GLYCOLYSIS

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GLYCOLYSIS
Definition from Greek glykys (sweet)
lysis (splitting)
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• 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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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

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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

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Background, cont

• Glucose is a precursor
• Supplies metabolic intermediates
• Three fates
• Storage
• Oxidation to pyruvate
• Oxidation to pentoses

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Background, cont

• beta D-Glucose is the major fuel
• Rich in potential energy
• Stored in bonds
• Is literally solar energy
• ?G01 -2840 kJ/mole
• Advantages to glucose
• Catabolism ? ATP
• Can be stored
• Eg Polysaccarides, sucrose
• Can be transported
• Blood glucose
• Organism to organism

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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The Reactions of Glycolysis

• 10 enzymes
• 9 Intermediates
• Cost (2 ATP)
• Payment
• 4 ATP
• 2 NADH H
• End products
• Metabolic crossroads

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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

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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

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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

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Reaction 1, cont
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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)
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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

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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

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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

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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

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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

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Reaction 3

• Phosphofructokinase second ATP utilization
• Phosphorylation of F6P to Fructose-1,6-bisphosphat
  e
• 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

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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 pathways rate-determining
  reactions
• Allosteric regulation of PFK in many organisms

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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

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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

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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

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Reaction 5, cont

• Only glyceraldehyde-3-P can continue in
  glycolysis
• Dihydroxyacetone-P is rapidly converted

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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

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Next

• Payoff phase Produces ATP
• One glucose ? 2 glyceraldehyde-3-P
• Conversion to pyruvate ? 4 ATP
• Also 2 reduced NADH

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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

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Reaction 6, cont

• Aldehyde is dehydrogenated to an acyl phosphate
  with a high standard free energy of hydrolysis
  (?G01 -49.3 kJ/mole)
• NAD serves as hydrogen acceptor
• NAD ? NADH H

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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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Reaction 8

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

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Reaction 9

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

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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

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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

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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

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Glycolosis, cont

• Dietary polysaccharides
• must by hydrolyzed to monosaccarides
• Dietary Disaccharides
• must by hydrolyzed to monosaccarides
• Disaccharides cannot enter glycolytic pathway

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Glycolosis, cont

• Hexoses can enter glycolysis
• Hydrolytic enzyes are attached to epithelial
  cells in intestines
• Monosaccharides ? intestinal cells ? blood ?
  liver (phosphorylation) ? glycolysis

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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

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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

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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

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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

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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

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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

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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

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Regulation of Carbohydrate Catabolism, cont

• Both sets of reactions are substrate limited
• Some glycolytic reactions are irreversible (3,
  catalyzed by regulatory enzymes)

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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

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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

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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

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