catabolism and anabolism. They are coupled by chemical energy cycle
Chemical energy ATP FADH2 NADH NADPH
Characteristics of metabolic pathways
Regulation of metabolic pathways
allosteric regulation covalent modification
G G RT lnproducts/reactants
G - R T lnKeq
G Values are Additive
High energy compounds
4 2 glycolysis-Anaerobic Metabolism of glucose 5 Things to Learn
Cellular function / localization
6 An overview on glucose metabolism
The major fuel of most organisms releasing large energy if completely oxidized to CO2 and H2O via the glycolysis () citric acid cycle() and oxidative phosphorylation () .
Can also be oxidized to make NADPH and ribose-5-P via the pentose phosphate pathway().
Can be stored in polymer form (glycogen or starch) or be converted to fat for long term storage.
Is also a versatile precursor for carbon skeletons of almost all kinds of biomolecules including amino acids nucleotides fatty acids coenzymes and other metabolic intermediates.
Glycolysisfrom the Greek glyk-sweet lysis-splitting
--the stepwise degradation of glucose the conversion of glucose into pyruvate()
Glycolysis means the anaerobic metabolism of glucose
8 1. The Development of Glycolysis
1897 Eduard Buchner (Germany) accidental observation sucrose (as a preservative) was rapidly fermented into alcohol by cell-free yeast extract. (1907 Nobel Prize laureate)
Metabolism became chemistry!
1900s Arthur Harden and William Young (Great Britain)separated the yeast juice into two fractions one heat-labile nondialyzable zymase (enzymes) and the other heat-stable dialyzable cozymase (metal ions ATP ADP NAD). (1929 Nobel Prize laureate)
1910s-1930s Gustav Embden and Otto Meyerhof (Germany) studied muscle and its extracts
Reconstructed all the transformation steps from glycogen to lactic acid in vitro revealed that many reactions of lactic acid (muscle) and alcohol (yeast) fermentations were the same!
Glycolysis was also known as Embden-Meyerhof pathway. (1922 Nobel Prize laureate)
The whole pathway of glycolysis (Glucose to pyruvate) was elucidated by the 1940s.
10 2. The overall glycolysis pathway can be divided into two phases
Ten steps of reactions are involved in the pathway.
The first 5 reactions are called the preparatory phase of glycolysis. The hexose is first activated and then cleaved to two three-carbon intermediates consuming ATP.
The remaining reactions are called the payoff phase of glycolysis. The three-carbon intermediates are then oxidized generating ATP and NADH.
All intermediates are phosphorylated (as esters or anhydrides).
Only a small fraction (5) of the potential energy of the glucose molecule is released and much still remain in the final product of glycolysis pyruvate.
All the enzymes are found in the cytosol.
12 3. Ten enzymes catalyze the ten reactions of glycolysis
The preparatory Phase of glycolysis
13 1. Hexokinase()
ATP binds to the enzyme as a complex with Mg.
The first priming reaction
Traps glucose inside cells
14 Glucose Hexokinase Induced fit 15 Isozymes Hexokinase Glucokinase Ubiquitous Liver Nonspecific Specific Product inhibited No product inhibition Low km(0.1mM) High km(10mM) 16
2. Phosphoglucose Isomerase()
Aldo to Keto isomerization 17 3. Phosphofructokinase 1 (PFK-1 -1)
The second priming reaction
Rate-limiting step of Glycolysis
Plays a major role in the regulation of glycolysis
18 ATP Low Affinity Allosteric Site Only binds when the ATP is very high. Catalytic Site Phosphofructo- kinase ATP High Affinity Binds at a low ATP Phosphofructokinase 1 is an allosteric enzyme. Allosterically inhibited by ATP citrate. AMP reverses the inhibition due to ATP. 19 4. Aldolase ()
Splits 6 carbon into two 3 carbon molecules
Note that carbons are renumbered in products of Aldolase.
Thermodynamically very unfavorable under standard conditions. Removal of glyceraldehyde-3-P allows throughput.
20 5. Triose Phosphate Isomerase (TIM) Glycolysis continues from glyceraldehyde-3-P. Keto to aldo isomerization 21 Energy investment phase 22 The payoff phase of glycolysis 23 6. Glyceraldehyde-3-phosphate Dehydrogenase (-3 - )
First production of high-energy intermediates
The only oxidative step in Glycolysis in which NAD is reduced to NADH.
A cysteine thiol at the active site has a role in catalysis.
iodoacetate inactivates the thioester intermediate
The high energy acyl thioester is attacked by Pi to yield the acyl phosphate (P) product. This step can be bypassed by Arsenate(analogous to phosphate).
25 7. Phosphoglycerate Kinase ()
First production of ATP.
Substrate Level Phosphorylation
26 8. Phosphoglycerate Mutase() Phosphate is shifted from the OH on C3 to the OH on C2. The process involves a 23-bisphosphate intermediate. 27 9. Enolase ()
Involves the dehydration and redistribution of energy within a molecule raising the phosphate on position 2 to the high-energy state
This Mg-dependent dehydration reaction is inhibited by fluoride. Fluoride forms a complex with Mg at the active site.
28 10. Pyruvate Kinase()
Second site of ATP production. Substrate level phosphorylation
PEP has a larger DG of phosphate hydrolysis than ATP.
29 Energy payoff phase 30 Glycolysis consists of 10 chemical reactions 7 reactions occur near equilibrium 3 are irreversible reactions 1 Glucose is converted to 2 pyruvate yields 2 ATP 31 (No Transcript) 32 (No Transcript) 33 Glycolysis continued. 34
glucose 2 NAD 2 ADP 2 Pi
2 pyruvate 2 NADH 2 ATP2H2H2O
14C13()14C 31()14C52 ()
36 4 Fate of Pyruvate and Regeneration of NAD Pyruvate Anaerobic condition lactate ethanol Aerobic condition acetylCoA Aerobic condition pyruvate is oxidized to acetate which enters the citric acid cycle and is oxidized to CO2 and H2O with ATP synthesis. Anaerobic condition pyruvate turns to the product of fermentation. They derive only 2 ATP from glucose catabolism. 37 Why should NAD be recycled
NAD is reduced to NADH during glycolysis
The amount of NAD in the cell is small
In order for glycolysis to continue the NADH formed must be reoxidised to NAD
38 Regeneration of NAD
Aerobic condition. NAD is recycled by electron transfer chain and 2 or 3 ATP are synthesized by oxphos().
Anaerobic condition. Hydrogen of NADH is transferred to the pyruvate.
39 This rxn would stop when NAD is depleted. Under anaerobic conditions the ETS doesnt work NAD lactate lactate dehydrogenase 40 Lactate Fermentation Some anaerobes lack a respiratory chain for reoxidizing NADH. They metabolize pyruvate to lactate to regenerate NAD needed for continuation of Glycolysis. Skeletal muscles function anaerobically in exercise when aerobic metabolism cannot keep up with energy needs. Total reaction C6H12O62ADP2Pi 2C3H6O32ATP2H2O 41 Ethanol Fermentation
Some anaerobic organisms metabolize pyruvate to ethanol The above pathway regenerates NAD needed for continuation of Glycolysis.
42 5 Energetics of glycolysis
How many ATP bonds expended
How many ATP produced in the pathway (Remember there are two 3C fragments from glucose.)
Net production of ATP per glucose in Anaerobic and aerobic conditions.
4ATP produced through substrate level phosphorylation
Net production of ATP
In anaerobic condition 2ATP
In aerobic condition 6-8ATP ( when NADH is oxidized through electron transfer chain 2 or 3 ATP are synthesized by oxphos)
44 6 Regulation of Glycolysis
a pathway is controlled at rate-limiting steps
Flux through the rate-determining steps may be altered by several mechanisms
1. Allosteric control
2. Covalent modifications
45 Regulation of the enzymes of Glycolysis3 irreversible reactions
HK is allosterically inhibited by the product G6P
ATP and alanine are allosteric inhibitors
F-16-2P is an allosteric activator
Feed forward activation
Inactivated by phosphorylation as a result of the activation of cAMP-dependent protein kinase (PKA) via Glucagon ()
46 Phosphofructokinase (PFK-1)
Most key rate-limiting step of Glycolysis
Allosterically inhibited by ATP citrate
Inhibition by ATP is relieved by AMP
Energy charge is an index of cellular energy status
E.C. (ATP1/2ADP) / (ATPADPAMP)
The most potent allosteric activator of PFK-1 in liver is fructose-26-bisphosphate (F-26-P)
47 7. Entry of other sugars into the glycolytic pathways
Other hexoses are also oxidized via the glycolysis
They are also first primed by phosphorylation (at C-1 or C-6).
48 Galactose Glucose galactokinase G6P Gal-1-P Fructose UDPG hexokinase uridilyltransferase epimerase F6P UDPGal fructokinase G1P F-16-BP aldolase F1P GAP Pyruvate 49 8. Dietary poly- and disaccharides are hydrolyzed to monosaccharides in the digestive system
Salivary a-amylase (a-) in the mouth hydrolyzes starch into short polysaccharides or oligosacchrides.
Pancreatic a-amylase (active at low pH) continue act to convert the saccharides to mainly maltoses and dextrins (from amylopectin ).
Specific enzymes (e.g. lactase sucrase maltaseetc.) on the microvilli of the intestinal epithelial cells finally hydrolyze all disaccharides into monosaccharides.
The monosacchrides are then absorbed at the intestinal microvilli and transported to various tissues for oxidative degradation via the glycolytic pathway.
51 9 Glycogen Breakdown
A high molecular weight glucose polysacharide comprised of a1-4 glucose linkages (mainly) and a1-6 linkages(at branches )
Found mainly in Muscle (1-2 by weight) and Liver (up to 10 by weight)
52 The Structure of Glycogen 53 LIVER MUSCLES
When blood glucose drops below normal (2-3 hours after the last meal)
Maintains blood glucose
Next meal or
Liver glycogen is depleated (12-24 hours)
To provide energy for muscles during strenuous exercise (i.e. when anaerobic conditions prevail)
Does NOT contribute significantly to blood glucose
By phosphorolysis Splits bonds by incorporating Pi
Continues until there are 4 glucose units on each side of the branch
Glycogen Pi Glycogen G1P n residues n-1 residues 55 Debranching Enzyme Has 2 activities 44 transferase a16-glycosidase H2O 56 Glycogenolysis 57
G1P produced in the Glycogen breakdown is converted to G6P in a reaction catalyzed by Phosphoglucomutase
G6P then has different fates in different tissues
glucose Blood (glucose-6-phosphatase) LIVER phosphoglucomutase Glucose-1- P MUSCLES Muscles do not possess the enzyme glucose-6-phosphatase Glycolysis in muscles for energy 58 Regulation of Glycogen breakdown
Glycogen Phosphorylase is allosterically activated by AMP and inhibited by ATP glucose-6-P
Glycogen Phosphorylase is regulated by covalent modification - phosphorylation
59 Covalent modification
Glucagon epinephrine activate cAMP cascades
The cAMP cascade results in phosphorylation of a serine hydroxyl of Glycogen Phosphorylase
phosphorylation promotes transition of b (less active ) state to the a(active) state.
The cAMP cascade activates glycogen degradation.
a is the form of the enzyme that is active
b is the form of the enzyme that is less active
60 LIVER ONLY Primarily MUSCLES but also in LIVER Glucagon or epinephrine Receptor Cytosol Adenylate cyclase b g a a GDP GTP GDP ATP cAMP GTP displaces GDP Cytoplasm Amplification 61 cAMP signal cascade 62 ATP ADP
13- ATP13-1 pOP
6-16- ATPAD PAMP26 -()ATP()
65 Home work
The cell uses many strategies to drive an energetically unfavorable reaction forward. Identify two such strategies and give an example from glycolysis of a reaction that demonstrates one of these strategies.
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