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GLYCOLYSIS Before glycolysis • Before we get to this step, carbohydrates such as starch are broken down into glucose via -amylase Glycolysis in β Cells • Glucose in the blood • • • • enters cells of the pancreas through GLUT2 transporters (KM = 15-20 mM) The cell metabolizes glucose to pyruvate generating ATP Increase in ATP/ADP ratio closes K+ channel This then opens a Ca2+ channel ultimately transporting insulin out of the cell Energy change results in insulin secretion GLUT2 transporter Before Glycolysis • Insulin interacts with insulin receptors which via protein chain transports GLUT4 to the cell surface to admit glucose into cell GLUT4 Glycolysis • Common to virtually all cells –prokaryotic and eukaryotic • Occurs anaerobically in cell cytoplasm • Break glycolysis down into 3 steps • Step 1 • Trap glucose in cell • Form a 6 membered ring fructose 1,6 biphosphate • Which can be broken into two 3-C compounds • Step 2 • Cleave fructose 1,6 biphosphate into 3-C compounds • Step 3 • Oxidize 3-carbon compounds to pyruvate (NAD+ reduced) • Generate ATP • Overall Glucose → 2 pyruvate + 2 ATP Overview of Glycolysis – Stage 1,2 Step 1 – Trapping Glucose in Cell Via Phosphoryl Group Transfer H+ from alcohol + H+ Mechanism Phosphorlyation Glucose • Magnesium ions in enzyme help make P more electrophilic • Enzyme needs a cofactor – Mg2+ Energy Considerations The enzyme - kinases • Kinases transfer phosphoryl groups from a donor (such as ATP above) to a receptor (such as glucose) • In particular if phosphoryl group transferred to a 6 membered ring it is called a hexokinase • The enzyme undergoes large conformational changes during reaction (induced fit—see earlier) The enzyme - kinases • The cleft holding the glucose is closed • Need to exclude water so that with hydrophobic R groups of the enzyme so that PO42- group can approach • Don’t want to hydrolyze ATP to ADP and Pi • Typical KM values for kinases are 0.01 to 0.1 mM so enzyme is typically at maximum reaction rate in vivo. Induced Fit and Water Exclusion Step 2 -Isomerization Reactions • Changing between molecules with same formula but different arrangements of atoms within the molecules • Glucose to Fructose isomerization practiced in industry to make sweeteners (Fructose is much sweeter than Glucose) and it goes into beverages • Isomerization example: C5H12 C5H12 C5H12 C5H12 Glucose and Fructose Glucose – aldehyde sugar Fructose – ketose sugar Cα Carbon Aldehyde-Ketone Isomerization • We will do this isomerization either forward or backward twice during the first two phases • Phase 1: G-6P → F-6P (aldehyde to ketone) • Phase 2: DHAP → DAP (ketone to aldehyde) More “electron pushing” -- in Isomerization a. the -carbon Hydrogen is acidic (electrophilic) due to the presence of the carbonyl oxygen (and alcohol) b. carbon can only have four bonds c. arrows are always drawn from the electron pair to the atom d. electrons are pushed to the oxygen since H can only share 2 electrons in its 1s orbital e. hydrogen on oxygen are acidic (electrophilic) f. charges must balance always g. no atoms can be “lost” since isomers have the same molecular formula – so H must reattach somewhere h. Need to regenerate the catalyst Aldose – Ketose Isomerization +B :H 2 :B2 B1: H:B + H:B11 • Properties of aldehydes due to electron-withdrawing effect of carbonyl oxygen • Makes carbonyl carbon susceptible to nucleophilic attack • Make H on -carbon electophilic • Even more electrophilic here due to presence of oxygen substituent on same carbon Enzyme Catalysts • Note that B1: and B2: are both bases • However, when protonated they are acids, B1:H+ and B2:H+ • Analogy is ammonia. When unprotonated it is a weak base; when protonated it is a weak ammonium acid. + Glucose 6 Phosphate → Fructose 6 phosphate Why the Isomerization? • Ultimately another phosphate group needs to be added to • • • • the molecule so that the 6-membered ring can be cleaved into two 3-membered rings It is easier to phosphorylate a primary alcohol than a secondary alcohol See table for free energy for this reaction At cellular conditions, it is slightly exergonic, so Keq not far from unity But the following phosphorylation reaction (to make F1,6BP) is heavily exergonic so the reaction will shift to the right Alcohols – 1o, 2o 3o Isomerization to F – 6P Simplified Isomerization +H:B 2 B1: +HB 1 :B2 Phosphoryl Group Transfer • Phosphofructokinase (PFK) is an allosteric enzyme • Its activity is very sensitive to the energy status of the cell • Carbon flux through glycolysis is largely controlled in this step • Need two phosphates to insure compound cleaves in middle to two 3-carbon fragments • Don’t want a two-carbon and a four carbon – would require more complex processing Overall summary Cleavage 96% in this form glycerin Forming DHAP and GAP How Do 3-Carbon Fragments Fit Into Glycolysis • GAP (only 4%) can be can be further oxidized to 1,3 BPG, whereas DHAP cannot • Don’t want to waste the DHAP (96%), so nature has provided an isomerization pathway to convert DHAP into GAP as shown below: Forming DHAP and GAP Acidic H Isomerization Mechanism-Another view Stage 3 G-3P Dehydrogenase Setting Up To Harvest Energy • So far we’ve invested 2 molecules of ATP –it’s time to get some back • Start with the transformation of GAP to 1,3 BPG: • (note both Pi = HPO42- and OPO32- have 32 e-) • Top half electrons 12 32 42 2 • NAD+ must be constantly supplied for Glycolysis to continue. Where do we get it? • From oxidizing NADH later in metabolism • How does this reaction work? 0 How’s this done? Two Step Scenario intermediate • First reaction (redox) spontaneous • Second reaction has ∆G > 0 (opposite of phosphoryl hydrolysis) G-3P to 1,3BPG His 176 The Intermediate is Stabilized • Intermediate stabilized by enzyme thioester bond • Coupling: free energy in oxidation reaction (1st reaction) drives the second reaction Why is Thioester bond so energetic? Harvesting Energy • For 1,3 BPG → 3-PG + Pi ΔGo’ = -49.3 • For ADP + Pi → ATP ΔGo’ = +30.5 • Since this is a 3-Carbon molecule, we form 2 moles of ATP for each 6-carbon glucose fed to Glycolysis Making Pyruvate • Reaction 1: rearrangement; Reaction 2: dehydration Phosphoenolpyruvate ∆Ghydrol = -15.6 kJ/mole Ghydrol = -61.9 kJ/mole Keto group more Stable than enol 3-PG to 2-PG Mechanism 2 PG to PEP Making Pyruvate • One PO32- is transferred to ADP (takes energy) get the enol • Enol is an unstable form of pyruvate which is why PEP has such a high Ghyd • Enol converts to more stable keto form (keto form) • Glycolysis has now yield a “profit” of two molecules of ATP/Glucose fed (Remember there are two pyruvates formed for each glucose fed) Keto – Enol Tautomerization H+ Metabolize Pyruvate to Get NAD+ • Use fermentations to oxidize NADH Yeast cells Humans Pyruvate to Ethanol (fermentation) • Carbon in ethanol is reduced –gains two electrons from NADH • Oxidation numbers for carbon • +2 +1 Total valence e-: 18 • Overall: Glucose → Ethanol no net redox -1 20 Pyruvate to Lactate – Our cells Under Anaerobic Exercise • We still must generate NAD+ in order to satisfy the needs of Glycolysis • +2 Total valence e-: 34 0 36 Summary Anaerobic - Lactate • Used by muscles for short bursts of energy if oxygen cannot be supplied rapidly enough to body • Buildup of lactate and lactic acid causes fatigue and “burning” Glycolysis Overall • Glucose + 2Pi + 2ADP+ 2NAD+ → 2 Pyruvate+ 2ATP +2NADH +2H+ + 2H2O • Thus 1 molecule of Glucose has yielded • Two molecules of pyruvate • 2 molecules (net) of ATP • Free energy change: -96 kJ/mole • Generated 2 molecules ATP at 30.5 kJ/mole and 2 moles of a reduced species, NADH • Potential total free energy change possible -2879 kJ/mole • Process thus far occurred anaerobically • In other words no O2 molecules were used as oxidants • For process to continue must oxidize NADH and send it back as NAD+ Steady-State Concentrations of Glycolytic Metabolites in Erythrocytes Metabolite mM Glucose 5.0 Glucose-6-phosphate 0.083 Fructose-6-phosphate 0.014 Fructose-1,60.031 bisphosphate Dihydroxyacetone 0.14 phosphate Glyceraldehyde-30.019 phosphate 1,30.001 Bisphosphoglycerate 2,34.0 Bisphosphoglycerate 3-Phosphoglycerate 0.12 2-Phosphoglycerate 0.030 Phosphoenolpyruvate 0.023 Pyruvate 0.051 Lactate 2.9 ATP 1.85 ADP 0.14 Pi 1.0 Regulation of Glycolysis ∆Go: offers little insight as to control points ∆G: Steps 2 and 4-9 ∆G ≈ 0 (at equilbrium) control points steps 1,3,10,11 Aerobic Metabolism • Pyruvate converted into acetyl CoA and enters the Citric Acid (aka Krebs) cycle. • The NAD+ required for glycolysis is regenerated from NADH in the Electron Transport Chain (ETC) Glycolysis Control – Muscle Cell • Three enzymes are used to control Glycolysis • Step 1: Hexokinase (phosphorylation of glucose) • Step 3: Phosphofructosekinase (mono to di phospho frutose) • Step 10: Pyruvatekinase (Phosphoenolpyruvate to pyruvate) • Irreversible and operate far from equilibrium • Glycolysis is stimulated when energy charge falls • Energy charge [ ATP ] 1 / 2[ ADP] Energy Ch arg e [ ATP ] [ ADP] [ AMP] Glycolysis Control – Muscle Cell • Hexokinase • Inhibited by product – glucose 6-phosphate (G-6P) • Glucose 6-phosphate in equilib with Fructose 6 –phosphate (F-6P) • Phosphofructokinase inhibition will cause F-6P to rise which also incr G-6P • However other sugars (such as fructose) bypass this step so it should not be only control • The inhibition of F-6P leads to inhibition in G-6P Glycolysis Control – Muscle Cell • Phosphofructokinase • Most important regulator in Glycolysis • ATP binds to an allosteric (Greek: other object) site on the enzyme • AMP competes for the site with ATP; AMP has no inhibitory effect • When ATP/AMP ratio lowered → increases enzyme activity • Also inhibited by a decrease in pH (such as when lactic acid is formed; don’t want to damage cell with acid buildup) Glycolysis Control Muscle • ATP allosterically hindered Glycolysis Control – Other Cells • Liver control steps are similar to those of muscle cell (steps 1,3,10) but mechanisms are different • Glucokinase in liver acts as hexokinase in muscle • Phosphofructokinase responds to signal molecule F-2,6,-BP • Pyruvatekinase inhibited by ATP at low glucose levels • But liver has additional functions • Glucose ↔ glycogen (a polymer of Glucose that can be utilized for rapid energy) • Cells have transporter molecules that bring Glucose into and out of cells (GLUT1-GLUT5) • Can bring glucose into different cells as a function of their KM value Glycolysis and Cancer • Tumor cells rapidly convert glucose to lactic acid even in the presence of oxygen (called the Warburg effect) • This glucose uptake is characteristic of aggressive cancers • What is the biochemistry reasoning behind this? • Lactic acid inhibits the immune system from attacking cancer cells • Cancer cells grow more rapidly than blood vessels supplying them so that they have adapted to grow under low oxygen conditions Industrial and Commercial Applications Food Industry ~ Beer ~ Bread ~ Cheese ~ Wine ~ Yogurt Pharmaceutical Industry ~ Insulin ~ Vaccine Adjuvants Energy ~ Fuel Ethanol Theory x Biomass Approx 1st order kinetics The plot showing the trends for yeast cell growth over time Lineweaver-Burk Rearrangement ss k s s s (s) m Michaelis-Menten like kinetics 1 (s) ks s ks 1 1 m s m s m Fermentation Overview • Yeast species: Saccharomyces cerevisiae converts carbohydrates (glucose: a monosaccharide sugar) into CO2 and EtOH • Follow cell growth with CO2 produced • More sophisticated ways are measuring yeast cells by light scattering in a spectrophotometer • Could also do studies on effect of glucose concentration on cell growth Experiment Resazurin turns pink in the presence of oxygen and colorless in the absence of oxygen