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GLYCOLYSIS

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).

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GLYCOLYSIS

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

  2. “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)

  3. 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

  4. 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

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

  6. 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

  7. Background, cont… • History • 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

  8. 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

  9. 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

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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

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

  18. Reaction 1 • 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

  19. 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

  20. 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

  21. Reaction 1, cont…

  22. 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)

  23. 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

  24. 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

  25. 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

  26. Reaction 2 • 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

  27. 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

  28. 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

  29. Reaction 3, cont… • 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

  30. Reaction 4 • 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

  31. 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+

  32. Reaction 5 • 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

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

  34. 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

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

  36. Reaction 6 • 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

  37. 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+

  38. Reaction 7 • 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

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