GLYCOLYSIS Definition: from Greek “glykys” (sweet) & “lysis” (splitting)

“Living organisms, like machines, conform to the law of the conservation of energy, and must pay for all their activities in the currency of catabolism” Ernest Baldwin, Dynamic Aspects of Biochemistry (1952)

I. BACKGROUND Glycolysis Carried out by nearly every living cell In cytosol of eukaryotes Catabolic process Releases energy stored in covalent bonds Stepwise degradation Glucose Other simple sugars

I. Background, cont… Anaerobic process Evolved in an environment lacking O2 Primitive earth … millions of years ago Early, important pathway Provided means to extract energy from nutrient molecules Central role in anaerobic metabolism For the first 2 billion years of biological evolution on earth Modern organisms Provides precursors for aerobic catabolic pathways Short term anaerobic energy source

Supplies metabolic intermediates Three fates Storage Background, cont… Glucose is a precursor Supplies metabolic intermediates Three fates Storage Oxidation to pyruvate Oxidation to pentoses

beta D-Glucose is the major fuel Rich in potential energy Background, cont… beta D-Glucose is the major fuel Rich in potential energy Stored in bonds Is literally solar energy ΔG01= -2840 kJ/mole Advantages to glucose Catabolism  ATP Can be stored Eg: Polysaccarides, sucrose Can be transported Blood glucose Organism to organism

History Background, cont… Began with Pasteur: Mid- nineteenth century Eduard Buchner: 1897 Fermentation in broken extracts of yeast cells Arthur Harden and William Young: 1905 Discover phosphate is required for glucose fermentation Gustov Embden, Otto Meyerhof and Jocob Parnas Seminal work Often called the Embden-Meyerhof-Parnas pathway Elucitated in 1940 Fritz Lipmann and Herman Kalckar: 1941 Metabolic role of high energy compounds like ATP

II. GLYCOLYSIS “Most completely understood biochemical pathway” Sequence of 10 enzymatic pathways 1 molecule of glucose is converted to 2 3-carbon pyruvate molecules Concomitant generation of 2 ATP Key role in energy metabolism Provides free energy for organisms Prepares glucose (and other molecules) for further oxidative degradation

Function, Glycolysis, cont… Most carbon in cells follows this pathway Only source of energy for many tissues Rates and Regulation vary among species Most significant difference is the way that pyruvate is utilized

The fates of pyruvate Aerobic Anaerobic Glycolosis, cont… Oxidative decarboxylation to acetyl 2-cabon molecule Forms acetyl-coenzyme A To Krebs cycle Electrons to ETS Anaerobic To lactate To ethanol

Glucose enters cells Overview of glycolysis in animal metabolism Glycolysis, cont… Overview of glycolysis in animal metabolism Glucose in the blood From breakdown of polysaccharides Liver glycogen Dietary sources Gluconeogenesis Synthesis from noncarbohydrate precursors Glucose enters cells Specific transporters

Enzymes of glycolysis in cytosol Glycolosis, cont… Enzymes of glycolysis in cytosol Glucose converted into 2 3-carbon unites (pyruvate) Free energy harvested to synthesis ATP from ADP and Pi Pathway of chemically coupled phosphorylation reactions 10 reactions broken into 2 phases Preparatory phase (energy investment) Reactions 1 – 5 Payoff phase (energy recovery) Reactions 6 - 10

Preparatory phase (energy investment) Glycolosis, cont… Preparatory phase (energy investment) Hexose glucose is phosphorylated by ATP C3-C4 bond broken yields 2 triose phosphates (glyceraldehyde -3-phosphate) Requires 2 ATP to “prime” glucose for cleavage

Payoff Phase (energy recovery) Each triose phosphate is oxidized Glycolosis, cont… Payoff Phase (energy recovery) Each triose phosphate is oxidized Energy is conserved by reduction of NAD+ Phosphate is transferred to ADP  ATP Net gain: 2 ATP 2 Glyceraldehyde-3-phosphate molecules are converted to 2 molecules of pyruvate NadH must be reoxidized

ATP formation is coupled to glycolysis Glycolosis, cont… ATP formation is coupled to glycolysis Glucose  pyruvate generates 2 ATP (net) Involves coupled reactions Makes glycolysis irreversible under intracellular conditions Most energy remains in pyruvate Glycolysis releases ~ 5% Oxidation via TCA cycle releases the rest

Phosphorylated intermediates are important Glycolosis, cont… Phosphorylated intermediates are important Each intermediate is phosphorylated Phosphate has 3 functions: Prevent diffusion of the intermediates out of the cell Can donate Pi to ADP  ATP Provide binding energy to increase specificity of enzymes

The Reactions of Glycolysis 10 enzymes 9 Intermediates Cost (2 ATP) Payment 4 ATP 2 NADH +H+ End products Metabolic crossroads

Reaction 1 Intermediate formed: Glucose-6-phosphate (G6P) Hexokinase: First ATP Utilization Transfer of a phosphoryl group From ATP To glucose (at C-6) Intermediate formed: Glucose-6-phosphate (G6P) Enzyme: Hexokinase Allosterically inhibited by product REGULATION SITE (one of three) Reaction is irreversible

Reaction 1, cont… Kinase: enzymes that transfers phosphoryl groups between ATP and a metabolite Name of metabolite acceptor is in prefix of the kinase name E.g.: glucokinase (in liver) is specific for glucose Hexokinase: ubiquitous, relatively nonspecific for hexoses D-glucose D-mannose D-fructose

Reaction 1, cont… Second substrate for kinases (including hexokinase) Mg2+ -ATP complex Mg2+ is essential Uncomplexed ATP is a potent inhibitor of hexokinase Mg2+ masks negative charge on phosphate oxygen atoms Makes nucleophilic attack by C6-OH group on gamma-phosphorus atom more possible

Reaction 1, cont…

Substrate induced conformational changes in yeast hexokinase Glucose (magenta) induces significant change … like jaws … this places ATP in close proximity to the C6-H2OH group and excludes water (which prevents ATP hydrolysis)

Reaction 1, cont… Begins glycolysis Is first of 2 priming reactions Reaction is favorable under cellular conditions Hydrolysis of ATP: liberates 30.5 kJ/mol Phosphylation of glucose: costs 13.8kJ/mol Delta G= -16.7 kJ/mol

Reaction 1, cont… Importance of phosphorylating glucose Keeps substrate in the cell Glucose enters cell via specific transporters The transporter does not bind to G6P G6P is negatively charged, thus can not pass through plasma membrane Rapid phosphorylation of glucose keeps intercellular concentrations of glucose low Favors diffusion into cell Regulatory control can be imposed only on reactions not at equilibrium Large negative free energy change make this an important site for regulation

Reaction 1, cont… Glucokinase In liver Carries out same reaction, but is glucose specific (high Km for glucose) Not inhibited by the product Important when blood glucose levels are high Glucose to G6P to stored glycogen Inducible by insulin When blood glucose levels are low, liver uses hexokinase

Reaction 2 Intermediate formed: Fructose-6-phosphate (F6P) Phosphoglucose Isomerase (PGI) Conversion of G6P to Fructose-6-phosphate Isomerization of an aldose to a ketose Intermediate formed: Fructose-6-phosphate (F6P) Enzyme: Phosphoglucose Isomerase Reversible reaction

Reaction 2, cont… Common reaction: isomerization of a sugar Requires ring of G6P to open Isomerization Ring of F6P closes Prep for next reactions R3: Phosphorylation at C-1 R4: cleavage between C-3 and C-4 PGI in humans Requires Mg2+ Highly specific for G6P Reaction is near equilibrium, easily reversible Small delta G value

Reaction 3 Phosphofructokinase: second ATP utilization Phosphorylation of F6P to Fructose-1,6-bisphosphate bis not di: phosphates not together) ATP donates a phosphate Intermediate formed: Fructose-1,6-bisphosphate (FBP or F1,6P) Enzyme: Phosphofructokinase (PFK-1) REGULATION SITE (two of three) Irreversible reaction

Reaction 3, cont… Allosteric regulation of PFK in many organisms Similar to Hexokinase reaction Nucleophilic attack by C1-OH of F6P on Mg2+ -ATP complex PFK plays central role in control of glycolysis Catalyzes one of the pathway’s rate-determining reactions Allosteric regulation of PFK in many organisms

Reaction 4 Intermediates formed: GAP and DHAP Enzyme: aldolase Cleavage of Fructose-1,6-bisphosphate Forms two trioses Glyceraldehyde-3-phosphate (GAP) Dihydroxyacetone phosphate (DHAP) Intermediates formed: GAP and DHAP Enzyme: aldolase Reversible reaction

Reaction 4, cont… A cleavage between C-3 and C-4 two molecules from one Requires: A carbonyl at C-2 A hydroxyl at C-4 Hence the “logic” at reaction 2 2 classes of aldolases Class I: in animal tissues Class II: in bacteria and fungi Require a active-site metal, normally zinc Zn 2+

Reaction 5 Intermediate formed: Glyceraldehyde-3-phosphate Triose phosphate isomerase Interconversion of DHAP and GAP (triose phosphates) Isomerization of aldose-ketose isomers Intermediate formed: Glyceraldehyde-3-phosphate Enzyme: Triose phosphate isomerase Reversible reaction

Reaction 5, cont … Only glyceraldehyde-3-P can continue in glycolysis Dihydroxyacetone-P is rapidly converted

Taking Stock so far Investment phase: Produces 2 triose phoshates One glucose  2 glyceraldehyde-3-P Costs 2 ATP Now, need a little chemical “artistry” to convert low energy GAP to high energy compounds and synthesis ATP

Next … Payoff phase: Produces ATP One glucose  2 glyceraldehyde-3-P Conversion to pyruvate  4 ATP Also 2 reduced NADH

Reaction 6 Intermediate formed: 1,3-bisphosphoglycerate Glyceraldehyde-3-phosphate Dehydrogenase: First “High-energy” Intermediate Formation Oxidation of GAP by NAD+ and Pi Intermediate formed: 1,3-bisphosphoglycerate Enzyme: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE Reaction is reversible Energy-conserving reaction

Reaction 6, cont … Aldehyde is dehydrogenated to an acyl phosphate with a high standard free energy of hydrolysis (ΔG01 = -49.3 kJ/mole) NAD+ serves as hydrogen acceptor: NAD+  NADH + H+

Reaction 7 Intermediate formed: 3-phosphoglycerate Phosphoglycerate kinase: first ATP generation Transfer of a phosphate to ATP Yields ATP & 3-phosphoglycerate Intermediate formed: 3-phosphoglycerate Enzyme: PHOSPHOGLYCERATE KINASE Energy-coupling reactions 6 & 7 A substrate-level phosphorylation

Enzyme: PHOSPHOGLYCERATE MUTASE (PGM) Reaction 8 Conversion of 3 PG to 2-phosphoglycerate (2PG) Intermediate formed: Enzyme: PHOSPHOGLYCERATE MUTASE (PGM) Reversible phosphate shift

Reaction 9 Enzyme: ENOLASE Intermediate formed: phosphoenol pyruvate Dehydration to Phosphoenol Pyruvate (PEP) Intermediate formed: phosphoenol pyruvate Enzyme: ENOLASE Energy-conserving reaction Reversible reaction

Enzyme: Pyruvate kinase Reaction 10 Pyruvate kinase: Second ATP generation Transfer of a phosphate to  ATP Product: pyruvate Enzyme: Pyruvate kinase Irreversible reaction Substrate-level phosphorylation “enol” spontaneously tautomerizes to “keto” form

Overall balance sheet: Glycolosis, cont… Overall balance sheet: Anaerobic: net gain of 2 ATP Must “free” reduced NAD from reaction 6 In humans: lactic acid pathway Aerobic: NADH re-oxidized to NAD+ via respiratory chain in mitochondria e- transfer provides energy for ATP synthesis 2.5 ATP/ reduced NAD Therefore: 5 more ATPs if go aerobic

Anaerobic alternatives for pyruvate Glycolosis, cont… Anaerobic alternatives for pyruvate Must oxidize NAD Lactic acid pathway Fermentation Aerobic alternatives for pyruvate Hydrogens from reduced NAD transported to ETS in mitochondria Transporters in mitochondrial membrane

Dietary polysaccharides: Dietary Disaccharides: Glycolosis, cont… Dietary polysaccharides: must by hydrolyzed to monosaccarides Dietary Disaccharides: Disaccharides cannot enter glycolytic pathway

Hexoses can enter glycolysis Glycolosis, cont… Hexoses can enter glycolysis Hydrolytic enzyes are attached to epithelial cells in intestines Monosaccharides  intestinal cells  blood  liver (phosphorylation)  glycolysis

III. REGULATION of CARBOHYDRATE CATABOLISM Regulatory enzymes act as metabolic valves Substrate-limited reactions are determined by [S] Enzyme-limited reactions are RATE-LIMITING STEPS Irreversible reactions Exergonic regulatory

Regulation of Carbohydrate Catabolism, cont… Regulation of glucose metabolism differs in muscle & liver Muscle: Object is ATP production Enzyme: GLYCOGEN PHOSPHORYLASE Enzyme is allosterically regulated Skeletal muscle signalled to  ATP by EPINEPHRINE Both enzyme & hormone influence ATP production

Regulation of Carbohydrate Catabolism, cont… Liver: object is maintenance of blood glucose levels Regulated by GLUCAGON & [blood glucose] Enzyme: GLUCOSE-6-PHOSPHATE ↓ GLUCOSE-6-P + H2O  GLUCOSE + Pi

Regulation of Carbohydrate Catabolism, cont… Other regulatory enzymes Hexokinase: catalyzes entry of free glucose into gycolysis Pyruvate kinase: catalyzes last step in glycolysis Inhibited by ATP, excess fuel

Regulation of Carbohydrate Catabolism, cont… Phosphofructokinase-1: commits cell to passage of glucose through glycolysis Irreversible reaction Allosterically inhibited by ↑ [ATP] When ATP levels are sufficiently high, glycolysis is turned down Inhibition relieved by allosteric action of ADP & AMP Rate of glycolysis increases when ATP levels are low

Regulation of Carbohydrate Catabolism, cont… Phosphofructokinase-1: links glycolysis and citric acid cycle (CAC) Allosterically inhibited by citrate An intermediate in CAC When citrate accumulates, glycolysis slows down Phosphofructokinase-1also regulated by beta-D-fructose-2,6-bisphosphate Allosteric activator Increases affinity of PFK for F6P

Regulation of Carbohydrate Catabolism, cont… Futile Cycling: simultaneous production & consumption of glucose by the cell Gluconeogenesis: conversion of pyruvate  glucose (opposite of glycolysis) Uses some of the same enzymes as glucolysis

Regulation of Carbohydrate Catabolism, cont… Both sets of reactions are substrate limited Some glycolytic reactions are irreversible (3, catalyzed by regulatory enzymes)

Regulation of Carbohydrate Catabolism, cont… These reactions are by-passed in gluconeogenesis by different enzymes To prevent FUTILE CYCLING, enzymes limited reactions are subject to reciprocal allosteric control

IV. SECONDARY PATHWAYS of GLUCOSE OXIDATION Pentose Phosphate pathway Produces NADPH & ribose-5-phosphate NADPH used in biosynthesis of fatty acids, steroids Pentoses used in nucleic acid synthesis

Secondary Pathways of Glucose Oxidation, cont… Transformation into Glucuromic Acid & Ascorbic Acid D-glucuronate: used to convert non-polar toxins to polar derivatives L-ascorbic acid: cannot be accomplished by humans