Chapter 17

Glycolysis

  • Glucose (sugar) is an excellent fuel - input of glycolysis

    • good amount of energy upon oxidation

    • efficiently stored in polymeric form

    • many organisms/tissues can meet energy needs on glucose only

  • Glucose is a versatile biochemical precursor (carbon source)

    • bacteria use glucose to build carbon skeletons

      • amino acids, membrane lipids, nucleotides, cofactors for metabolism

    • synthesis of structural polymers, storage, oxidation via pentose phosphate pathway, and oxidation via glycolysis

  • Glycolysis: sequence of enzyme-catalyzed reactions

    • glucose is converted into pyruvate

      • pyruvate can be further aerobically oxidized if oxygen present

      • pyruvate can be used as precursor in biosynthesis

    • oxidation free energy is captured by synthesis of ATP and NADH

  • Research of glycolysis plays large role in development of modern biochemistry

    • role of coenzymes and ATP (energy), methods for enzyme purification

  • No requirement for oxygen

    • anaerobic metabolism was most important for first years of life on earth

    • aerobic organisms generate precursors for aerobic metabolism

    • useful for short-term energy production when oxygen is limiting

      • during intense activity, the heart cannot supply sufficient oxygen

      • glucose is converted anaerobically to lactate

        • lactate: acid - buildup causes muscles to be sore

  • Preparatory phase: conversion of glucose to glyceraldehyde-3-phosphate

    • phosphorylation of glucose to give glucose-6-phosphate

      • irreversible due to large ∆G’º with a negative value

      • driven in direction of G6P by 2ATP

      • hexokinase binds ATP and glucose to prime glucose-6-phospate

      • first regulatory step of glycolysis

    • isomerization of glucose-6-phosphate to give fructose-6-phosphate

      • reversible due to positive ∆G’º

      • glucose becomes fructose via phosphohexase isomerase

    • phosphorylation of fructose-6-phosphate to give fructose-1,6-biphosphate

    • cleavage of fructose-1,6-biphosphate to give glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)

    • isomerization of dihydroxyacetone phosphate to give glyceraldehyde-3-phosphate

  • Reaction 1: ATP-dependent phosphorylation of glucose

    • nucleophilic oxygen at C-6 of glucose attacks y-phosphorus of ATP

      • traps glucose in the cell

      • lowers intracellular glucose concentration

        • facilitates glucose into the cell

    • Hexokinase: shows structural change upon binding of glucose

      • two domains of the protein move closer together

        • bring ATP closer to C-6 of glucose

        • exclude water from active site

          • prevent hydrolysis of ATP

      • four hexokinase isozymes are found in humans

        • I-III have similar properties

          • high affinity for glucose

          • hill number = 1.0 (positive cooperativity)

          • allosterically inhibited by product (glucose-6-phosphate)

        • IV (glucokinase) found in liver with different properties

          • low affinity for glucose

          • hill number = 1.5 (positive cooperativity)

          • no product inhibition

            • liver does not need to feed glucose in glycolysis when blood glucose is normal

            • when blood glucose is high, liver can function to feed extra glucose into glycolysis

  • Reaction 2: Formation of fructose-6-phosphate from glucose-6-phosphate

    • reversible - positive ∆G’º

  • Reaction 3: ATP-dependent phosphorylation of fructose-6-phosphate

    • ATP is donor of second phosphate group

    • second priming reaction with phosphofructokinase-1 (PFK-1) enzyme

    • irreversible - committed step in glycolysis

    • product (fructose-1,6-biphosphate) is committed to become pyruvate

    • complex regulatory pattern - PFK-1

      • PFK-1 is inhibited by ATP and citrate (citric acid cycle)

      • PFK-1 is activated by AMP, ADP, and fructose-2,6-biphosphate

    • PFK-1 is inhibited when ATP is high and activated when ATP is low

  • Reaction 4: Cleavage of fructose-1,6-biphosphate into two triose phosphates

    • fructose-1,6-bisphosphate: aldolase enzyme

      • cleaves aldol into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate

  • Reaction 5: Interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P)

    • only glyceraldehyde-3-phosphate is a substrate for the remaining reactions of glycolysis

      • important to interconvert DHAP and G3P

    • triose phosphate isomerase enzyme converts DHAP to G3P

  • Summary for first phase of glycolysis

    • glucose converted to 2 molecules of glyceraldehyde-3-phosphate

    • key intermediate is ATP-dependent phosphorylation of fructose-6-phosphate to fructose-1,6-biphosphate (reaction 3)

      • phosphofructokinase-1 enzyme catalyzes reaction

        • subject to allosteric control

Payoff phase of glycolysis: 2 simultaneous processes

  • Reaction 6: Formation of 1,3-biphosphoglycerate

    • first energy-yielding step in glycolysis

    • glyceraldehyde-3-phosphate dehydrogenase enzyme

      • oxidation of aldehyde with NAD+ gives NADH

    • Arsenic poisoning: arsenate is a phosphate analog and can serve as an alternate substrate (can attack same spot as phosphate)

      • uncouples glucose oxidation from phosphorylation

  • Reaction 7: Substrate level phosphorylation of ADP and formation of 3-phosphoglycerate

    • 1,3-biphosphoglycerate is a high energy compound that can donate the phosphate group to ADP to make ATP

    • phosphoglycerate kinase enzyme

    • glucose is cleaved into 2 3-carbon fragments

      • net ATP yield from glycolysis is zero to this point

      • subsequent reactions generate a net GAIN in ATP

  • Reaction 8: Isomerization of 3-phosphoglycerate (transfer phosphate to C-2)

    • isomerization to form 2-phosphoglycerate

    • active site phosphorylated His required for catalysis

    • phosphoglycerate mutase enzyme

  • Reaction 9: Dehydration of 2-phosphoglycerate (create double bond)

    • a,ß-elimination of H2O

    • enolase enzyme

    • chemical logic: create a better phosphoryl donor

  • Reaction 10: Substrate level phosphorylation #2 and conversion of PEP to pyruvate (phosphoenolpyruvate to pyruvate)

    • net ATP yield is 2ATP/glucose

    • pyruvate kinase enzym

    • subsequent aerobic oxidation of glucose by other metabolic pathways generates additional ATPs/glucose

Glycolysis summary

  • 10 reactions

  • three of the 10 exhibit large negative ∆G’ and are the sites of regulation

    • hexokinase (reaction 1)

    • PFK-1 (reaction 3)

    • pyruvate kinase (reaction 10)

Pyruvate kinase is subject to regulation

  • allosterically activated by AMP and fructose-2,6-biphosphate

  • allosterically inhibited by ATP and acetyl-CoA

  • requires divalent metals for activity

    • limited by level of Mg++

    • when there is plenty of ATP, Mg ions are sequestered by ATP

      • slows pyruvate kinase

Glycolysis and Cancer

  • Metabolism in tumor cells is largely anaerobic

    • glycolysis occurs at elevated rates in tumor cells

      • yields far less ATP than complete oxidation to carbon dioxide that takes place in healthy cells under aerobic conditions

        • tumor cell must consume more glucose to produce same amount of ATP

    • glucose transporters and glycolytic enzymes are overproduced

    • compounds that inhibit hexokinase, glucose 6-phosphate dehydrogenase, or transketolase block ATP production by glycolysis

      • depriving cancer cell of energy and killing it

    • Phosphorylation of 18F-labeled 2-fluoro-2-deoxyglucose by hexokinase traps FdG in cells where is can be detected by positron emission from 18F

      • CT shows cancer in various areas of the body including the bladder which excretes 18F-labeled compounds

Entry of galactose into glycolysis

  • inability to convert galactose to glucose leads to human disease

    • galactosemia - characterized by high blood galactose concentration

      • symptoms: failure to thrive, mental retardation, cataracts, and death from liver disease

      • treatment: eat galactose-free diet

    • defects in all three enzymes are known

      • most common and most severe is mutation in UDP-glucose:galactose 1-phosphate uridyltransferase

Metabolic fates of pyruvate

  • regeneration of NAD+

    • if NADH is not recycled back to NAD+, glycolysis cannot run

    • NAD+ is required in the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate (reaction 6)

    • if oxygen is available, NADH is re-oxidized by the electron transport chain leading to complete oxidation to carbon dioxide and reduction of oxygen to water

    • if oxygen is unavailable, NADH is re-oxidized by reduction of pyruvate to either lactate or ethanol

      • pyruvate to lactate - animal tissues

        • catalyzed by lactate dehydrogenase

        • carbonyl of pyruvate is transformed to lactate

        • lactate builds up in muscles during strenuous exercise

          • acidification prevents continuous strenuous work

          • lactate can be transported to liver and converted to glucose there

      • pyruvate to ethanol - yeast

        • requires Mg++, thiamine pyrophosphate with pyruvate decarboxylase

        • requires Zn++ and NAD+ with alcohol dehydrogenase

        • ester group of pyruvate is transformed to ethanol

Summary

  • pyruvate is converted to lactate in anaerobic tissues

    • actively metabolizing muscle

    • NAD+ is recycled

  • pyruvate is converted to ethanol in yeast

    • requires thiamine pyrophosphate as coenzyme

    • NAD+ is recycled