BMS100 Glycolysis and Gluconeogenesis Student Notes PDF

Catabolism Glycolysis Dr. Ian Fraser Dr. Rhea Hurnik BMS100 Glycolysis Enzymes Enolase Aldolase Triose Phosphate Isomerase Phosphoglucose isomerase Hexokinase Phosphoglycerate mutase Phosphoglycerate kinase Glyceraldehyde-3-phosphate dehydrogenase Pyruvate kinase Phosphofructokinase 1 Learning...

Catabolism Glycolysis Dr. Ian Fraser Dr. Rhea Hurnik BMS100 Glycolysis Enzymes Enolase Aldolase Triose Phosphate Isomerase Phosphoglucose isomerase Hexokinase Phosphoglycerate mutase Phosphoglycerate kinase Glyceraldehyde-3-phosphate dehydrogenase Pyruvate kinase Phosphofructokinase 1 Learning Outcomes • Draw out glycolysis, including structures of reactants & products, enzyme names, and any regulators of the reaction. List which steps of glycolysis are irreversible • Identify the reaction steps that require an input of energy & the reaction steps that produce energy (indirect vs. direct?) and calculate the energy yield from a cycle of glycolysis Glycolysis - Introduction • As we go through glycolysis there are a few things to make note of: § 1. Reactants, products, & enzyme names • For quiz 2 and exam 1 you will be required to know all the reactants, products and enzymes of glycolysis § 2. Is the reaction reversible or irreversible? § 3. Is the reaction spontaneous? • If not, how is the reaction able to move forward during glycolysis § 𝚫G0 values are FYI to help guide your understanding of thermodynamics Overview – phases • Glycolysis can be divided into two main phases: Glucose Phase I: Preparative Phase ATP ATP Fructose 1,6-bisphosphate DHAP Phase II: ATP - generating phase Glyceraldehyde-3-P 2 NADH + 2 H+ 2 ATP 2 ATP 2 Pyruvate Glycolysis Overview Overview – Reactions 1 à 3 Glycolysis – Reaction 1 • Enzyme: Hexokinase/ Glucokinase 𝚫G0’ = -16.7 kJ/mol • Q: Glucose is now trapped in the cell, why? What is the role of Mg2+ cofactor? Glycolysis – Reaction 2 • Enzyme: Phosphoglucose isomerase H2C - OH 1 𝚫G0’ = +1.67 kJ/mol • Since the 𝚫G0’ is positive, how does the reaction proceed in the forward direction? Glycolysis – Reaction 3 • Enzyme: Phosphofructokinase 1 (PFK-1) 𝚫G0’ = -14.2 kJ/mol • Rate-limiting & committing step of glycolysis • Most important regulated reaction § More to come later Glycolysis – Reaction 3 continued • After the addition of the second phosphoryl group the straight chain form is favoured Overview – Reactions 4 & 5 Glycolysis – Reaction 4 • Enzyme: Aldolase + 𝚫G0’ = +23.9 kJ/mol Glycolysis – Reaction 5 • Enzyme: Triose phosphate isomerase 𝚫G0’ = +7.56 kJ/mol • What would be the effect on glycolysis if triose phosphate isomerase was not present/ able to function? Overview – Reactions 6 & 7 Glycolysis – Reaction 6 • Enzyme: Glyceraldehyde-3-phosphate dehydrogenase Glycolysis - Reaction 7 • Enzyme: Phosphoglycerate kinase 𝚫G0 = -18.8 kJ/mol Overview – Reactions 8 à 10 Glycolysis – Reaction 8 • Enzyme: Phosphoglycerate mutase 𝚫G0 = 4.4 kJ/mol Glycolysis – Reaction 9 • Enzyme: Enolase 𝚫G0 = +7.5 kJ/mol Glycolysis – Reaction 10 • Enzyme: Pyruvate kinase 𝚫G0 = -31.4 kJ/mol Review – phases • Glycolysis can be divided into two main phases: Glucose Phase I: Preparative Phase ATP ATP Fructose 1,6-bisphosphate DHAP Phase II: ATP - generating phase Glyceraldehyde-3-P 2 NADH + 2 H+ 2 ATP 2 ATP 2 Pyruvate Review – direct energy • 2 ATP are required during the preparatory phase of glycolysis • 4 ATP are created during the generating phase of glycolysis § Substrate level phosphorylation • Net of 2 ATP/ glucose molecule Glucose ATP ATP Fructose 1,6-bisphosphate DHAP Glyceraldehyde-3-P 2 NADH + 2 H+ 2 ATP 2 ATP 2 Pyruvate Review – indirect energy • 2 NADH is generated per glucose molecule § Where does the NADH go? Glucose ATP ATP Fructose 1,6-bisphosphate DHAP • Under aerobic conditions, where does pyruvate go next? Glyceraldehyde-3-P 2 NADH + 2 H+ 2 ATP 2 ATP 2 Pyruvate Summary – irreversible reactions • There were three irreversible reactions: § 1) Glucose à Glucose-6-phosphate § 3) Fructose-6-phosphate à Fructose 1,6-bisphosphate § 10) Phosphoenolpyruvate à pyruvate • Preview: § These reactions are the three main regulated steps of glycolysis • Much more to come later in the term Anabolism - GNG Gluconeogenesis Dr. Ian Fraser Dr. Rhea Hurnik BMS100 Learning outcomes • Define the following anabolic processes, including where they occur, their purpose, overall energy input, and the connections between them: Gluconeogenesis, Fatty acid synthesis, Citric acid cycle, Glycogenesis, Lipogenesis, Ketogenesis, Pentose phosphate shunt. § Covered last last class • Outline the pathway for gluconeogenesis and be able to identify the steps of that are not simply a reverse of glycolysis (the bypass reactions): Pyruvate to OAA, F-1,6-bisP to F-6-P, Glucose-6-P to glucose • Identify the GNG substrates and how they feed into gluconeogenesis: lactate, glycerol, glucogenic amino acids Introduction to Gluconeogenesis • Gluconeogenesis is the synthesis of glucose de novo from non carbohydrate precursors • Gluconeogenesis occurs in the liver and kidney § Provides a source of glucose for other tissues in the body • Particularly important during times of fasting Glycolysis review – Thinking question • Is Gluconeogenesis simply a reversal of Glycolysis? Why or why not? Gluconeogenesis overview Glycolysis Gluconeogenesis Gluconeogenesis pathway: 1 Glycolysis Gluconeogenesis Gluconeogenesis – Reaction 1a • By-pass reaction 1: § A) Enzyme: Pyruvate carboxylase • Biotin is coenzyme, helps add a CO2 (carboxylation) Biotin Gluconeogenesis – Reaction 1b • By-pass reaction 1: § B) Enzyme: Phosphoenolpyruvate carboxykinase Gluconeogenesis pathway: 3à7 Glycolysis Gluconeogenesis Glucogeneogenesis – Reactions 2 à 7 • Phosphoenolpyruvate is a glycolytic intermediate and can continue through the reversible reactions of glycolysis 4 3 2 Glucogeneogenesis – Reaction 2 à 7 • Reversible reactions continue until we reach Fructose 1,6bisphosphate 7 6 5 Gluconeogenesis pathway: 8 à 10 Glycolysis Gluconeogenesis Gluconeogenesis – Reaction 9 • Bypass reaction 2 § Enzyme: Fructose 1,6-bisphosphatase *Note: no ATP is generated Gluconeogenesis – Reaction 10 • Enzyme: phosphoglucose isomerase § Reversal of glycolysis Gluconeogenesis – Reaction 10 • Bypass reaction 3 § Enzyme: Glucose-6 Phosphatase *Note: no ATP is generated Gluconeogenesis: Bypass reactions Bypass 3 Glycolysis Gluconeogenesis Bypass 2 Bypass 1 Thinking Question – partners (5 minutes) • Consider the very rare inherited disorders of gluconeogenesis: § § § § PEPCK deficiency Glucose-6-phosphatase deficiency Pyruvate carboxylase deficiency Fructose 1,6-bisphosphatase deficiency • They typically present very early in life with poor growth and development, drowsiness, tremors, & seizures. • What do you think is the underlying cause of the main symptoms of these conditions? § Can you think of a possible treatment for these condition? Substrates for gluconeogenesis • Remember gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors. § Let’s consider some of the precursors: • Lactate • Glycerol • Glucogenic amino acids § Alanine Substrates - Lactate • Lactate is a product of anaerobic glycolysis § Review: How does conversion of pyruvate to lactate under anaerobic conditions support glycolysis? § In addition: Lactate travels from tissues to the liver via the blood. • Once in the liver it is converted back to pyruvate and used to build glucose via gluconeogenesis § Cori Cycle Lactate dehydrogenase Substrates – Lactate – Cori Cycle Liver Muscle Glucose Glucose Glycolysis Gluconeogenesis 2 NADH 2 NAD+ 6 ATP Blood 2 ATP 2 pyruvate 2 NADH Glucose 2 pyruvate LDH 2 lactate 2 NAD+ LDH 2 lactate 2 lactate Gluconeogenesis: Lactate substrate Glycolysis Gluconeogenesis Lactate Substrate - Glycerol • Glycerol can enter gluconeogenesis pathway by being converted to DHAP. § Where might the body get glycerol? Glycerol kinase Glycerol-3-phosphate dehydrogenase Gluconeogenesis: Glycerol substrate Glycolysis Glycerol Gluconeogenesis Substrate – Glucogenic amino acids • Amino acids that can serve as a substrate for gluconeogenesis are called glucogenic amino acids • All amino acids except leucine and lysine are glucogenic § These glucogenic amino acids can either be converted directly into pyruvate or into a citric acid cycle intermediate • TCA cycle intermediates are eventually converted into oxaloacetate to serve as substrate for gluconeogenesis § Glucogenic amino acids that are particularly important are alanine and glutamine. Glucogenic amino acids Glucose Substrate – Alanine • Alanine can be converted to pyruvate • Enzyme: Alanine Transaminase (ALT) § Transamination reaction (transfer of an amino group) • Requires pyridoxal phosphate (PLP) as coenzyme § PLP is derived from B6 PLP Substrate - Alanine • Transamination involves transferring an amino group from an amino acid to an alpha ketoacid NH2 and O switch places Alanine (amino acid) Alpha ketoglutarate (ketoacid) Pyruvate (amino acid) Glutamate (ketoacid) Glucogenic amino acids Glucose Gluconeogenesis: Alanine substrate Glycolysis Gluconeogenesis Alanine Substrates: summary Glycolysis Gluconeogenesis Glycerol Alanine Lactate Shuttle systems • Gluconeogenesis begins in the mitochondria: § Alanine is converted to pyruvate inside the mitochondria § Lactate is converted to pyruvate in the cytosol and pyruvate is immediately shuttled into the mitochondria § Once inside the mitochondria, pyruvate is converted to oxaloacetate • What does that tell us about the cellular location of pyruvate carboxylase? • However, oxaloacetate cannot cross the inner mitochondrial membrane to get back into the cytosol to feed into gluconeogenesis. • Requires shuttle systems • Shuttle systems are slightly different based on the substrate 1. Malate shuttle • Also called malate-aspartate shuttle • Used when the starting substrate is alanine (or any other glucogenic amino acid) • Malate shuttle: § Oxaloacetate is converted to malate § Malate crosses the inner mitochondrial membrane (with the help of an antiporter) 1. Malate shuttle continued • Malate shuttle continued § Once in the cytosol malate is converted back to oxaloacetate § Oxaloacetate be converted to phosphoenolpyruvate to continue gluconeogenesis 2. Shuttle for lactate substrate • Lactate is converted to pyruvate in the cytosol • Pyruvate enters the mitochondria & is converted to oxaloacetate • Oxaloacetate is converted to phosphoenolpyruvate (PEP) § Phosphoenolpyruvate is shuttled out of the mitochondria to continue with gluconeogenesis Test yourself • Glycerol can enter gluconeogenesis by being converted to: § A) Pyruvate § B) Oxaloacetate § C) Dihydoxyacetone phosphate § D) Glyceraldehyade-3-phosphate § C) None of the above References - Glycolysis • Abali, Emine E; Cline, Susan D; Franklin, David S; Viselli, Susan M. Lippincott Illustrated Reviews: Biochemistry (Lippincott Illustrated Reviews Series) (Chapter 8). Wolters Kluwer Health. • Lehninger's Principles of Biochemistry, 6th ed., Figure 14-2, p. 545 References - GNG • Abali, Emine E; Cline, Susan D; Franklin, David S; Viselli, Susan M. Lippincott Illustrated Reviews: Biochemistry (Lippincott Illustrated Reviews Series) (p. 105). Wolters Kluwer Health. • Lehninger's Principles of Biochemistry, 6th ed., Figure 14-2, p. 545 • Lehninger's Principles of Biochemistry, 4thed., Figure 14-16, p. 547 • Images: § BiochemistryProf, CC0, via Wikimedia Commons. https://upload.wikimedia.org/wikipedia/commons/8/86/Glycolysis%26Gluconeogen esis.jpg § Mikael Häggström, CC0, via Wikimedia Commons. https://upload.wikimedia.org/wikipedia/commons/1/16/Amino_acid_catabolism_re vised.png