Ch 06 - Glycolysis Gluconeogenesis & Fates of Pyruvate.pdf

Chapter 6 Carbohydrates I: Mono- and Disaccharides, Glycolysis, Gluconeogenesis, and the Fates of Pyruvate Biochemistry First Edition John Tansey Lecture PowerPoints Tanea Reed Chapter 6 Outline 6.1 Properties, Nomenclature, and Biological Functions of Monosaccharides 6.2 Properties, Nomenclature, a...

Chapter 6 Carbohydrates I: Mono- and Disaccharides, Glycolysis, Gluconeogenesis, and the Fates of Pyruvate Biochemistry First Edition John Tansey Lecture PowerPoints Tanea Reed Chapter 6 Outline 6.1 Properties, Nomenclature, and Biological Functions of Monosaccharides 6.2 Properties, Nomenclature, and Biological Functions of Complex Carbohydrates 6.3 Glycolysis and an Introduction to Metabolic Pathways 6.4 Gluconeogenesis 6.5 The Fates of Pyruvate Copyright © 2019 John Wiley & Sons, Inc. 2 Section 6.3 Glycolysis and An Introduction to Metabolic Pathways Figure 6.11 Pyruvate in metabolism. Glucose and other monosaccharides are catabolized through glycolysis into pyruvate. Pyruvate can be decarboxylated and enter the citric acid cycle, producing other metabolic intermediates or contributing to ATP production. Copyright © 2019 John Wiley & Sons, Inc. 3 Section 6.3 Learning Objective § Understand the metabolic reactions and importance of the glycolytic pathway. Copyright © 2019 John Wiley & Sons, Inc. 4 Dynamic Figure 6.1 Copyright © 2019 John Wiley & Sons, Inc. All rights reserved. Metabolism 6 Metabolic Pathway Defined § Metabolic pathway is the process when a small molecule (a metabolite) is transformed into a different molecule through a series of enzymatic reactions. Can be catabolic or anabolic Can be linear or branched Copyright © 2019 John Wiley & Sons, Inc. 7 Overview of Metabolic Pathways Figure 6.10 Overview of metabolic pathways. Any metabolic pathway consists of intermediates (A, B, C, D, and F) converted to one another by enzymes (E1, E2, E3). Some steps may be reversible, others may not. Pathways may branch or be linear. Copyright © 2019 John Wiley & Sons, Inc. 8 Metabolic Regulation § Genes can be upregulated or downregulated § Enzymes are key in regulation: Presence, location, and activity of enzymes Differential expression of genes encoded for enzymes Compartmentalization of processes Key enzymes can be post-translationally modified Key enzymes can be allosterically regulated § Reaction direction depends on [substrate] and [product] Simple chemistry is still important! Copyright © 2019 John Wiley & Sons, Inc. 9 Overview of Glycolysis Figure 6.12 Reactions of glycolysis. There are ten reactions in glycolysis. The first five are the investment stage, and the final five are where energy is yielded. Copyright © 2019 John Wiley & Sons, Inc. 10 Glycolysis § Overall, it is an oxidative process § One molecule of glucose (six carbons) is catabolized into two pyruvates (three carbons) § Provides energy Carried in ATP and NADH (e- carrier) § Provides entry points for other molecules into metabolism e.g. fructose and glycerol § Pyruvate can be oxidized to acetyl-CoA goes into the citric acid cycle Copyright © 2019 John Wiley & Sons, Inc. 11 Metabolic Breakdown of Carbohydrates Dynamic Figure 6.1 Carbohydrates can be simply classified as monosaccharides (single building blocks) or polysaccharides (polymers of monosaccharides). Polysaccharides can serve structural roles or can serve as a stored form of energy. Monosaccharides can be catabolized into pyruvate. Likewise, pyruvate can be used to synthesize new glucose through gluconeogenesis. Copyright © 2019 John Wiley & Sons, Inc. 12 Glycolysis § What to know for each step? § Enzyme name(!) and whether it catalyzes a reversible reaction § Substrate (usually the product of the previous rxn) Structure (key = number of carbons & phosphates) § Product § Energetics – is it considered favourable? § Extras – cofactors, coenzymes, etc. Copyright © 2019 John Wiley & Sons, Inc. 13 Reactions in Glycolysis § This is going to move quickly, so pay attention! § Ten reactions total § Energy investment phase ATP are “invested” into the process first five steps § Energy yielding phase ATP are produced last five steps Copyright © 2019 John Wiley & Sons, Inc. 14 Step 1 – Hexokinase § Committed (irreversible) step § Phosphorylation § Glucose is phosphorylated to glucose-6-phosphate § Glucose-6-phosphate (G6-P) is trapped in the cell § One molecule of ATP is consumed ∆G’° = -16.7 kJ mol-1 (very favourable) FIRST IRREVERSIBLE REACTION Rule of thumb: if ATP/GTP is part of a reaction, look for Mg2+ as a cofactor! Copyright © 2019 John Wiley & Sons, Inc. 15 Mg2+-Mediated Phosphorylation Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Step 2 – Phosphoglucose Isomerase (PG) § Isomerization § Glucose-6-phosphate is isomerized to fructose-6phosphate (F-6-P) § F-6-P aids in subsequent phosphorylation ∆G’° = 1.7 kJ mol-1 (near zero) Easily reversible Driven forward by product removal by next step Copyright © 2019 John Wiley & Sons, Inc. 17 Step 3 – Phosphofructokinase (PFK) § Committed (irreversible) step § Rate-limiting step § Phosphorylation § Fructose-6-phosphate is phosphorylated to fructose-1,6-bisphosphate (F-1,6bP) § One molecule of ATP is consumed ∆G’° = -14.2 kJ mol-1 (very favourable) SECOND IRREVERSIBLE REACTION Again with the Mg2+ to stabilize charges Copyright © 2019 John Wiley & Sons, Inc. 18 Step 4 – Aldolase § Cleavage § Fructose-1,6-bisphosphate is cleaved into glyceraldehyde3-phosphate (GAP) and dihydroxyacetonephosphate (DHAP) § Retro aldol reaction Schiff base formation § One 6-carbon molecule is cleaved into two 3-carbon molecules ∆G’° = 23.8 kJ mol-1 (unfavourable!) Driven forward by product removal Copyright © 2019 John Wiley & Sons, Inc. 19 Step 5 – Triose Phosphate Isomerase (TIM) § Isomerization § Dihydroxy acetonephosphate (DHAP) is isomerized to glyceraldehyde-3-phosphate (GAP or G3P) § All carbons of glucose can be oxidized § Enediol intermediate § End of energy-investment phase of glycolysis ∆G’° = 7.5 kJ mol-1 (near zero) Easily reversible Driven forward by product removal Copyright © 2019 John Wiley & Sons, Inc. 20 End of First Stage § Entering Pathway: 1 glucose 2 ATP § Enzymes Used: Hexokinase (irreversible), Mg2+ Phosphohexose Isomerase (reversible), Mg2+ Phosphfructokinase (irreversible), Mg2+ Aldolase (reversible) Triose phosphate isomerase (reversible) § Yield: 2 Glyceraldehyde 3-phosphate (GAP) 2 ADP (no ATP made yet!) Step 6 – Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) § Oxidation-reduction § Glyceraldehyde-3 phosphate is oxidized and phosphorylated to form 1,3bisphosphoglycerate (1,3bPG) § NADH + H+ is generated NAD(H/+) is a common cofactor NAD+ accepts two electrons (and a proton) ∆G’° = 6.3 kJ mol-1 (near zero) Easily reversible Driven forward by product removal Copyright © 2019 John Wiley & Sons, Inc. 22 Common Molecules in Redox Chemistry Figure 6.16 Numerous molecules participate in redox chemistry in biochemistry. NAD+ is a small redox active organic molecule that is a critical cofactor in many enzymatic reactions. Electrons transferred to NAD+ are used in subsequent reactions, such as the electron transport chain. Copyright © 2019 John Wiley & Sons, Inc. 23 Step 7 – Phosphoglycerate Kinase (PGK) § Substrate-level phosphorylation § 1,3-bisphosphoglycerate transfers a phosphate to generate 3phosphoglycerate (3-PG) § FIRST ATP molecule is generated § Coupled reaction that causes –ΔG ∆G’° = -18.5 kJ mol-1 (favourable) Reversible even though ATP is made! Copyright © 2019 John Wiley & Sons, Inc. 24 Step 8 – Phosphoglycerate Mutase (PGM) § Isomerization— sort of § 3-phosphoglycerate is converted to 2phosphoglycerate (2-PG) § Why not call this “isomerase”? ∆G’° = 4.4 kJ mol-1 (near zero) Easily reversible Driven forward by high substrate concentration Copyright © 2019 John Wiley & Sons, Inc. 25 FYI: Phospho-His Residue in PGM Active Site Yeast phosphoglycerate mutase PDBid 1QHF Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Mechanism of Mutase Figure 6.17 The mechanism of mutase. Mutase catalyzes the isomerization of 2phosphoglycerate to 3-phosphoglycerate. Exchange of a phosphate between C-2 or C-3 and the active site histidine results in the formation of 2,3-bisphosphoglycerate (an intermediate in this reaction and an important regulator of hemoglobin). Copyright © 2019 John Wiley & Sons, Inc. 27 Step 9 – Enolase § Dehydration § 2-PG is dehydrated to generate phosphoenolpyruvate (PEP) § PEP is better phosphate donor than 2-PG ∆G’° = 7.5 kJ mol-1 (near zero) Easily reversible Driven forward by product removal Copyright © 2019 John Wiley & Sons, Inc. 28 Step 10 – Pyruvate Kinase (PK) § Committed (irreversible) step § Substrate-level phosphorylation § Phosphoenolpyruvate transfers its phosphate to form pyruvate (final product) § One molecule of ATP is generated § End of energy-yielding stage ∆G’° = -16.7 kJ mol-1 (very favourable) THIRD & FINAL IRREVERSIBLE REACTION Mg2+ or Mn2+ required K+ required as an activator of PK Copyright © 2019 John Wiley & Sons, Inc. 29 Pyruvate Tautomerization Drives ATP Production Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Second Stage of Glycolysis § Entering Second Stage: 2 glyceraldehyde-3 phosphate 2 NAD+ 4 ADP § Enzymes Used: GAPDH, NAD+ (reversible) Phosphoglycerate kinase, Mg2+ (reversible) Phosphoglycerate mutase (reversible) Enolase (reversible) Pyruvate kinase, Me2+ (irreversible) § Yield (per glucose): 2 Pyruvate 4 ATP, 2 NADH Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Glycolysis Summary § Ten-step anaerobic process § One glucose molecule is converted into two pyruvate molecules § Two ATP molecules are generated § Two NADH + H+ are generated Copyright © 2019 John Wiley & Sons, Inc. 32 Energetics of Glycolysis Figure 6.18 Energetics of glycolysis. A. In the standard state six of the steps of glycolysis are energetically unfavorable and would proceed in the reverse direction. B. Due to the concentrations of metabolites found in the cell, these reactions proceed favorably with much lower DG values. Copyright © 2019 John Wiley & Sons, Inc. 33 Energetics of Glycolysis Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Regulation of Glycolysis Figure 6.19 Regulation of glycolysis. The regulation of glycolysis occurs at several points in the pathway and through a variety of mechanisms, including both post translational modification and allosteric regulation. Key to learning regulation principles: Ask: does it make sense (chemically and metabolically)? How does the process you are looking at fit into the bigger picture of metabolism? (Think of those maps…) Copyright © 2019 John Wiley & Sons, Inc. 35 Regulation of PFK-2 § Rate-limiting step for glycolysis Figure 6.20 Regulation of phosphofructokinase-2 (PFK-2). PFK-2 has both kinase and phosphatase activities. The activity of PFK-2 is regulated by PKA and AMPK. Copyright © 2019 John Wiley & Sons, Inc. 36 Feed Forward Activation Defined § Feed forward activation is a type of regulation where the products of a reaction act to further activate the pathway. This is observed in PFK-2 and mediated by PKA Copyright © 2019 John Wiley & Sons, Inc. 37 Entry Points for Glycolysis Figure 6.21 Other monosaccharides catabolized through glycolysis. Mannose, fructose, and galactose are also catabolized through glycolysis. Glycolysis is a major thoroughfare in carbon and energy metabolism in cells “Metabolic Main Street” Copyright © 2019 John Wiley & Sons, Inc. 38 6.3 Concept Check (1 of 2) Which step(s) of glycolysis must be coupled by ATP hydrolysis to produce a spontaneous reaction? Copyright © 2019 John Wiley & Sons, Inc. 39 Section 6.4 Gluconeogenesis § Synthesis of glucose from various metabolites § Not the opposite of glycolysis six common reactions (but some different ones) § Can be made from pyruvate, lactate, glycerol, and several amino acids § Energetically expensive requires six ATP per glucose (glycolysis only yields four) Copyright © 2019 John Wiley & Sons, Inc. 40 Section 6.4 Learning Objective § Understand the metabolic reactions and pathway for gluconeogenesis. Copyright © 2019 John Wiley & Sons, Inc. 41 Reactions of Gluconeogenesis Figure 6.23 Reactions of gluconeogenesis. Gluconeogenesis differs from glycolysis by four different enzymes (three steps). Pyruvate is converted to oxaloacetate by pyruvate carboxylase. Phosphoenolpyruvate is formed from oxaloacetate by phosphoenolpyruvate carboxykinase (PEPCK). The steps of glycolysis now proceed in the reverse direction until fructose-1,6-bisphosphate is synthesized. Removal of the last two phosphates to yield glucose is accomplished by fructose bisphosphatase and glucose-6phosphatase. Copyright © 2019 John Wiley & Sons, Inc. 42 Reactions of Gluconeogenesis Copyright © 2019 John Wiley & Sons, Inc. 43 Novel Reactions in Gluconeogenesis (1 of 4) § Pyruvate carboxylase point of control pyruvate is converted to oxaloacetate one ATP is consumed Copyright © 2019 John Wiley & Sons, Inc. 44 Novel reactions in Gluconeogenesis (2 of 4) § Phosphoenolpyruvate carboxykinase (PEPCK) point of control oxaloacetate is converted to phosphoenolpyruvate one GTP is consumed (ATP equivalent) Copyright © 2019 John Wiley & Sons, Inc. 45 Reactions of Gluconeogenesis Copyright © 2019 John Wiley & Sons, Inc. 46 Novel reactions in Gluconeogenesis (3 of 4) § Fructose-1,6bisphosphatase regulated by allosteric control F-1,6-bP is converted to F-6-P Copyright © 2019 John Wiley & Sons, Inc. 47 Novel reactions in Gluconeogenesis (4 of 4) § Glucose-6phosphatase Glucose-6-phosphate is converted to glucose Copyright © 2019 John Wiley & Sons, Inc. 48 Pathways That Flow Through Gluconeogenesis Figure 6.22 Pathways that flow through gluconeogenesis. Glucogenic precursors include lactate, pyruvate, glycerol, and glucogenic amino acids. Copyright © 2019 John Wiley & Sons, Inc. 49 Regulation of Gluconeogenesis § Occurs through compartmentalization § Allosteric regulation § Phosphorylation § Changes in gene expression Copyright © 2019 John Wiley & Sons, Inc. 50 Regulation of Gluconeogenesis Figure 6.24 Regulation of gluconeogenesis. The regulation of gluconeogenesis is in many ways the reciprocal of glycolysis. Additionally, in eukaryotic cells the first two reactions of gluconeogenesis are compartmentalized to the mitochondria. Copyright © 2019 John Wiley & Sons, Inc. 51 Control of Gluconeogenesis § Control of gluconeogenesis and glycolysis is reciprocal Can you think of examples of reciprocal regulation in other cellular pathways? Why is this a good idea? § When glycolysis proceeds, gluconeogenesis is inhibited energetic level of cell is low § When gluconeogenesis proceeds, glycolysis is inhibited plasma glucose is low Copyright © 2019 John Wiley & Sons, Inc. 52 6.4 Concept Check If glucose levels are low, which process is more prone to occur? a. b. c. d. glycolysis gluconeogenesis fructolysis glycogenesis Copyright © 2019 John Wiley & Sons, Inc. 53 Time for a Break! Stop, breathe, and stretch. Let glucose metabolism sink in before moving forward. Work with your Team and review this information to prepare for the Module 01 RATs next week! And later, we will talk about pyruvate…. J 54 Metabolism 55 Section 6.5 Fates of Pyruvate § Aerobic conditions – converted to acetyl-CoA oxidative process (extracts electrons from pyruvate) yields one NADH + H+ catalyzed by pyruvate dehydrogenase § Anaerobic conditions reductive process (adds electrons to pyruvate) yields lactate or ethanol (or other products) § Other molecules made from pyruvate: alanine and oxaloacetate Copyright © 2019 John Wiley & Sons, Inc. 56 Section 6.5 Learning Objective § Understand the biological consequences of pyruvate. Copyright © 2019 John Wiley & Sons, Inc. 57 Fates of Pyruvate Figure 6.25 Fates of pyruvate. There are five fates of pyruvate discussed in this section, four of which are carried out in animals (decarboxylation to acetaldehyde is only carried out by microbes, such as some yeast and bacteria). Copyright © 2019 John Wiley & Sons, Inc. 58 Pyruvate Dehydrogenase § Multienzyme complex found in mitochondrial matrix § Consists of three enzymes E1 - pyruvate dehydrogenase E2 - dihydrolipoyl transacetylase E3 - dihydrolipoyl dehydrogenase § Committed step between carbohydrate metabolism and lipid metabolism (one-way!) § One NADH/H+ is generated (+ one CO2) Copyright © 2019 John Wiley & Sons, Inc. 59 Mechanism of Pyruvate Dehydrogenase 60 Acetyl-CoA & Biological Tethers Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Regulation of Pyruvate Dehydrogenase (1 of 2) § Product inhibition (NADH and acetyl-CoA) and phosphorylation (phosphorylation inhibits E1 of PDH; dephosphorylation activates E1) § Phosphatase is regulated by insulin, AMP, ATP, phosphoenolpyruvate, NADH, and acetyl-CoA Copyright © 2019 John Wiley & Sons, Inc. 62 Regulation of Pyruvate Dehydrogenase (2 of 2) Figure 6.28 Regulation of pyruvate dehydrogenase. E1, E2, and E3 indicate the subunits of the pyruvate dehydrogenase complex. The numbers on arrows represent the individual reactions. Pyruvate dehydrogenase is regulated through substrate availability and product inhibition and through phosphorylation of the E1 subunit. Pyruvate dehydrogenase kinase and phosphatase regulate the activity of the E1 subunit and are regulated by many small molecules including ATP, pyruvate, and acetyl-CoA. Copyright © 2019 John Wiley & Sons, Inc. 63 Lactate Dehydrogenase § Pyruvate is reduced to lactate anaerobically one molecule of NAD+ is generated two isoforms: muscle and heart Copyright © 2019 John Wiley & Sons, Inc. 64 Alanine Production from Pyruvate § Keto acids can be converted to amino acids by adding an amino group § Pyruvate is transaminated with glutamate to form alanine and a-ketoglutarate by alanine aminotransferase (ALT) § Alanine is shuttled into the bloodstream into the liver Copyright © 2019 John Wiley & Sons, Inc. 65 Anaplerotic Reactions Defined § Anaplerotic reactions are reactions that build up levels of citric acid cycle intermediates, and restore levels of oxaloacetate. Example is pyruvate carboxylase Copyright © 2019 John Wiley & Sons, Inc. 66 Pyruvate Carboxylase § Part of gluconeogenesis pathway § Oxaloacetate formation from pyruvate + CO2 ATP hydrolysis provides energy § Oxaloacetate is also transaminated into aspartate Copyright © 2019 John Wiley & Sons, Inc. 67 Alcohol Production from Pyruvate § Pyruvate decarboxylase forms acetaldehyde and CO2 § Alcohol dehydrogenase generates alcohol from acetaldehyde § NADH/H+ cofactor § Microbes must be included-they do this! § Important in the brewing process Copyright © 2019 John Wiley & Sons, Inc. 68 Copyright Copyright © 2019 John Wiley & Sons, Inc. All rights reserved. Reproduction or translation of this work beyond that permitted in Section 117 of the 1976 United States Act without the express written permission of the copyright owner is unlawful. Request for further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. The purchaser may make back-up copies for his/her own use only and not for distribution or resale. The Publisher assumes no responsibility for errors, omissions, or damages, caused by the use of these programs or from the use of the information contained herein. Copyright © 2019 John Wiley & Sons, Inc. 69