Acetyl-CoA: A metabolic crossroads

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• Acetyl-CoA A metabolic crossroads

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Your perspective?

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Glycolysis
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D-glucose is prominent in biology

• Glucose is an excellent fuel with its oxidation
  liberating 2,840 kJ/mol of free energy
• Serves as a precursor in synthesis of numerous
  molecules
• Has three major fates in plants and animals, it
  can be stored in polymers, oxidized to pentoses
  via pentose phosphate pathway, or oxidized to
  pyruvate via glycolysis

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Pathways for glucose utilization
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Glucose is degraded in glycolysis

• Glycolysis is a series of sequential reactions
  that yield two molecules of the three carbon
  compound pyruvate
• During these reactions some of the free energy
  released from glucose is conserved in the form of
  ATP and NADH
• This pathway is seemingly universal, even in
  microbes that do not utilize externally supplied
  glucose

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Lehninger breaks down glycolysis into two phases

• Preparatory phase energy investment through ATP
  dependent phosphorylation. These reactions
  prime the glucose molecule for the second
  phase. Cost 2 ATP
• Payoff phase Net yield of 2 ATP molecules and 2
  NADH molecules per molecule of glucose

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The preparatory phase
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Cleavage results in two phosphorylated
three-carbon compounds

• In cells, DHAP and G3P are quickly removed,
  lowering their concentration thus driving this
  reaction towards the right. Standard free energy
  is misleading

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Only glyceraldehyde 3 phosphate can be used in
subsequent glycolytic steps

• As a result, DHAP is rapidly converted to G3P

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Glyceraldehyde 3 Phosphate dehydrogenase reaction
mechanism

• This reaction is a source of NADH and protons for
  the cell

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Iodoacetate is a potent (suicide) inhibitor of
G3P dehydrogenase
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Phosphoglycerate mutase works through a
phosphorylated intermediate
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An example of substrate level phosphorylation
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Glycolysis accounting

• Glucose 2NAD 2ADP 2 Pi ? 2 pyruvate 2
  NADH 2 ATP 2 H2O
• Chemical transformations that occur during
  glycolysis include 1) degradation of glucose to
  pyruvate 2) phosphorylation of ADP to ATP and 3)
  transfer of hydride ion with its electrons to NAD
  to form NADH

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Cells tightly regulate levels of ATP

• This regulation is achieved by the regulation of
  key enzymes in catabolism.
• For glycolysis, these include
• Hexokinase
• Phosphofructokinase
• Pyruvate kinase

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Regulation of glycolysis

• Flux through biochemical pathways depends on the
  activities of enzymes within the pathway
• For some steps, the reactions are at or near
  equilibrium in the cell
• The enzyme activity is sufficiently high that
  substrate equilibrates with product as fast as
  substrate is supplied.
• Flux is thus substrate limited

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Flux through a multi-step pathway
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Glycolysis has a bottleneck at the
phosphofructokinase catalyzed step

• The rate of fructose 6 phosphate to fructose 1,6
  bisphosphate is limited by PFK-1 activity
• Can produce as much fructose 6 phosphate as you
  want, but still wont push glycolysis
• PFK-1 acts as a valve
• This is an enzyme-limited reaction, and also the
  rate-limiting step in glycolysis

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Glycolytic enzyme and metabolite balances

• Table 15-2

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Trademarks of rate-limiting steps

• Rate-limiting steps are very exergonic reactions,
  essentially irreversible under cellular
  conditions
• Typically, the enzymes that catalyze these
  reactions are under allosteric control
• Often, these enzymes are situated at critical
  branch points in metabolism
• For glycolysis, the first committed step is the
  PFK-1 mediated reaction

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PFK-1 is under complex allosteric regulation

• Glucose-6-phosphate can flow into glycolysis or
  other pathways, PFK-1 commits substrate to
  glycolysis.
• PFK-1 is first unique step, not hexokinase.
• Several allosteric sites on PFK-1
• ATP is not only a substrate but a product of the
  metabolic pathway in question and inhibits PFK-1
  by lowering affinity for fructose-6-P
• ATP effect countered by ADP and AMP
• Citrate, a key TCA cycle intermediate, enhances
  ATP effect. High citrate, more inhibition
• PFK-1 is inhibited by protons, thus senstive to
  pH change
• Fructose 2,6 bisphosphate activates the enzyme

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Regulation of PFK-1

• Fig 15-18

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Fructose 2,6 bisphosphate?

• This metabolite has an important role in
  switching glycolysis and gluconeogenesis
• Fructose 2,6 bisphosphate is synthesized from
  fructose-6-phosphate by phosphofructokinase-2
  (PFK-2)
• PFK-2 is a unique enzyme, because this
  polypeptide also acts as fructose bisphosphatase
  2 (FBPase2) which converts Fructose 2,6
  bisphosphate to fructose-6-phosphate
• A bifunctional enzyme

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Hexokinase is a site for regulation in glycolysis

• Catalyzes the entry of free glucose into
  glycolysis
• When PFK-1 is inhibited both Fructose-6-phosphate
  and glucose 6-P build up. Glucose-6-phosphate
  inhibits hexokinase.
• Many distinct forms of hexokinase, which all
  convert glucose to glucose-6-phosphate.
• These multiple forms are called isozymes

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Why isozymes?

• Isozymes resulting from gene duplication events
  allow evolution to tune the metabolic potential
  of cells
• Different metabolic patterns in different tissues
• Different locations and metabolic roles for
  isozymes in the same cell
• Different stages of development
• Different responses of isozymes to allosteric
  modulators

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For instance,

• Hexokinase expressed in liver has distinct
  properties from the enzyme expressed in muscles
• Higher Km for glucose
• Inhibited by Fructose-6-phosphate, not
  glucose-6-phosphate
• Inhibition is mediated by a regulatory protein

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Another regulatory step Pyruvate kinase

• Again, multiple isoforms or isozymes, which
  respond to distinct metabolic cues
• Pyruvate kinase found in muscle is activated by
  Fructose 1,6 bisphosphate (pulling intermediates
  through the pathway)
• Inhibited by ATP and alanine (feedback
  inhibition alanine serves as a monitor for
  biosynthetic precursors)
• Also under hormonal control - glucagon

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Fate of pyruvate

• In animal cells, pyruvate can go to mitochondria
  and be metabolized by the TCA, citric acid, or
  Krebs cycle (same cycle)
• However, when oxygen is limiting, cells ferment
  pyruvate to lactic acid or ethanol
• Fermentation allows the oxidation of NADH to NAD
  (Protons are conserved among metabolites during
  fermentation)
• Pyruvate acts or supplies a terminal electron
  acceptor for fermentative processes
• In addition to ethanol and lactate, some microbes
  make useful solvents or products through
  fermentation.

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Why your muscles hurt after running.

• The resulting
• NAD can then
• be used for
• glycolysis
• Also used in
• yogurt production

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Other cells (i.e. yeast) ferment pyruvate to
ethanol

• Note, in all fermentations
• The CH ratio of reactants
• And products remain the same.
• Glucose HC 12/6 2
• 2 ethanol and 2 CO2
• HC 12/6 2

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Making acetyl-CoA from pyruvate
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Entry into the citric acid cycle occurs through
formation of acetyl-CoA

• Carbon skeletons of sugars (and fatty acids) are
  degraded to the acetyl group of acetyl-CoA to
  enter the citric acid cycle
• For pyruvate, this is accomplished via the
  pyruvate dehydrogenase complex, a cluster of
  three enzymes in the mitochondria of eukaryotic
  cells (cytosol of prokaryotes)

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The complexity of pyruvate dehydrogenase

• Five cofactors participate in the reaction
  mechanism
• Enzyme is subject to covalent modification and
  allosteric regulation
• Pyruvate dehydrogenase is similar to other enzyme
  complexes
• a-ketoglutarate dehydrogenase (citric acid cycle)
• a-ketoacid dehydrogenase (amino acid oxidative
  pathway)

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The cofactors of pyruvate dehydrogenase complex

• CoA
• TPP also, cofactor of pyruvate decarboxylase
  and transketolase
• FAD electron carrier
• NAD electron carrier
• Lipoate

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CoA has nucleotide character, pathothenate, and
importantly a reactive thiol
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Lipoate acts both as an electron carrier and acyl
carrier
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Pyruvate dehydrogenase complex is comprised of
three enzymes

• Pyruvate dehydrogenase (E1)
• 24 copies attached to E2 core, each contains
  bound TPP
• Dihydrolipoyl transacetylase (E2)
• Forms the core of the complex, 24 polypeptides,
  each containing 3 covalently bound lipoate
  molecules (E. coli enzyme).
• Lipoate is attached to the end of lysine chains
  providing long flexible arms of acyl group
  transfer
• Dihydrolipoyl dehydrogenase (E3)
• 12 copies attached to E2 core, Each contains
  bound FAD

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And two regulatory proteins

• A specific protein kinase phosphorylates a serine
  residue on one of two subunits of E1
• A second enzyme, a phosphatase, removes this
  phosphate to activate the enzyme
• Another example of allosteric regulation
• This enzyme complex is regulated by ATP,
  acetyl-CoA levels, NADH, and fatty acids
• More on this later

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Microscopic biochemistry
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Five steps in the decarboxylation and
dehydrogenation of pyruvate

• 1. Pyruvate is decarboxylated (similar to
  pyruvate decarboxylase reaction) the C1 of
  pyruvate is released as CO2, while C2 and C3
  remain fixed to TPP of E1
• 2. The group attached to TPP is oxidized to a
  carboxylic acid (acetate) the removed electrons
  reduce the disulfide bond of a lipoyl group on
  E2 the acetate is transferred to one of the
  resulting sulfhydryl groups on the lipoyl
  molecule

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First steps of pyruvate dehydrogenase complex
reaction
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Further steps

• 3. The acetate is then transferred to CoA to
  from Acetyl-CoA subsequent reactions in this
  cycle regenerate the oxidized lipoyl group of E2
• 4. Dihydrolipoyl dehydrogenase (E3) promotes the
  transfer of two Hydrogen atoms from the reduced
  lipoyl groups of E2 to the FAD of E3
• 5. The reduced FADH2 of E3 transfers a hydride
  ion to NAD forming NADH.

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Swinging arms

• The long lipoyllysl arms of E2 are central to the
  catalytic mechanism of pyruvate dehydrogenase
  complex
• They accept two electrons and the acetyl group
  from pyruvate and pass them to E3
• Importantly note that the intermediates along
  this pathway are never released (channeling)

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Acetyl-CoA is a feedback regulator of glycolysis
and gluconeogenesis
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Citric acid cycle a hub for intermediary
metabolism and energy generation
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The citric acid cycle generates ATP and reducing
power (NADH, FADH2)

• There are eight steps in this cycle, four of
  which are oxidations (forming NADH and FADH2)
• In each turn of the cycle, one acetyl group
  enters as acetyl-CoA, two molecules of CO2 leave,
  one molecule of OAA is used to make citrate, but
  the OAA is regenerated
• Various intermediates are siphoned off for
  biosynthetic pathways, and replenished by
  anaplerotic reactions

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Aconitase is a moonlighting protein

• In addition to its role in glycolysis, aconitase
  also acts as a mRNA regulatory factor
• Aconitase, an iron-sulfur containing protein,
  binds to the mRNA of transferrin, whose gene
  product pulls in iron from the environment
• The mRNA binding protects the mRNA from
  degradation, allowing for increased transferrin
  production

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a-ketoglutarate dehydrogenase complex resembles
pyruvate dehydrogenase complex

• Three enzymes homologous to E1, E2, and E3
• Requires TPP, bound lipoate, FAD, NAD and
  coenzyme A
• E1 components of these two complexes have
  distinct binding properties

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The citric acid cycle is so long and complicated

• Why?

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Citric acid cycle intermediates are used to
synthesize other biomolecules

• a-ketoglutarate and oxaloacetate serve as
  precursors for aspartate and glutamate (simply by
  transamination), which can subsequently be used
  for other molecules
• Oxaloacetate is converted to glucose via
  gluconeogenesis
• Succinyl-CoA ? porphryin rings

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Summary of citric acid cycle and anabolism
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Amino Acid biosynthesis

• Amino acids are derived from intermediates in
  glycolysis, citric acid cycle, and PPP pathway
• Ten of the amino acids have relatively simple
  pathways compared to say aromatic amino acids
• Although many organisms can synthesize all 20,
  mammals can synthesize only about ½. Those they
  can synthesize are called non-essential amino
  acids. (You do not need to distinguish between
  essential and non-essential)

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Removed intermediates are replenished via
anaplerotic reactions

• Among other reactions, oxaloacetate can be
  generated from CO2 and pyruvate catalyzed by
  pyruvate carboxylase

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Acetyl-CoA is a major product of amino acid
catabolism, not just glycolysis
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Acetyl-CoA is derived from several (ten) amino
acids

• Pyruvate can be a common intermediate

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Cofactors of amino acid catabolism
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Tetrahydrofolate

• Intracellular carrier of methyl groups (can also
  can carry a methylene, or a formimino, formyl or
  methenyl different oxidative states (fig 18-16)
• Major source of these one carbon units is serine
• Although versatile, most methyl group transfers
  are performed by adoMet

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AdoMet

• Synthesized from ATP and methionine
• Displacement of triphosphates only observed in
  one other known reaction involved in coenzyme B12
  synthesis

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Regulation of citric acid cycle

• Point of entry, pyruvate dehydrogenase complex,
  is tightly regulated
• When high levels of acetyl-CoA, or high ratios of
  ATP/ADP and NADH/NAD this complex is
  turned off by allosteric inhibition
• Vertebrates also exhibit covalent protein
  modification via phosphorylation

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Three valves are present in the citric acid cycle

• Three factors govern flux through citric acid
  cycle substrate availability, product
  inhibition, and allosteric feedback inhibition of
  early enzymes in pathway
• Regulated at its three exergonic steps steps
  catalyzed by citrate synthase, isocitrate
  dehydrogenase and a-ketoglutarate dehydrogenase

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Each of these steps can be rate-limiting

• Substrates for citrate synthase (acetyl-CoA and
  OAA) can vary and limit citrate formation
• NADH accumulation inhibits isocitrate and
  a-ketoglutarate oxidation
• Product accumulation inhibits all three limiting
  steps of the cycle.

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Summary of citric acid cycle regulation
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Linking anabolic and energy yielding pathways
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Citric acid cycle and glyoxylate cycle

• Isocitrate conversion is the point of control
  between these two pathways
• Accumulation of citric acid cycle intermediates
  activate isocitrate dehydrogenase
• Accumulation of citric acid cycle intermediates
  inhibits isocitrate lyase

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The glyoxylate cycle also involves acetyl-CoA

• The glyoxylate cycle converts acetate to
  carbohydrate
• Acetate can be a prevalent carbon source in the
  environment for several organisms (plants,
  invertebrates and some microbes)
• Acetate is also a result of lipid breakdown

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The first steps look like the citric acid cycle

• Acetyl-CoA condenses with OAA to form citrate
• Citrate is converted to isocitrate
• Next, instead of isocitrate dehydrogenase, an
  enzyme, isocitrate lyase converts isocitrate to
  succinate and glyoxylate

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The glyoxylate cycle
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Glyoxylate then is used to regenerate OAA

• Glyoxylate condenses with a second molecule of
  acetyl-CoA to yield malate (catalyzed by malate
  synthase)
• Malate dehydrogenase oxidizes the malate to OAA
  generating NADH, as well.
• And the cycle can begin again.

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Glyoxylate cycle produces

• One molecule of succinate with concomitant
  condensation of 2 molecules of acetyl-CoA
• The succinate can then be used as a point of
  entry for glucose production or other
  biosynthetic purposes

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Converting fat into energy (in plants)

• Intermediates are exchanged between glyoxysome,
  lipid body, mitochondria and cytosol
• Four distinct pathways participate
• Fatty acid breakdown to acetyl-CoA
• Glyoxylate cycle
• Citric acid cycle
• gluconeogenesis
• Resulting hexoses and sucrose can be transported
  to other cells for breakdown

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Relationship between glyoxylate and citric acid
cycle
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Isocitrate reactions are a target for regulation
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Isocitrate dehydrogenase is regulated by covalent
modification

• A protein kinase and phosphatase (separate
  activities on the same polypeptide) control
  isocitrate dehydrogenase (Phosphorylation
  inactivates the enzyme)
• Accumulation of citric acid cycle and glycolytic
  intermediates stimulates the phosphatase activity
  ? activating isocitrate dehydrogenase
• When intermediates fall ? kinase and inactivation
• This is a switch for isocitrate between the
  citric acid cycle and glyoxylate cycle

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This switch includes inhibition of isocitrate
lyase

• Intermediates of citric acid cycle and glycolysis
  are allosteric inhibitors of isocitrate lyase
• When these pathways proceed fast enough and the
  concentration of these intermediates are low,
  isocitrate dehydrogenase is phosphorylated and
  inhibited while isocitrate lyase is uninhibited
• Conversely, when intermediates are high,
  isocitrate lyase is allosterically inhibited and
  isocitrate dehydrogenase is activated by the
  phosphatase

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Acetyl-CoA is a central player in fatty acid
breakdown and synthesis
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Storage to structural
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Getting energy from fat

• Oxidation of long-chain fatty acids to acetyl-CoA
  is another central energy generating pathway
• Electrons from this process pass to the
  respiratory chain, while acetyl-CoA produced
  during this process is further oxidized by the
  citric acid cycle

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Fatty acids are activated and transported into
the mitochondria
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Fatty acid breakdown

• The oxidation of fatty acids
• proceeds in three stages

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b-oxidation

• b-oxidation is catalyzed by four enzymes
• Acyl-CoA dehydrogenase
• Enoyl-CoA hydratase
• b-hydroxyacyl-CoA dehydrogenase
• Acyl-CoA acetyltransferase (thiolase)

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First step

• Isozymes of first enzyme
• confers substrate specificity
• FAD-dependent enzymes
• Reaction analogous to succinate
• dehydrogenase in citric acid
• cycle

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b-oxidation bottomline

• The first three reactions generate a much less
  stable, more easily broken C-C bond subsequently
  producing
• two carbon units
• through thiolysis

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The process gets repeated over and over until no
more acetyl-CoA can be generated

• 160-CoA CoA FAD NAD H2O ? 140-CoA
  acetyl-CoA FADH2 NADH H
• Then..
• 140-CoA CoA FAD NAD H2O ? 120-CoA
  acetyl-CoA FADH2 NADH H
• Ultimately..
• 160-CoA 7CoA 7FAD 7NAD 8H2O ?
  8acetyl-CoA 7FADH2 7NADH 7H

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Acetyl-CoA can be fed to the citric acid cycle
resulting in reducing power
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Lipid Biosynthesis

• Fatty acid biosynthesis and oxidation proceed by
  distinct pathways, catalyzed by different
  enzymes, using different cofactors (NADPH instead
  of NAD and FAD), and take place in different
  places in the cell.
• Notably, a three carbon intermediate,
  malonyl-CoA is involved in biosynthesis but not
  breakdown (except as a regulatory molecule)

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Step one

• Enzyme primed
• by acetyl-CoA

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Steps 2, 3, and 4
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Why common lipids contain even of carbons
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Fatty acid synthase brings new meaning to enzyme
complex

• Contains seven proteins, seven activities

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Acyl carrier protein

• Contains the prosthetic group 4-phosphopantethein
  e
• Forms a thioester linkage with
  fatty acid, serving as a flexible
  arm tethering fatty acyl chain
  to surface of enzyme and
  passes
  intermediates between
  active sites

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To initiate fatty acid synthesis, the two thiol
groups on the enzyme must be charged

• The acetyl group of acetyl-CoA is transferred to
  the cysteine of b-ketoacyl-ACP synthase
• In a second reaction, the malonyl of malonyl-CoA
  to the SH group of ACP (catalyzed by
  malonyl-CoA-ACP transferase)

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Charging fatty acid synthase
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Condensation of acetyl-CoA and malonyl-CoA

• Condense to form acetoacetyl-ACP (bound to
  phosphopantetheine thiol group)
• The acetyl group of acetyl-CoA becomes the
  terminal residues on the fatty acid intermediate
• Catalyzed by b-ketoacyl-ACP synthase
• Produces a molecule of carbon dioxide (same
  carbon atom introduced into malonyl-CoA through
  bicarbonate reaction)

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Step 1
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Step 2, reduction of the carbonyl group

• The acetoacetyl-ACP undergoes reduction (using
  NADPH) b-ketoacyl-ACP reductase

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Step 3 dehydration

• b-hydroxyacyl-ACP dehydratase catalyzes the
  formation of trans-D2-butenoyl-ACP

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Step four Reduction of the double bond

• Butyryl-ACP is formed by
• enoyl-ACP reductase using
• NADPH

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To allow next cycle, butyryl group is transferred
to cysteine of b-ketoacyl-ACP synthase
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Next cycle
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Protein interactions and reaction channeling
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Distinctions among isozymes
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Paralogous Isozymes

• Add parallelism through
• Isozymes
• Can modulate flux
• Sequential feedback
• inhibition

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Getting energy from oxidative pathways
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Summary of electron transport

• There can be branches, at terminal electron
  acceptor, at terminal oxidase, at entry point of
  NADH

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NADH, a great source of energy

• NADH 11 H ½ O2 ? NAD 10 H
  H2O
• Highly exergonic DGo -220 kJ/mol
• Actually in cell, much NADH than NAD, making the
  available free energy more negative
• Much of this energy is used to pump protons out
  of the matrix

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Cytosolic-derived NADH must be shuttled into the
mitochondria

• Although citric acid cycle and fatty acid
  oxidation occur in the right place
  (mitochondrial matrix), glycolysis is cytoplasmic
  and NADH from this pathway must be shuttled into
  the matrix of the mitochondria (membrane is
  impermeable to this compound no transporter)
• Glycerol-3-phosphate shuttle
• Malate-Aspartate shuttle

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Pumping protons lowers the pH and generates an
electrical potential
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Generation of a proton-motive force

• In an actively respiring mitochondria, the pH is
  0.75 units lower outside than in the matrix
• Also generates an electrical potential of 0.15 V
  across the membrane, because of the net movement
  of positively charged protons outward across the
  membrane (separation of charge of a proton
  without a counterion)
• The pH difference and electrical potential both
  contribute to a proton motive force

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Really, what does that mean?

• Energy from electron transport drives an active
  transport system, which pumps protons across a
  membrane. This action generates an
  electrochemical gradient through charge
  separation, and results in a lower pH outside
  rather than in. Protons have a tendency to flow
  back in to equalize the pH and charge. This flow
  is coupled to ATP synthesis.

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Measuring the proton motive force

• DmH Dy 2.3RTDpH/F
• (different in Lehninger)
• mH is the resulting proton motive force
  (sometimes p)
• y is the electrochemical membrane potential
• pH has a negative value, thus contribution is
  positive in this equation

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So what is protonmotive force used for?
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ATP synthase A molecular machine
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ATP synthase has two functional domains

• This enzyme has two distinct parts, one a
  peripheral membrane protein (F1) and one a
  integral membrane protein (Fo) ( the o stands for
  oligomycin sensitive)
• These parts can be separated biochemically, and
  isolated F1 catalyses ATP hydrolysis (it has the
  site for ATP synthesis and hydrolysis)

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The F1 component

• This component is made up of nine proteins of
  five different types with a composition of
  a3b3gde
• Each of the three b subunits have a catalytic or
  active site where the reaction occurs
• ADP Pi ? ATP H2O

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The a and b subunits make a cylinder with the g
subunit as an internal shaft
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Conformational changes

• Although the b subunits have the exact same amino
  acid sequence and composition, they are in
  different conformations due to the g subunit.
• These conformational differences affect how the
  enzyme binds ATP and ADP

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The Fo component forms a proton pore in the
membrane
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Rotation of the g subunit by H translocation
drives ATP synthesis

• Passage of protons through the Fo component
  causes g to rotate in that internal chamber
• Each rotation of 120o causes g to contact another
  b subunit, this contact forces b to drop ATP and
  stay empty
• The three b subunits interact so that when one is
  empty, one has ADP and Pi, while another has ATP.

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Proton transfer is converted to mechanical
energy, then chemical energy
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ATP synthase at work

• http//nature.berkeley.edu/hongwang/Project/ATP_s
  ynthase/
• http//www.sciencemag.org/feature/data/1045705.shl
  

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ATP exits the mitochondria through active
transport

• P N
• Side Side