Glycolysis, Gluconeogenesis, and Pentose Pathway PDF

Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway Marc A. Ilies, Ph. D. Lehninger - Chapter 14 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the disc...

Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway Marc A. Ilies, Ph. D. Lehninger - Chapter 14 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©MAIlies2024 1  Glucose: central position in metabolism - glucose is an energy rich fuel: C6H12O6 + 6O2 → 6 CO2 + 6 H2O G = - 2840 kJ/mol - potent osmolyte; can be stored under polymeric forms (starch, glycogen); structure? - roles: - energy generation (ATP) via glycolysis (Embden-Meyerhof Pathway) - precursor for synthesis of aminoacids, nucleotides, coenzymes, fatty acids, etc 2  Glucose dynamics in living organisms - the amount of glucose in blood (glycemia) is tightly regulated and is the result of several processes in which glucose is produced or consumed (for regulation of these processes see next chapter): - note different energy storage systems in organisms: - triglycerides (high energy, main energy reserve long-term) - glucose and its polymers (glycogen, starch); enough for 1-2 days - ATP, phosphocreatin (muscles) and other energy rich phosphophate esters, Acetyl-CoA (very dynamic) 3  Glycolysis (Embden-Meyerhof Pathway) - glycolysis, from gr. glykis = “sweet, sugar” + lysis = “splitting”, is a biochemical multistep process (10 steps) in which one molecule of glucose (6C) is cleaved/split into two molecules of pyruvate (3C), harvesting some energy as ATP and NADH: Glycolysis + + energy - breaking a C-C bond requires a lot of energy (activation energy), therefore we distinguish two main phases: - a preparatory phase (A), in which the molecule A Overall: of glucose is activated (with energy consumption) + = - a payoff phase (B), in which the glucose molecule is cleaved, while harvesting energy: B 4 Glycolysis (Embden-Meyerhof Pathway) PFK-1 5 6  Glycolysis: energetics - the overall ecuation of glycolysis Glucose + 2NAD+ + 2ADP + 2Pi 2 Pyruvate + 2NADH + 2H+ + 2ATP + 2H2O can be resolved into two separate processes in order to find the total energetic effect: Glucose + 2NAD+ 2 Pyruvate + 2NADH + 2H+ G10'= -146 kJ/mol 2ADP + 2Pi 2ATP + 2H2O G20'= + 61 kJ/mol GGly0' = - 85 kJ/mol Glycolysis is an essential irreversible process, driven to completion by a net decrease in free energy Glycolysis is not very efficient in terms of energy generation: Glucose full oxidation to CO2 and H2O G0' = - 2,840 kJ/mol Glucose to 2 Pyruvate = - 146 kJ/mol so only 146/2840 x 100 = 5.14 % of total energy is harvested 7  Glycolysis steps: details Step 1 - Glucose enters cells via glucose transporters (GLUT) - Glucose molecule is marked for degradation by phosphorylation: (makes reaction irreversible) - Human genome encodes four hexokinases (I→IV), which catalyze the same reaction (isoenzymes); in hepatocytes: hexokinase IV, which differ from other isoforms (hexokinases I, II in muscles) in kinetic and regulatory properties (see regulation) 8 Step 2 - see slide 8 Bioenergetics for the mechanism Step 3 - the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is essentially irreversible under cellular conditions and it is the first “comitted” step in the glycolytic pathway; fructose 1,6-bisphosphate is targeted for glycolysis 9 Step 4 Step 5 (see “Bioenergetics” for mechanism) 10 11  - beginning of energy harvesting: NADH and ATP are formed; these two steps are Step 6 coupled through the energy rich intermediate 1,3-bisphosphoglycerate: - oxidative phosphorylation (redox + phosphorylation): Step 7 - energy transfer to ATP: - Step 7 is bypassed in RBCs through bypassing the phosphoglycerate kinase and the formation of 2,3-bisphosphoglycerate (BPG), so that there is no NET ATP synthesis in RBCs: 2,3-bisphosphoglycerate bisphosphoglycerate phosphatase mutase 1,3-bisphosphoglycerate 2,3-bisphosphoglycerate 3-phosphoglycerate 12  Step 8 - isomerization: Step 9 - dehydration: Step 10 Energy harvesting (see also slides 13-20,13-26) 13  Regulation of glycolysis - achieved at the level of the 3 irreversible steps of the process: 1. Glucokinase/Hexokinase: Glucose to Glucose-6P 2. Phosphofructokinase: Fructose-6-P to Fructose-1,6-bis-P 3. Pyruvate Kinase: Phosphoenolpyruvate to Pyruvate (Note: deficiencies of this enzyme in humans is the most common genetic defect in glycolytic enzymes.) - the rate of glycolysis is adjusted by a complex interplay by metabolites with regulatory function: Enzyme Activator Inhibitor Hexokinase AMP, ADP, Pi, Glucose-6-phosphate Fructose-6-phosphate PFK-1 Fructose-2,6-bisphosphate NADH, Citrate, ATP Pyruvate kinase AMP, Fructose-1,6-bisphosphate ATP, Acetyl-CoA 14  Feeder pathways for glycolysis - energy from various mono- and disaccharides can be harvested through glycolysis: note that all pathways are convergent 15  Feeder Pathways: details - endogenous glycogen (starch in plants) is degraded by phosphorolysis: Starch structure the product of glycogen degradation is glucose-1-phosphate ! 16  Feeder Pathways: details Galactose is converted to glucose-1-phosphate: - conversion involves two nucleotide intermediates - UDP-galactose and UDP-glucose - genetic defects (usually inherited) in any of the three enzymes results in galactosemias of various severity 17  Fates of pyruvate - pyruvate produced by glycolysis is usually metabolized further: Pasteur effect: glucose is more rapidly consumed when oxygen is absent  (hypoxic) tumors consume more glucose than normal tissues 18  Tissue glucose consumption and tumor detection - many fast-growing tumors lack enough oxygen (are hypoxic) due to insufficient vascularization; in these tumors glucose consumption occurs 10-15X faster than in normal tissues; - glucose consumption can be monitored in vivo using a modified glucose molecule (FdG, a PET probe) and can serve to localize malignancies (both primary tumor and distant metastases): (trapped in cell) (patient ingests FdG, which is quickly absorbed and metabolized to 6-phospho-FdG; a CT scan (left) and a PET scan (middle) are made; superimposing + false coloring of PET density gives the tumor map) 19  Fate of pyruvate under anaerobic conditions - under aerobic conditions, NAD+ is regenerated via electron transfer to O2 in mitochondria: 2NADH + 2H+ + O2 → 2NAD+ + 2H2O - when O2 is not available or it is insufficient, then NAD+ is regenerated in other ways: CH3 CH 2 OH NAD+ Alcohol Dehydrogenase H++ NADH O CH3 C H Acetaldehyde NAD+ Acetaldehyde Dehydrogenase H++ NADH O (skeletal muscles, erythrocytes) CH3 C OH Acetate In yeast In Human Liver 20  Decarboxylation of pyruvate by thiamine (Vit B1) - TPP carries an “active” aldehyde: - lack of B1: Beri-beri (severe lethargy, fatigue, swelling, pain, paralysis, death) 21 22 Gluconeogenesis: carbohydrate synthesis from simple precursors 23  Gluconeogenesis and glycolysis share several steps but not all - irreversible enzymatic degradation steps in glycolysis must be replaced with different enzymatic synthetic steps in gluconeogenesis: 24  Gluconeogenesis: carbohydrate synthesis Step 1 (in mitochondria) moved into cytosol via malate Step 2 (in cytosol) 25  Oxaloacetate is moved from mitochondria to cytoplasm via malate - oxaloacetate synthesized inside mitochondria - mitochondrial membrane has no transporter for oxaloacetate - mitochondrial malate dehydrogenase reduces oxaloacetate to malate at NADH expense (reversible reaction) - malate leaves mitochondria via a specific transporter - in cytoplasm is reconverted to oxaloacetate regenerating NADH 26  Gluconeogenesis: carbohydrate synthesis Step 9 + H2O, (Mg2+) + Fructose 1,6-bisphosphatase (FBPase-1) - 16.3 KJ/mol Step 11 + H2O, (Mg2+) Glucose 6-phosphatase + (simple phosphoric ester hydrolysis) - 13.8 KJ/mol - Glucose 6-phosphatase found only in the luminal side of the ER of hepatocytes, renal cells, and epithelial cells of the small intestine 27 Gluconeogenesis: energetics 28 29  Pentose Phosphate Pathway of Glucose Oxydation = Pentose Pathway or Shunt = Hexose Monophosphate Pathway or Shunt (HMP) - conversion of glucose 6-phosphate into ribose 5-phosphate and NADPH: - the non-oxidative phase recycles pentose phosphates to glucose 6-phosphate: 30 Oxidative Branch Products Precursors for nucleic acids in the form of pentose phosphates NADPH formed: Glu-6P + 2NADP+ + H2O → Ribose-5P + CO2 + 2NADPH + 2H+ Nucleic Acids 31  Pentose pathway vs Glycolysis The amount of NADPH determines the fate of G-6P. The HMP is turned off by high NADPH conc. 32  Oxidative “Stress” and the role of glutathione O O H O N O N O NH 3 H O CH2 SH Reduced glutathione H 2O 2 GSH NADP + Glutathione peroxidase Glutathione reductase 2H 2 O NADPH + H+ O O H From N HMP O N O NH3 H O CH2 S S CH2 NH 3 H O HMP O N N O O O H O GS-SG In RBCs the HMP is the only source for NADPH. Since there is no nucleus G6PD can’t be continually synthesized. 33 34  O O H O CH3 O N N O CH (CH2)3 N Pentose pathway NH N H3 H CH 2 O H N and malaria SH Reduced glutathione O CH3 Oxidized pamaquin H 2 O2 + NADP NADPH + H Glutathione peroxidase Glutathione reductase nonenzymatic 2H2O NADPH + H + NADP O O H O N O N O CH3 NH3 H O CH (CH2)3 N CH 2 NH S H In RBCs, Pamaquin will use NADPH N and get reduced spontaneously. S This use of NADPH may be its O mechanism of action. The decrease CH 2 H H CH3 in NADPH levels leads to a decrease NH3 H O in GSH. Therefore, levels of H2O2 O N O N Reduced will increase and this oxidizer is believed O O H O pamaquin to be what kills the Plasmodium. This is a problem for those with already low NADPH levels from G-6-PD deficiency, Oxidized glutathione they cannot maintain glutathione in the reduced state (GSH) and hemolysis results. 35 Polyol pathway – another alternative to glycolysis – converts glucose to sorbitol Ovaries, Liver Effect of hyperglycemia (uncontrolled diabetes) on sorbitol metabolism: - insulin is NOT needed for glucose entry into cells of lens, nerve, and kidneys - in addition, these cells lack sorbitol dehydrogenase -sorbitol is hydrophilic, it is stuck inside cell and its accumulation causes an osmotic swelling of the cells. - aldose reductase inhibitors have been used experimentally to decrease sorbitol accumulation in the Glycolysis ← lens of diabetics 36 37  Goals and Objectives Upon completion of this series of lectures at minimum you should be able to answer the following: ►What is the role of glucose in metabolism and which are the main pathways involved? ►What pathways are involved in glucose dynamics in living organism? ►Which are the main steps, intermediates and enzymes involved in glycolysis, what are their particularities, what is the energetic balance of the process? ►What fates can have pyruvate in various tissues and organisms, when, and why? ►How is glycolysis regulated, what activators and inhibitors are known, what feeder pathways are known, what are the main characteristics, intermediates, enzymes and their implications in various diseases? ►What is gluconeogenesis, what is its physiologic role, what substrates can be processed, what steps it involves, which steps are common with glycolysis and which are not, what is its energetic balance, what intermediates and vitamines are involved in the steps not common with glycolysis ? ►What is the pentose pathway, which are its main phases, what is its outcome, what compounds are involved, how is regulated and where is this pathway important? ►What is the polyol pathway, what representatives and enzymes are involved, what consequences has the accumulation of sorbitol in various tissues? 38  Drugs and diseases ►Diseases: galactosemia (in infants), cancer, Beri-beri, malaria, diabetes ►Drugs/imaging agents: FdG (a PET probe), thiamine (vitamin B1), pamaquin 39