Title: GLYCOLYSIS
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)
3I. 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
4I. 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
5Background, cont
- Glucose is a precursor
- Supplies metabolic intermediates
- Three fates
- Storage
- Oxidation to pyruvate
- Oxidation to pentoses
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7Background, 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
8Background, 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
9II. 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
10Function, 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
11Glycolosis, 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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13Glycolysis, 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
14Glycolosis, 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
15Glycolosis, 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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17Glycolosis, 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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19Glycolosis, 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
20Glycolosis, 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
21The Reactions of Glycolysis
- 10 enzymes
- 9 Intermediates
- Cost (2 ATP)
- Payment
- 4 ATP
- 2 NADH H
- End products
- Metabolic crossroads
22Reaction 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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24Reaction 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
25Reaction 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
26Reaction 1, cont
27Substrate 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)
28Reaction 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
29Reaction 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
30Reaction 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
31Reaction 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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33Reaction 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
34Reaction 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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36Reaction 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
37Reaction 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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39Reaction 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
40Reaction 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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43Reaction 5, cont
- Only glyceraldehyde-3-P can continue in
glycolysis - Dihydroxyacetone-P is rapidly converted
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45Taking 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
46Next
- Payoff phase Produces ATP
- One glucose ? 2 glyceraldehyde-3-P
- Conversion to pyruvate ? 4 ATP
- Also 2 reduced NADH
47Reaction 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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49Reaction 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
50Reaction 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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52Reaction 8
- Conversion of 3 PG to 2-phosphoglycerate (2PG)
- Intermediate formed
- Enzyme PHOSPHOGLYCERATE MUTASE (PGM)
- Reversible phosphate shift
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54Reaction 9
- Dehydration to Phosphoenol Pyruvate (PEP)
- Intermediate formed phosphoenol pyruvate
- Enzyme ENOLASE
- Energy-conserving reaction
- Reversible reaction
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56Reaction 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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59Glycolosis, 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
60Glycolosis, 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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63Glycolosis, cont
- Dietary polysaccharides
- must by hydrolyzed to monosaccarides
- Dietary Disaccharides
- must by hydrolyzed to monosaccarides
- Disaccharides cannot enter glycolytic pathway
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65Glycolosis, 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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67III. 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
68Regulation 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
69Regulation 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
70Regulation 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
71Regulation 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
72Regulation 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
73Regulation 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
74Regulation of Carbohydrate Catabolism, cont
- Both sets of reactions are substrate limited
- Some glycolytic reactions are irreversible (3,
catalyzed by regulatory enzymes)
75Regulation 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
76IV. 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
77Secondary 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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