Carbohydrate Metabolism PDF

CARBOHYDRATE METABOLISM October 2024 Van Nguyen 1  1 Metabolism 3 1 Carbohydrate Metabolism 2 Energy and electron transfer 3 Lipid Metabolism...

CARBOHYDRATE METABOLISM October 2024 Van Nguyen 1  1 Metabolism 3 1 Carbohydrate Metabolism 2 Energy and electron transfer 3 Lipid Metabolism 2 Summary of Energetic pathways in humans 4 Viel & Gaudet, Principle of Biochemistry, 2015 Van Nguyen 2 Carbohydrate Metabolism Polysaccharide digestion Glycolysis: glucose à pyruvate Anaerobic catabolism of pyruvate (fermentation) Other monosaccharides catabolism Gluconeogenesis Pentose phosphate pathways Van Nguyen 3 Glycolysis sweet breaking down DIGESTION 1 Carbohydrates (next chapters) Glycogen (glucose storage in liver) 2 3 4 Glycolysis resources Polysaccharides (from food) hydrolysis à oligosaccharides à monosaccharides (galactose, glucose, fructose, mannose) Glycogen (in liver) degradation à glucose Van Nguyen 4 1 Polysaccharide digestion Glycolysis takes place in the cytosol Cleavage α-1,4-bonds Salivary a-amylase inhibited by HCl α-Dextrinase cleavage α-1,6-bonds in dextrins Sucrase for sucrose (glucose & fructose) Lactase for lactose (glucose & galactose) Maltase for maltose (2 glucoses) Van Nguyen 5  Monosaccharides’ pathways Monosaccharides can enter glycolysis by converting to glycolytic intermediates Fructose (and sucrose - contains fructose): major sweeteners in many foods & beverages Galactose from milk; honey Mannose from dietary polysaccharides, glycoproteins Van Nguyen 6 Glucose is an Important Fuel for Most Organisms Almost all organisms use glucose as a fuel. In mammals, glucose is the only fuel the brain uses under nonstarvation conditions and the only fuel red blood cells are able to use at all. Why is glucose such a prominent fuel in all life-forms? 1.Glucose may have been available for primitive biochemical systems because it can form under prebiotic conditions. 2.Glucose is the most stable hexose. 3.Glucose has a low tendency to non-enzymatically glycosylate proteins.  Glycolysis Glycolysis is also called the Embden-Meyerhof (or Warburg) Pathway 10 steps/reactions – Essentially all cells carry out glycolysis but with different rates Investment phase: converts glucose to two glyceraldehyde-3-P Paid off phase: produces two pyruvates Products are pyruvate, ATP and NADH 3 possible fates for Pyruvate Reduced to lactate Reduced to ethanol Oxidized to eventually form CO2 and H2O Van Nguyen 8  2 Glycolysis Pathway First of four slides Phosphorylation of glucose to give glucose-6-phosphate Investment à Isomerization of glucose-6- phosphate to give fructose-6- phosphate Van Nguyen 9 1 Hexokinase Traps Glucose in the Cell and Begins Glycolysis Upon entering the cell through a specific transport protein, glucose is phosphorylated at the expense of ATP to form glucose 6-phosphate. Hexokinase, which requires Mg2+ or Mn2+ as a cofactor, catalyzes the reaction. Hexokinase, like most kinases, employs substrate-binding induced fit to help exclude water and minimize undesired hydrolysis of ATP.  Induced Fit in Hexokinase The two lobes of hexokinase are separated in the absence of glucose (left). The conformation of hexokinase changes markedly on binding glucose (right). Notice that two lobes of the enzyme come together, creating the necessary environment for catalysis. Glycolysis Pathway Second of four slides Investment à Phosphorylation of fructose-6-phosphate to yield fructose-1,6-bisphosphate Cleavage of fructose-1,6,-bisphosphate to give glyceraldehyde-3-phosphate and dihyroxyacetone phosphate Isomerization of dihyroxyacetone phosphate to give glyceraldehyde-3- phosphate Van Nguyen 12 3 Phosphofructokinase Catalyzes the Second Irreversible Step in Glycolysis The carbohydrate is trapped in the fructose form by the addition of a second phosphate to form fructose 1,6 bisphosphate. This irreversible reaction is catalyzed by the allosteric enzyme phosphofructokinase (PFK). Glycolysis Pathway Third of four slides Oxidation of glyceraldehyde-3-phosphate to give 1,3-bisphosphoglycerate Paid-off à Transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP to give 3-phosphoglycerate Paid-off à Van Nguyen 14 6 Catalyzed by Glyceraldehyde 3-phosphate Dehydrogenase The formation of glyceraldehyde 1,3-bisphosphate can be thought of as occurring in two steps: 1. The highly exergonic oxidation of carbon 1 in GAP to an acid 2. The highly endergonic formation of glyceraldehyde 1, 3-bisphosphate from the acid These two reaction are linked by the formation of an energy-rich thioester in the active site of glyceraldehyde 3-phosphate dehydrogenase. (A thioester intermediate involves)  Glycolysis Pathway Fourth of four slides Isomerization of 3-phosphoglycerate to give 2-phosphoglycerate Dehydration of 2- phosphoglycerate to give phosphoenolpyruvate Transfer of a phosphate group from phosphoenolpyruvate Paid-off to ADP to give pyruvate Van Nguyen 16 Glycolysis summary 2 NAD+ 2 NADH 1 glucose 2 pyruvate 2 ADP 2 ATP Van Nguyen 17 3 Fates of Pyruvate In Anaerobic Conditions Alcoholic Fermentation Yeast Sugar in fruits wine, beer CO2: gas released Overall Lactic Acid Fermentation Bacteria Milk cheese, yogurt Overall Van Nguyen 18 NAD+ regeneration In Anaerobic Conditions (no O2) Van Nguyen 19 Summary on Anaerobic catabolism of glucose Formed NADH is used; not available at the end Van Nguyen 20  Aerobic Metabolism Through the Citric Acid Cycle and Electron Transport Chain The third possible fate of pyruvate, rather than a type of fermentation, is to completely oxidize the pyruvate through pyruvate processing (to generate acetyl CoA) and the citric acid cycle (to oxidize the acetyl group). The electrons are transferred to the final acceptor in the electron transport chain (i.e., O2) via aerobic metabolism. As the electrons are delivered from NADH to the electron transport chain, NAD+ is restored. Study guide Memorize the 10 reactions of glycolysis (name the reactions by the enzymes involve) For all 10 reactions of glycolysis identify and show the relationship between Name and structure of the substances Enzyme’s name and the reaction it catalyzes Identify the oxidation state change in each step of the glycolysis Is glucose oxidized or reduced in glycolysis (forming pyruvate)? How many molecules of ATP, NAD+, and NADH have been consumed/produced in glycolysis? Explain the importance of reaction 6 and reaction 9. Van Nguyen 22 Other Monosaccharides’ pathways Mannose metabolism ATP ADP Van Nguyen 23 Fructose metabolism High glucose concentration Low glucose concentration Van Nguyen 24 Galactose metabolism Sum up: Van Nguyen 25 Glycogen degradation Cleavage by Pi: phosphorolysis; NOT hydrolysis No ATP involved Van Nguyen 26 Many Adults are Intolerant of Milk Because They are Deficient in Lactase Lactose intolerance (hypolactasia) occurs because most adults lack lactase, the enzyme that degrades lactose. Northern Europeans have a mutation that prevents the decline of lactase activity after weaning. In lactase-deficient individuals, gut bacteria ferment lactose to lactic acid, also generating methane (CH4) and hydrogen gas (H2); the products cause discomfort and disrupt water balance in the intestine. Scanning Electron Micrograph of Lactobacillus The anaerobic bacterium Lactobacillus is shown here. As suggested by its name, this genus of bacteria ferments glucose into lactic acid and is widely used in the food industry. Lactobacillus is also a component of the normal human bacterial flora of the urogenital tract where, because of its ability to generate an acidic environment, it prevents the growth of harmful bacteria. [SPL/Science Source.] Galactose is Highly Toxic if the Transferase is Missing Classic galactosemia results if galactose 1-phosphate uridyl transferase activity is deficient Symptoms include failure to thrive, jaundice, and liver enlargement that can lead to cirrhosis. Cataract formation may also occur. The most common treatment is to remove galactose (and lactose) from the diet.  Cataracts can Form in the Eye When Galactose is Converted to Galactitol Cataracts, a clouding of the lens, form because galactose is converted into galactitol, which is poorly metabolized and accumulates in the lens. Water diffuses into the lens to maintain osmotic balance, causing cataract formation. Glycogen debranching Complete breakdown requires debranching enzymes to degrade the b (1->6) linkages Van Nguyen 31 Energy harvest from Glycogen Van Nguyen 32 Carbohydrates catabolism ATP ADP Galactose G-1-P G-6-P Mannose F-6-P ATP ADP Fructose ADP ATP Acetyl-CoA Van Nguyen 33 Gluconeogenesis Gluconeogenesis glucose new generation (from common metabolites) Humans consume 160 g of glucose per day (75% by the brain) Body fluids ~ 20 g glucose; Glycogen stores yield ~ 180-200 g: depleted (at least partially) in strenuous exercise Gluconeogenesis: body makes its own glucose (for activity and storage) from noncarbohydrate precursors: lactate, amino acids, and glycerol The major site of gluconeogenesis is the liver, although some gluconeogenesis can occur in the kidney. Gluconeogenesis is especially important during fasting or starvation, as glucose is the primary fuel for the brain and the only fuel for red blood cells. Van Nguyen 34 Glycerol as a precursor Glycerol may enter either the gluconeogenic or the glycolytic pathway at dihydroxyacetone phosphate (DHAP) Van Nguyen 35  Gluconeogenesis Pathway Red: unique to gluconeogenesis The enzymes for gluconeogenesis are located in the cytoplasm, except for pyruvate carboxylase (in the mitochondria) and glucose 6-phosphatase (membrane bound in the endoplasmic reticulum) Van Nguyen 36  Gluconeogenesis steps Gluconeogenesis retains 7 steps (2, 4-9) of glycolysis Replace the enzymes and substrates in 3 steps Pyruvate carboxylase and PEP carboxykinase replace the pyruvate kinase reaction of glycolysis (step 10) [change enzymes and substrates ] Fructose-1,6-bisphosphatase replaces the phosphofructokinase reaction of glycolysis (step 3) [change enzyme only] Glucose-6-phosphatase replaces the hexokinase reaction of glycolysis (step 1) [change enzyme only] à Gluconeogenesis has overall ΔG < 0 à New mechanisms of regulation Van Nguyen 37 Gluconeogenesis Is Not a Reversal of Glycolysis The ΔG of glycolysis is -74 kJ/mol. If gluconeogenesis were just the reverse of glycolysis, its ΔG would be positive Van Nguyen 38 Summary of Gluconeogenesis Reactions Van Nguyen 39 Pyruvate à Oxaloacetate Details: (1) (2) (3) Pyruvate carboxylase uses the vitamin biotin as a cofactor, and its reaction mechanism occurs in three stages: 1. The biotin carboxylase domain catalyzes the formation of carboxyphosphate 2. The carboxylase then transfers the CO2 to the biotin carboxyl carrier protein (BCCP). 3. The BCCP carries then activated CO2 to the pyruvate carboxylase domain, where the CO2 is transferred to pyruvate. Acetyl CoA is a required cofactor for carboxylation of biotin. Van Nguyen 40 A Subunit of Pyruvate Carboxylase Biotin, covalently attached to the BCCP, transports CO2 from the BC active site to the CT active site of an adjacent subunit. (BC) biotin carboxylase; (BCCP) biotin carboxyl carrier protein; (CT) carboxylase transferase; (PT) pyruvate carboxylase tetramerization domain Structure of Biotin and Carboxybiotin Van Nguyen 41 Oxaloacetate à Phosphoenolpyruvate 1 2a Guanine base The formation of oxaloacetate by pyruvate carboxylase occurs in the mitochondria. 1 2b Oxaloacetate is reduced to malate 2a and transported into the cytoplasm, 2b where it is reoxidized to oxaloacetate with the generation of cytoplasmic NADH 2c 2c PEP is then synthesized from oxaloacetate by phosphoenolpyruvate carboxykinase. 3 3 PEP Van Nguyen 42 Pyruvate to Phosphoenolpyruvate The sum of 2 steps: Van Nguyen 43 Fructose 1,6-bisphosphate to Fructose 6-phosphate Phosphoenolpyruvate is metabolized by the enzymes of glycolysis in the reverse direction until the next irreversible step, the hydrolysis of fructose 1,6-bisphosphate. The enzyme catalyzing this reaction is fructose 1,6- bisphosphatase, an allosteric enzyme. Van Nguyen 44 Generation of Glucose from Glucose 6-phosphate Occurs essentially only in liver Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum by T1 Glucose 6-phosphatase, an integral membrane on the inner surface of the endoplasmic reticulum, catalyzes the formation of glucose from glucose 6-phosphate. T2 and T3 transport Pi and glucose, respectively, back into the cytoplasm In tissues that do not dephosphorylate glucose, glucose 6-phosphate is converted into glycogen for storage. Van Nguyen 45 6 NTPs needed to Form Glucose from Pyruvate The formation of glucose from pyruvate is energetically unfavorable unless it is coupled to reactions that are favorable. The stoichiometry of gluconeogenesis is Note how many more NTPs (nucleoside triphosphate) were hydrolyzed, compared with the number that are hydrolyzed when looking at purely the reverse of glycolysis: 2 Van Nguyen 46  Glycolysis and gluconeogenesis have reciprocal regulation When glycolysis is active, gluconeogenesis is turned off When gluconeogenesis is proceeding, glycolysis is turned off. Van Nguyen 47 The fate of glucose molecule in the cell Synthesis of glycogen Glucose Pentose phosphate pathway Glucose-6- Ribose, Glycogen phosphate NADPH Degradation of glycogen Gluconeogenesis Glycolysis Pyruvate Van Nguyen 48 Pentose phosphate pathway Glycolysis The Calvin cycle: Like glycolysis and gluconeogenesis, CC and PPP are mirror images of each other: the Calvin cycle uses NADPH to reduce carbon dioxide to generate hexoses, whereas the pentose phosphate pathway oxidizes glucose to carbon dioxide to generate NADPH. Van Nguyen 49  The Role of Pentose Phosphate Pathway (Phosphogluconate pathway) (1) Synthesis of NADPH (for reductive reactions in biosynthesis of fatty acids and steroids) (2) Synthesis of Ribose 5-phosphate (for the biosynthesis of ribonucleotides (RNA, DNA) and several cofactors) (3) Pentose phosphate pathway also provides a means for the metabolism of “unusual sugars”, 4, 5 and 7 carbons. Pentose phosphate pathway does not function in the production of high energy compounds like ATP. Van Nguyen 50 Occurrence of the pentose phosphate pathway Liver, mammary and adrenal glands, and adipose tissue Red blood cells (NADPH maintains reduced iron) NOT present in skeletal muscles. All enzymes in the cycle occur in the cytosol Van Nguyen 51 Oxidative Steps Glucose-6-P Dehydrogenase Irreversible 1st step - highly regulated! Gluconolactonase The uncatalyzed reaction happens, too 6-Phosphogluconate Dehydrogenase An oxidative decarboxylation (in that order) Van Nguyen 52  Dehydrogenation The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway. Van Nguyen 53 Gluconolactonase Reaction The gluconolactonase reaction. The uncatalyzed reaction also occurs. Van Nguyen 54  Oxidative Steps The 6-phosphogluconate dehydrogenase reaction. Initial NADP+- dependent dehydrogenation yields a β-keto acid, 3-keto-6- phosphogluconate, which is very susceptible to decarboxyation (the second step). The resulting product, D-ribulose-5-P, is the substrate for the nonoxidative reactions of the pentose phosphate pathway. Van Nguyen 55 Regulation of the oxidative phase Van Nguyen 56  Net reaction for the oxidative phase of pentose phosphate pathway Glucose + ATP + 2NADP+ + H2O ribose 5-phosphate + CO2 + 2NADPH + 2H+ + ADP Van Nguyen 57 Nonoxidative Steps Five steps, but only 4 types of reaction... Phosphopentose isomerase converts ketose to aldose Phosphopentose epimerase epimerizes at C-3 Transketolase ( a TPP-dependent reaction) transfer of two-carbon units Transaldolase (uses a Schiff base mechanism) Transfer of a three-carbon unit Van Nguyen 58 The Nonoxidative Steps of the Pentose Phosphate Pathway The phosphopentose isomerase reaction converts a ketose to an aldose. The reaction involves an enediol intermediate. Van Nguyen 59 The Nonoxidative Steps of the Pentose Phosphate Pathway The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3. Van Nguyen 60 The Nonoxidative Steps of the Pentose Phosphate Pathway The transketolase reaction of step 6 in the pentose phosphate pathway. This is a two-carbon transfer reaction that requires thiamine pyrophosphate as a coenzyme. Van Nguyen 61 The Nonoxidative Steps of the Pentose Phosphate Pathway The transketolase reaction of step 8 in the pentose phosphate pathway. This is another two-carbon transfer, and it also requires TPP as a coenzyme. Van Nguyen 62 The Nonoxidative Steps of the Pentose Phosphate Pathway The mechanism of the TPP-dependent transketolase reaction. The group transferred is really an aldol. Despite this, the name “transketolase” persists. Van Nguyen 63 The mechanism of the TPP- dependent transketolase reaction. The group transferred is really an aldol. Despite this, the name “transketolase” persists. Van Nguyen 64 The Nonoxidative Steps of the Pentose Phosphate Pathway The transaldolase reaction. In this reaction, a 3-carbon unit is transferred, first to an active-site lysine, and then to the acceptor molecule. Van Nguyen 65 The transaldolase mechanism involves attack on the substrate by an active-site lysine. Departure of erythrose-4-P leaves the reactive enamine, which attacks the aldehyde carbon of Gly-3-P. Van Nguyen 66 Nonoxidative phase summary Step 6 Step 7 Step 4 Step 8 Step 5 Van Nguyen 67 Carbon transformation during nonoxidative phase Van Nguyen 68 ATP synthesis vs Nucleotide synthesis Van Nguyen 69 In epithelial cell Van Nguyen 70 In adipocyte Van Nguyen 71 In hepatocyte Van Nguyen 72  The Warburg Effect and Cancer https://www.cancer.gov/news-events/cancer-currents-blog/2016/cancer-metabolism-lactate Otto Warburg observed in 1924 that rapidly proliferating cancer cells metabolize glucose mainly to lactate, even when O2 is plentiful Lewis Cantley has suggested that this behavior arises because cells need more than ATP – they must synthesize large amounts of nucleotides, amino acids, and lipids This requires lots of NADPH for biosynthesis as well as intermediates for building blocks Cancer cells divert large amounts of glucose to the pentose phosphate pathway to produce NADPH Van Nguyen 73 Glycolysis in red blood cell (RBC) Red blood cell function and properties Transport and deliver O2 à deformed shape, no nuclei, no mitochondria (mitochondria consumes O2); No mitochondria à needs glycolysis to generate ATP Glycolysis in RBC: No O2 à forms lactate; Lactate accumulation à acidify cytoplasm à affect Hb function à lactate is transported out to Liver In Liver, lactate is converted back to glucose (then glycogen) via Gluconeogenesis Van Nguyen 74 Fermentation Wine, beer production, muscle sore, … Muscles of higher organisms and humans lack pyruvate decarboxylase and cannot produce ethanol from pyruvate Muscle contain lactate dehydrogenase. During intense activity when the amount of oxygen is limiting the lactic acid can be accumulated in muscles (lactic acidosis). Lactate formed in skeletal muscles during exercise is transported to the liver. Liver lactate dehydrogenase can reconvert lactate to pyruvate. Van Nguyen 75 Preventing ROS Glutathione GSH normally helps to control the amounts of harmful peroxides that are generated in the course of metabolism. Peroxides are a form of ROS. Oxidized glutathione (GSSG) is converted back into reduced glutathione by NADPH. NADPH generated by glucose 6- phosphate dehydrogenase in the pentose phosphate pathway is required to maintain adequate levels of reduced glutathione. Cells with reduced levels of glucose 6- The Reduced Form of the phosphate dehydrogenase are especially Tripeptide Glutathione sensitive to oxidative stress. Van Nguyen 76 Glucose 6-phosphate Dehydrogenase Deficiency Causes a Drug-induced Hemolytic Anemia In red blood cells, which lack mitochondria, the main role of NADPH is to regenerate the reduced form of GSH. Example: fava beans contain a purine glycoside that causes oxidative damage to red blood cells – People deficient in glucose 6-phosphate dehydrogenase suffer hemolysis (destruction of red blood cells) from consuming fava beans or inhaling pollen of the fava flowers (called a favism response). Also, glutathione is required to maintain the normal structure of hemoglobin because it helps to maintain the cysteine residues in their reduced form. – In the absence of glutathione, sulfhydryl bonds occur among hemoglobin molecules, forming aggregates called Heinz bodies, and the red blood cells may lyse. Van Nguyen 77 Vicia faba, the Source of Fava Beans Van Nguyen 78  Red Blood Cells with Heinz Bodies The light micrograph shows red blood cells obtained from a person deficient in glucose 6-phosphate dehydrogenase. The dark particles, called Heinz bodies, inside the cells are clumps of denatured hemoglobin that adhere to the plasma membrane and stain with basic dyes. Red blood cells in such people are highly susceptible to oxidative damage Van Nguyen 79 Make Use of Glucose 6-phosphate Dehydrogenase Deficiency Glucose 6-phosphate dehydrogenase deficiency protects against malaria by depriving the parasites of NADPH that they require for growth. Because the pentose phosphate pathway is compromised, the cell and parasite die from oxidative damage. Van Nguyen 80  Hummingbirds and the Pentose Phosphate Pathway All aerobic animals that are highly active face the problem of damage by ROS, and this problem increases with increasing activity. The hummingbird is an example of one such highly active animal. Van Nguyen 81  Hummingbirds and the Pentose Phosphate Pathway When organisms are using carbohydrates (nectar) only as a fuel, the respiratory quotient (RQ) is 1. When hummingbirds are using carbohydrates as a fuel, the RQ is greater than 1. The extra CO2 comes from the pentose phosphate pathway. Van Nguyen 82 Hummingbirds and the Pentose Phosphate Pathway Due to the high activity of the pentose phosphate pathway (both its oxidative and nonoxidative phases), hummingbirds are using carbohydrate-rich nectar to produce ATP to power muscle activity. While doing so, they are also able to generate NADPH, which protects against ROS. Human athletes who consume carbohydrates during intense, extended exercise may also use the pentose phosphate pathway for ROS protection. Van Nguyen 83 List of phenomena to explain Pentose phosphate pathway in different cell, regulation mechanism Warburg Effect and cancer Glycolysis in RBC and how it affect RBC function Lactose intolerance Fermentation related processes Roles of glucose 6-phosphate dehydrogenase Pentose phosphate pathway and intensive activity Glycolysis regulation in muscle, liver Reciprocal regulation of glycolysis and gluconeogenesis Glycolysis and gluconeogenesis cooperation in a sprint Trio phosphate isomerase deficiency Pyruvate carboxylase deficiency Van Nguyen 84 References Garry Blankenship http://slideplayer.com/slide/8527280/ Viel & Gaudet, Principle of Biochemistry, 2015 Garrett & Grisham, Biochemistry, 2017 Campbell & Farrell, Biochemistry, 2016 Lehninger, Principle of Biochemistry, 2013 Biochemistry, Berg, 2019 Van Nguyen 85