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Glycolysis Andy Howard Introductory Biochemistry 25 March 2008 25 Mar 2008 What we’ll discuss Glycolysis Overview Steps through TIM Steps to pyruvate Fate of pyruvate Glycolysis Glycolysis (continued) Free energy Regulation Other sugars Entner-Doudoroff Pathway* p. 2 of 56 25 Mar 2008 Glycolysis Now we’re ready for the specifics of metabolism Why glycolysis first? Well-understood (?) early on Illustrates concepts used later Inherently important Glycolysis p. 3 of 56 25 Mar 2008 The big picture Conversion of glucose to pyruvate Catabolic, ten steps, energy-yielding Overall reaction: glucose + 2 ADP + 2 NAD+ + 2Pi 2pyruvate + 2ATP + 2NADH + 2H+ + 2H2O Glycolysis p. 4 of 56 25 Mar 2008 Significance Why is this important? Energy production (ATP and NADH) Pyruvate as precursor to various metabolites Some steps require energy So it isn’t all energy-yielding The net reaction yields energy Glycolysis p. 5 of 56 25 Mar 2008 The reactions See fig. 11.2 and the table in the HTML notes Wide variety of enzyme sizes Most structures have been determined by X-ray crystallography Glycolysis p. 6 of 56 25 Mar 2008 The pathway through TIM Fig. courtesy U.Texas Glycolysis p. 7 of 56 25 Mar 2008 Pathway to pyruvate Bottom half of same graphic Glycolysis p. 8 of 56 25 Mar 2008 G-6-P Hexokinase Transfers γ-phosphoryl group of ATP to oxygen atom at C-6 of glucose, producing glucose 6phosphate and ADP. Coupling between ATP hydrolysis and an energy-requiring reaction is very close: phosphate is transferred directly from ATP to the recipient molecule, in this case glucose. The reaction catalyzed by hexokinase is energetically favored: Go’ = -22.3 kJ/mol Glycolysis p. 9 of 56 PDB 2YHX Yeast 52kDa monomer 25 Mar 2008 Hexokinase isozymes various isozymes (functionally related but structurally slightly distinct) forms of hexokinase in humans liver form has Km in millimolar range, perhaps a factor of 1000 higher than the Km of hexokinase found in other tissue Liver form is therefore much less active than the other forms unless the liver glucose concentration is high Glycolysis p. 10 of 56 25 Mar 2008 Activity and complexity Hexokinase is active on sugars besides glucose; activity against mannose is comparable to the activity on glucose Hexokinase has the highest molecular mass per monomer of any of the glycolytic enzymes; given that it is the first enzyme in an important pathway, it makes sense that it is large and complex. Glycolysis p. 11 of 56 25 Mar 2008 Phosphoglucomutase Interconverts phosphorylated forms of glucose— glucose 1-P and glucose 6-P. Intermediate is bisphosphorylated equilibrium between the 1-P and 6-P forms is determined by relative concentrations. Active on other phosphorylated aldoses in addition to glucose. This enzyme doesn’t appear on the chart: not part of the linear pathway from glucose to pyruvate. Glycolysis p. 12 of 56 PDB 1ZOL Lactococcus 24 kDa monomer 25 Mar 2008 Glucose 6-phosphate isomerase F-6-P interconverts two monophosphorylated sugars— glucose 6-phosphate and fructose 6-phosphate. Interconversion proceeds through (1,2) ene-diol intermediate with enzyme present the energy barriers around this ene-diol are lowered enough to speed the interconversion. Also called phosphohexoseisomerase or phosphoglucose isomerase Glycolysis p. 13 of 56 25 Mar 2008 Properties of G6P isomerase Dimeric enzyme plays roles extracellularly as well as intracellularly: it can function as a nerve growth factor. Each monomer contains two unequal-sized domains, and the active site is formed by the association of the two subunits. Glycolysis PDB 1U0F mouse 124 kDa dimer p. 14 of 56 25 Mar 2008 Phosphofructokinase-1 catalyzes phosphorylation at the 1 position of fructose 6-phosphate. F-1,6-bisP example of a kinase that acts on an already-phosphorylated form, creating a bisphosphorylated compound. ADP sometimes acts as an allosteric activator on this enzyme as well as being a product of the reaction. We’ll discuss PFK-2 later Glycolysis p. 15 of 56 25 Mar 2008 PFK-1 structures Of all the enzymes in this pathway it appears to be the one for which the least structural information is available Best structure determined to date for the allosteric enzyme was Phil Evans's 2.4 Å structure from 1988, and there have not been many other structures done. Glycolysis PDB 4pfk E.coli 140 kDa tetramer p. 16 of 56 25 Mar 2008 Lactobacillus PFK This one isn’t allosteric No MgADP binding observed (> 20 mM) Yet it’s highly homologous! Effector binding site is very different Glycolysis PDB 1zxx Lactobacillus bulgaricus 140 kDa tetramer p. 17 of 56 25 Mar 2008 + Aldolase Catalyzes actual C-C bond cleavage: fructose 1,6-bisphosphate D-glyceraldehyde-1-phosphate + dihydroxyacetone phosphate large and important enzyme Some bacterial and yeast forms require a divalent cation as a cofactor; eukaryotic aldolases do not. The non-cationic forms proceed through an imine (Schiff-base) intermediate. Glycolysis p. 18 of 56 25 Mar 2008 Secondary activity Enzyme is active on fructose 1-phosphate as well as its "standard" substrate, fructose 1,6bisphosphate; in this context it forms part of catabolic pathway by which fructose itself can be used as an energy and carbon source. Glycolysis PDB 1zah Rabbit muscle 80 kDa dimer p. 19 of 56 25 Mar 2008 Triosephosphate isomerase Interconverts two 3-C phosphosugars possibly the most efficient enzyme known, in terms of the rate acceleration afforded by the enzyme relative to the uncatalyzed reaction. Glycolysis p. 20 of 56 25 Mar 2008 TIM Barrels TIM is an enzyme with a characteristic structure in which alpha helical stretches alternate with beta strands such that the beta strands curve around to form a barrel-like structure with the helices outside. This structural motif appears in many other enzymes, and has become known as a "TIM barrel." Glycolysis p. 21 of 56 PDB 1YPI Saccharomyces 27 kDa monomer 25 Mar 2008 Glyceraldehyde 3phosphate dehydrogenase medium-sized dimeric or tetrameric enzyme responsible for the conversion of Glyc-3P to 1,3bisphosphoglycerate. Somewhat allosteric Glycolysis PDB 1GD1 Bacillus stearothermophilus 74 kDa dimer p. 22 of 56 25 Mar 2008 Phosphoglycerate kinase Glycolysis catalyzes dephosphorylation of 1,3-bisphosphoglycerate to 3phosphoglycerate with production of ATP from ADP named for reaction running in opposite direction relative the one shown in chart. In the direction shown in the table it produces ATP rather than consuming it. p. 23 of 56 25 Mar 2008 PGK Structural Notes Has a hinge motion about a point near the center of the molecule; the open and closed forms of the enzyme involve movements as large as 17Å in the residues farthest from the hinge point. Enzyme is primarily alphahelical in conformation. Glycolysis PDB 16pk Trypanosoma brucei 184 kDa tetramer; monomer shown p. 24 of 56 25 Mar 2008 Phosphoglycerate mutase interconverts 3phosphoglycerate and 2phosphoglycerate Mechanism of reaction involves formation of 2,3bisphosphoglycerate via transient phosphorylation of a histidine residue of the enzyme. Glycolysis p. 25 of 56 2-phosphoglycerate PDB 1e59 E.coli 55 kDa dimer; monomer shown 25 Mar 2008 PG Mutase: a problem! 2,3BPG can diffuse from phosphoglycerate mutase, 2,3however, leaving the enzyme bisphosphoglycerate trapped in an unusable state. Cells make excess 2,3BPG (using the enzyme bisphosphoglycerate mutase) in order to drive 2,3BPG back to phosphoglycerate mutase, so the reaction can go to completion. Glycolysis p. 26 of 56 25 Mar 2008 Enolase interconverts 2phosphoglycerate & phosphoenolpyruvate This reaction plays a role in gluconeogenesis as well as glycolysis. PDB 4enl Saccharomyces 97 kDa dimer; monomer shown Glycolysis p. 27 of 56 25 Mar 2008 Enolase details Mg2+ ions are required for activity, at least in some forms of the enzyme. Vertebrate genes code for two slightly different forms of the monomer of enolase, alpha and beta. Most of the enolase in fetal tissue is alpha-alpha; mature skeletal muscle contains beta-beta; some alpha-alpha remains in smooth muscle tissue. Glycolysis p. 28 of 56 25 Mar 2008 Pyruvate Kinase transfers a phosphate from phosphoenolpyruvate to ADP, producing pyruvate and ATP The reaction is essentially irreversible (Go’ ~ -30 kJ mol-1) Fructose 1,6bisphosphate, the substrate for the aldolase reaction, is a feed-forward activator of the reaction Glycolysis PDB 1PKM Cat muscle 236 kDa tetramer monomer shown p. 29 of 56 25 Mar 2008 So we’ve gotten to pyruvate This is conventionally seen as the endpoint of glycolysis It’s worthwhile, though, to see what can happen to the products Pyruvate (memorize that structure!) is an important intermediate in several pathways Glycolysis p. 30 of 56 25 Mar 2008 What happens to pyruvate? Four paths: Pyruvate + CoASH acetylCoA + CO2; this leads to Krebs cycle, to fatty acid biosynthesis, and amino acids Pyruvate + CO2 oxaloacetate; this is an anapleurotic mechanism for Krebs cycle Pyruvate + NADH + H+ lactate + NAD+ Pyruvate + H+ acetaldehyde + CO2 acetaldehyde + NADH + H+ ethanol + NAD Glycolysis p. 31 of 56 25 Mar 2008 Pyruvate to Lactate Lactate dehydrogenase catalyzes pyruvate + NADH + H+ lactate + NAD+ Occurs in some anaerobic bacteria and in mammals (e.g. in muscles) if oxygen is not plentiful: anaerobic glycolysis Net glycolysis reaction under these conditions: glucose + 2 Pi2- + 2 ADP3- 2 lactate- + 2 ATP4- + 2H2O Can result in drop in blood pH until reverse reaction (in liver) restores pH and regenerates pyruvate Glycolysis p. 32 of 56 25 Mar 2008 Lactate dehydrogenase Typical tetrameric Rossmann-fold NADdependent dehydrogenase Structural homology to other NAD-binding enzymes Glycolysis p. 33 of 56 PDB 1xiv Plasmodium 140 kDa tetramer 25 Mar 2008 Pyruvate to ethanol Pyruvate decarboxylated to acetaldehyde: pyruvate + H+ acetaldehyde + CO2 Acetaldehyde is reduced to ethanol: acetaldehyde + NADH + H+ ethanol + NAD Net glycolytic reaction is glucose + 2 Pi2- + 2 ADP3- + 2H+ 2 ethanol + 2CO2 + 2 ATP4- + 2H2O Yeast depend on this pathway Glycolysis p. 34 of 56 25 Mar 2008 Pyruvate decarboxylase Catalyzes first reaction in pathway to ethanol TPP-dependent reaction: see section 7.7, especially fig. 7.15 Related to the pyruvate dehydrogenase complex that we will meet in chapter 13 Glycolysis p. 35 of 56 PDB 1pvd Saccharomyces 62 kDa monomer 25 Mar 2008 Alcohol dehydrogenase Second reaction in fermentation path Reaction itself is reversible: ethanol acetaldehyde direction leads to detox in humans Often unselective: can be used to oxidize other primary alcohols Glycolysis p. 36 of 56 PDB 2hcy Saccharomyces 156 kDa tetramer 25 Mar 2008 Free energy in glycolysis Cliché: G matters, not Go’! See fig. 11.11: Several reactions are endergonic as far as Go’ are concerned, but they’re flat or exergonic with G. Glycolysis p. 37 of 56 25 Mar 2008 Hamori’s data Step Reactant 0 1 2 3 4 5 6 7 8 9 Glucose, ATP G6P F6P+ATP FDP Glyc3P+NAD+Pi+ADP 3PG PG2 PEP+ADP PYR+NADH Products DGo' G6P, ADP F6P FDP + ADP 2 Glyc-3P 3PG+ATP+NADH 2PG PEP PYR+ATP Lac+NAD 0 -5.1 0.49 -4.3 7.4 -6.5 2.1 -1.3 -12.2 -11.9 DG Cum DGo' 0 -9.5 -0.06 -6.2 -0.17 -0.56 -0.27 -0.64 -7.4 0 0 -21.3 -19.3 -37.3 -6.3 -33.5 -24.7 -30.2 -81.2 -131.0 Sum DG 0 -39.7 -40.0 -65.9 -66.7 -69.0 -70.1 -72.8 -103.8 -103.8 E. Hamori (1975) J.Chem.Ed. 52: 370 Individual values in kcal mol-1 Cumulative values in kJ mol-1 Glycolysis p. 38 of 56 25 Mar 2008 My version of fig. 11.11 Standard and actual free energy 0 0 1 2 3 4 5 6 7 8 9 10 Cumulative free energy changes, kJ mol-1 -20 -40 Cum DGo' Sum DG -60 -80 -100 -120 Data from Hamori (1975), J.Chem.Ed.52:370 -140 Step in glycolysis Glycolysis p. 39 of 56 25 Mar 2008 Which steps are irreversible? Just three: Glucose to G-6P (G ~ -40 kJ mol-1) Fructose-6-P to Fructose-1,6-bisP (-26) PEP to pyruvate (-31) All the others are reversible So the controls are likely to be at those three points: and they are! Glycolysis p. 40 of 56 25 Mar 2008 Regulation of glycolysis Two ways to study this: Enzymology (know thy enzymes) Metabolic biochemistry (know concentrations and fluxes under cellular conditions) Sometimes enzymology gives interesting but cellularly unrealistic results (e.g., inhibitors that only inhibit at 100 * actual cellular concentrations) Glycolysis p. 41 of 56 25 Mar 2008 Regulators of glycolysis See fig. 11.12: Glucose-6-P inhibits hexokinase ATP and citrate inhibit PFK-1 AMP, Fructose 2,6-bisP activate PFK1 F 1,6-bisP activates pyruvate kinase ATP inhibits pyruvate kinase Glycolysis p. 42 of 56 25 Mar 2008 Control at the transport level [glucoseintracellular] < [glucoseblood] (except in liver); passive transport aided by transporters All mammalian cells have transporters Na+ dependent cotransport: SGLT1 in intestinal & kidney cells GLUT family (1-7) found in other cells Glycolysis p. 43 of 56 25 Mar 2008 Insulin and Glut4 (fig. 11.13) When insulin binds to tyr-kinase receptors, they dimerize and promote fusion of intracellular vesicles with the plasma membrane Vesicles carry Glut4 transporters This happens only in striated muscle and adipose tissue—that’s where the Glut4 transporters are This is only one of several roles that insulin plays in glucose and lipid metabolism Glycolysis p. 44 of 56 25 Mar 2008 How does glucose get in? SGLT1 and GLUT4 stories (above) GLUT1,3 provide basal intake levels GLUT2 brings glucose in & out of liver GLUT5: fructose in small intestine GLUT7: G6P from cytoplasm to ER Doesn’t stay neutral long: once it gets into the cell, it gets 6phosphorylated with help of hexokinase Glycolysis p. 45 of 56 25 Mar 2008 Regulation of hexokinase Isozymes I,II,III: Km ~ 0.1mM; G6P allosterically inhibits the enzyme Glucokinase (IV): unregulated, high Km … found in liver & islet cells Pileup of G6P occurs if downstream steps are inhibited; allostery in hexokinase I-III alleviates that Glycolysis p. 46 of 56 25 Mar 2008 GKRP and F-6P: regulators of liver glucokinase Glucokinase regulatory protein binds glucokinase in presence of F-6-P and F-1-P Lowers affinity to ~ 10mM sigmoidal kinetics With high [glucose], GKRP pulls GK into nucleus; low [glucose] makes GKRP release GK so it can phosphorylate glucose Glycolysis p. 47 of 56 25 Mar 2008 Regulation of PFK-1 Nucleotides: ATP is both substrate and (usually) allosteric inhibitor ATP increases apparent Km for F6P AMP is activator: relieves ATP inhibition ADP’s effects vary [ATP] fairly constant; [AMP] varies Citrate (Krebs cycle component) inhibits it [H+] is also an inhibitor (lactic acid debt) Glycolysis p. 48 of 56 25 Mar 2008 F-2,6-bisP and PFK-1, PFK-2 Potent activator of PFK-1 Absent in prokaryotes F-2,6-bisP Formed by action of PFK-2 Fructose 2,6-bisphosphate (n.b.: drawn backward from text) ATP + F-6-P F-2,6-bisP + ADP Stimulated by Pi, inhibited by citrate Same enzyme is also fructose 2,6bisphosphatase at different active site See fig. 11.16! Glycolysis p. 49 of 56 25 Mar 2008 PFK-2 and glucagon High [glucagon] turns on adenylyl cyclase pathway in liver Protein kinase A then phosphorylates a serine in PFK-2 That turns on phosphatase activity, turns off PFK-2 activity Thus [F-2,6-bisP] , PFK-1 less Phosphofructokinase-2 active, glycolysis is depressed PDB 2AXN 57 kDa monomer Glycolysis p. 50 of 56 25 Mar 2008 What if glucose is being rapidly metabolized? [glucagon] , [F-6-P], [F-2,6-bisP] F-6-P is a substrate for PFK-2 F-6-P is a potent inhibitor of F-2,6bisphosphatase That activates a phosphatase that dephosphorylates PFK-2 PFK-2 activity , phosphatase activity ! See figure 11.17 Glycolysis p. 51 of 56 25 Mar 2008 Pyruvate kinase regulation Four isozymes in mammals Liver, kidney, blood forms have sigmoidal kinetics for [PEP] Activated by F-1,6-bisP, inhibited by ATP Low [F-1,6-bisP]: ATP almost completely inhibits enzyme High [F-1,6-bisP]: ATP almost irrelevant Feed-forward activation Glycolysis p. 52 of 56 25 Mar 2008 Pyruvate kinase, phosphorylation, and glucagon One isozyme (liver, intestine) is sensitive to [glucagon]: Protein kinase A (see PFK-2!) phosphorylates pyruvate kinase, inactivating it somewhat Glucagon stimulates protein kinase A, so it tends to inactivate pyruvate kinase Glycolysis p. 53 of 56 25 Mar 2008 Pasteur effect Definition: increase in glycolysis under anaerobic conditions Relevant to yeast behavior Also to muscle metabolism when exercising, since not enough [O2] is getting to the muscles to maintain oxidative phosphorylation Reason: less ATP per glucose molecule with anaerobic metabolism, so you need to use more glucose to get the same amount of ATP out Modulation at PFK-1 level, others Glycolysis p. 54 of 56 25 Mar 2008 Fructose Transported with GLUT5 Ordinarily phosphorylated to F-1-P by ATPdependent fructokinase F-1-P cleaved to DHAP and glyceraldehyde by fructose 1-P aldolase Glyceraldehyde is 3-phosphorylated by ATPdependent triose kinase DHAP, Glyc-3-P then enter glycolysis as usual Glycolysis p. 55 of 56 25 Mar 2008 Fructosemetabolizing enzymes Fructokinase Fructokinase F-1P aldolase PDB 2hlz (now considered a subset human of ordinary F-1,6-bisP 136 kDa tetramer aldolase) Triose kinase (no structures yet!) Glycolysis p. 56 of 56 25 Mar 2008