FTP: 202'114'31'88, userpassword: bio

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• FTP 202.114.31.88, user/password bio

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Metabolism

• 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

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Bioenergetics

• ?G' ?G?' RT lnproducts/reactants
• ?G?' - R T lnKeq
• ?G ' Values are Additive
• High energy compounds

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2 glycolysis-Anaerobic Metabolism of glucose
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Things to Learn

• Pathway
• Energetics
• Regulation
• Cellular function / localization

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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.

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Glycolysis

• 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

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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)

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• 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.

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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.

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• 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.

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3. Ten enzymes catalyze the ten reactions of
glycolysis

• The preparatory Phase of glycolysis

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1. Hexokinase(????)

• ATP binds to the enzyme as a complex with
  Mg.
• The first priming reaction
• Traps glucose inside cells
• Irreversible

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Glucose
Hexokinase
Induced fit
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Isozymes
Hexokinase
Glucokinase
Ubiquitous
Liver
Nonspecific
Specific
Product inhibited
No product inhibition
Low km(0.1mM)
High km(10mM)
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• 2. Phosphoglucose Isomerase(????????)

Aldo to Keto isomerization
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3. Phosphofructokinase 1 (PFK-1, ??????-1)

• The second priming reaction
• Irreversible
• Rate-limiting step of Glycolysis
• Plays a major role in the regulation of
  glycolysis

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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.
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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.

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5. Triose Phosphate Isomerase (TIM) Glycolysis
continues from glyceraldehyde-3-P.
Keto to aldo isomerization
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Energy investment phase
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The payoff phase of glycolysis
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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.

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• 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).

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7. Phosphoglycerate Kinase (???????)

• First production of ATP.
• Substrate Level Phosphorylation

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8. Phosphoglycerate Mutase(???)
Phosphate is shifted from the OH on C3 to the OH
on C2. The process involves a 2,3-bisphosphate
intermediate.
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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.

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10. Pyruvate Kinase(?????)

• Second site of ATP production. Substrate level
  phosphorylation
• Irreversible.
• PEP has a larger DG of phosphate hydrolysis than
  ATP.

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Energy payoff phase
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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
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Glycolysis continued.
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• total reaction
• glucose 2 NAD 2 ADP 2 Pi ?
• 2 pyruvate 2 NADH 2 ATP2H2H2O

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???14C???????1??3??5?? ?????????????????????

• ???????????????????????????????????,?????????????
  ?????????,????????123456?????321123?
• ??,??14C???????1??,????????3?(???)?????14C??????
  ?3??,????????1?(???)?????14C???????5??,????????2?
  (???)????

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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.
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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

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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.

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This rxn would stop when NAD is depleted.
Under anaerobic conditions the ETS doesnt work
NAD
lactate
lactate dehydrogenase
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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
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Ethanol Fermentation

• Some anaerobic organisms metabolize pyruvate to
  ethanol, The above pathway regenerates NAD,
  needed for continuation of Glycolysis.
• Total reaction
• C6H12O62ADP2Pi? 2C2H6O2CO22ATP2H2O

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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.

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• 2ATP expended
• 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)

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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

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Regulation of the enzymes of Glycolysis3
irreversible reactions

• Hexokinase
• HK is allosterically inhibited by the product,
  G6P
• Pyruvate Kinase
• ATP and alanine are allosteric inhibitors
• F-1,6-2P is an allosteric activator
• Feed forward activation
• Inactivated by phosphorylation as a result of the
  activation of cAMP-dependent protein kinase (PKA)
  via Glucagon (?????)

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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-2,6-bisphosphate (F-2,6-P)

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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).

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Galactose
Glucose
galactokinase
G6P
Gal-1-P
Fructose
UDPG
hexokinase
uridilyltransferase
epimerase
F6P
UDPGal
fructokinase
G1P
F-1,6-BP
aldolase
F1P
GAP
Pyruvate
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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, ????).

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• Specific enzymes (e.g., lactase, sucrase,
  maltase,etc.) 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.

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9 Glycogen Breakdown

• Glycogen
• 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)

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The Structure of Glycogen
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LIVER MUSCLES

• When blood glucose drops below normal (2-3 hours
  after the last meal)
• Maintains blood glucose
• Continues until
• 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

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Glycogenolysis

• Glycogen Phosphorylase
• 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
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Debranching Enzyme
Has 2 activities
44 transferase
a1,6-glycosidase
H2O
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Glycogenolysis
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• 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
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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

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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

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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
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cAMP signal cascade
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?????????????????ATP??????????????????????????????
?ADP?????

• ??????????????????,???1,3-???????????????,???????
  ????????ATP?1,3-????????1????????????????,????,???
  ??????,??????????????????p??O??P????,????????,??
  ???????

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??????,???????????????????????????NADH??????

• ????????????????????????????????????NADH?????NAD?
  ??????????,??????,NADH?????????,??????????????????
  ???
• ????,???????????,???NADH?????,????????????????NADH
  ???????????????3-????????????????????1,3-???????

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???????,????????????????????????????????

• ?????????????????????????,???????????????????????
  ???????,????????????(ATP)???????,?????????????????
  ?????????????????????????????,?????????????,??????
  ??????????
• ???????????????????6-???????1,6-?????,??????????
  ?????????????????,?ATP???,???????????,???????,?AD
  P,AMP???,?????,????????????????????????,???????2,6
  -?????(??)????????????,??ATP???,???????????????(?)
  ?????,????????????????

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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.