glucose 6-Phosphate whereas the PDH pathway is compartmented within the Since glucose 6-phosphate is also a product of gluconeogene- mitochondrial matrix. These locations provide for more focused regulation of both pathways. Cells that rely on sis, it serves as a substrate for glucose-6-phosphatase in the anaerobic glycolysis for their energy, such as fast-twitch liver. The action of this enzyme releases free glucose into muscle fibers and RBCs, have few to no mitochondria. the bloodstream. Conversion of glucose 6-phosphate to glucose 1-phosphate by phosphoglucomutase provides for interchange between PHYSIOLOGY Glucose Function of Glucokinase in B cells Glycogen The B cells in the pancreatic islets of Langerhans contain Pentose glucokinase instead of hexokinase to prevent the inappropriate Galactose G1P G6P phosphate secretion of insulin, which would lead to a persistent pathway hypoglycemia. Since the elevated G6P serves as the insulin release signal, insulin is released only when blood glucose Uronic acid pathway rises above the normal fasting levels. Amino F6P Mannose sugars G3P DHAP Glycerol-3P Multienzyme Complexes The PDC is an example of a large multienzyme unit that has a TG highly coordinated function. It is composed of multiple copies of three enzymes in a geometric arrangement that allows 2 NADH/ethanol transfer of each reaction product to the next enzyme. This pre- produced vents intermediate products from diffusing, ensuring that the reaction goes to completion. Other examples of multienzyme Alanine Ethanol complexes are a-ketoglutarate dehydrogenase, branched- chain ketoacid dehydrogenases, and fatty acid synthase. Lactate Pyruvate OAA Acetyl-CoA FFA Energy Production The amount of ATP produced in the oxidation of glucose de- Gluconeogenesis Citric pends on the availability of O2. acid cycle Under aerobic conditions, the complete conversion of glu- cose to CO2 and water produces 36 to 38 ATP/glucose. Figure 6-7. Intersection of glycolysis and pyruvate dehydro- Under anaerobic conditions, the complete conversion of genase reactions with other major metabolic pathways. OAA, glucose to lactate (with regeneration of NADþ) produces 2 oxaloacetate; FFA, free fatty acids. See text for expansion of ATP/glucose (see Fig. 6-6). other abbreviations.  54 Glycolysis and Pyruvate Oxidation glycogen, galactose, and uronic acid metabolism (see (massive blood loss), since oxygen is required for oxidation Chapters 8 and 9). First, glucose 1-phosphate is activated to of NADH in the mitochondrial electron transport chain (see the uridine diphosphate precursor, which then contributes Chapter 7). Slower electron transport also is accompanied to glycogen polymerization, to galactose metabolism, or to by a drop in ATP production (thus an increase in AMP), caus- glucuronic acid formation. ing an acceleration of glycolysis. This further increases the If glucose 6-phosphate is oxidized by glucose 6-phosphate production of NADH. dehydrogenase, it enters the pentose phosphate pathway Excess consumption of ethanol also will elevate NADH, (see Chapter 9). since 2 NADH are produced for every molecule of ethanol that is catabolized to acetate. An excess of pyruvate can result from PDH deficiency or Fructose 6-Phosphate pyruvate carboxylase deficiency (see Chapter 8). In addition, F6P is the precursor for the synthesis of amino sugars, such as hypoxia-induced acceleration of glycolysis will produce galactosamine and glucosamine. These amino sugars serve as pyruvate faster than it can be metabolized through the citric precursors for glycoproteins and glycosaminoglycans (see acid cycle. Chapters 9 and 17). In addition, F6P can be converted to mannose 6-phosphate, Pyruvate Kinase Deficiency also a precursor for glycoprotein synthesis. Pyruvate kinase deficiency is the most common enzyme defi- ciency in the glycolytic pathway. Patients have only 5% to Dihydroxyacetone Phosphate 25% of the normal level of the pyruvate kinase isoform found DHAP is converted to glycerol 3-phosphate by glycerol-3- in erythrocytes. Since RBCs cannot use fats for metabolism, phosphate dehydrogenase. This provides a source of glycerol there is a severe reduction in the ability to produce ATP that 3-phosphate for triglyceride and phospholipid metabolism leads to premature destruction of RBCs and a condition from the glycolytic pathway. It also provides a source of car- known as hemolytic anemia. bons for gluconeogenesis, since triglycerides are mobilized and the free glycerol is transported to the liver. Pyruvate Dehydrogenase Deficiency Deficiencies have been identified in each of the three enzyme Pyruvate components of the PDC. A deficiency in the conversion of py- When pyruvate is not being actively converted to acetyl-CoA, ruvate to acetyl-CoA leads to an increase in lactate (see pre- it is being converted to oxaloacetate by pyruvate carboxylase vious discussion) and lactic acidosis. Since the amount of as a precursor for gluconeogenesis. pyruvate entering the citric acid cycle is dramatically reduced, Pyruvate also is interconverted with alanine by alanine ami- the overall energy supply to the cell is reduced, leading to my- notransferase (see Chapter 12); when these processes are oc- opathy (e.g., movement disorders) and neuropathy (e.g., curring between skeletal muscle and liver, the process is called encephalopathy). the alanine cycle. Pyruvate is interconverted with lactate in both skeletal GENETICS & PATHOLOGY muscle and liver during the Cori cycle. Pyruvate Kinase Deficiency Pyruvate kinase deficiency is the most common Acetyl-Coenzyme A glycolytic enzyme deficiency. Since the conversion of PEP to pyruvate is critical for the net production of ATP, a reduction Acetyl-CoA is both a precursor for fatty acid synthesis and the in the energy needed for electrolyte balance leads to an product of fatty acid b-oxidation. osmotic imbalance and to RBC swelling and rupture and Acetyl-CoA is also a product of ethanol catabolism and produces hemolytic anemia. ketone body catabolism. lll RELATED DISEASES PHYSIOLOGY Lactic Acidosis Lactic Acidosis Lactic acidosis is the result of an increase of lactate in the blood PDH deficiency prevents the oxidation of pyruvate, leading to due to overproduction, generally occurring either in the liver its accumulation in the cytoplasm. This increases the or skeletal muscle. It is usually caused by an increase in the conversion of pyruvate to lactate and produces an increase in supply of NADH, but it may also be due to an increase in py- both blood lactate and pyruvate. The protons that accompany these anions are neutralized by the serum bicarbonate, ruvate. Lactic acidosis refers to the increased production of creating a metabolic acidosis with an increased anion gap. lactic acid, whereas lactic acidemia refers to the presence of Lactic acidosis is one of several metabolic acidosis conditions excess lactate in the blood. caused by the accumulation of organic acids in the blood (e.g., Increased NADH can result from hypoxia (e.g., as a result ketoacidosis, methylmalonic acidemia). of exercise), acute respiratory distress syndrome, or shock  Related diseases 55 Arsenate and Arsenite Poisoning KEY POINTS ABOUT METABOLIC PATHWAYS Arsenate poisoning is due to the uncoupling of substrate level AND CLINICAL DISEASES phosphorylation by G3P dehydrogenase. Arsenate is a struc- tural analog of phosphate and is incorporated into 3PG to n Interchange with other major pathways occurs with G6P, F6P, DHAP, pyruvate, and acetyl-CoA. form an unstable mixed anhydride. The end result is the re- lease of arsenate without the formation of ATP. This elimi- n Pyruvate kinase deficiencies produce hemolytic anemia as a nates the net gain of ATP from anaerobic glycolysis and is result of lower intracellular concentrations of ATP. therefore most damaging to the RBC. Aerobic cells are affected when arsenate is incorporated into ATP during oxi- dative phosphorylation with the subsequent spontaneous hydrolysis to produce ADP and free arsenate. In both cases, Self-assessment questions can be accessed at www. the heat associated with ATP hydrolysis is also released. StudentConsult.com. Arsenite poisoning is due to the covalent reaction of arse- nite with lipoic acid, thus preventing it from transferring the hydroxyethyl group from thiamine to CoA.