Bioenergetics and Metabolism

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Citrate CycleEnergy conversion through redox
reactions, a cycle with eight renewable
reactants, and regulation
Bioc 460 Spring 2008 - Lecture 28 (Miesfeld)
The Citrate Cycle is spinning out of control!
If the citrate cycle is an engine, what is the
fuel, the energy output, and the exhaust?
Compound 1080 contains fluoracetate, a poison
that blocks the activity of aconitase
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Key Concepts in Citrate Cycle

• The primary function of the citrate cycle is to
  convert energy available from the oxidization
  acetyl-CoA into 3 moles of NADH, 1 mole of FADH2
  and 1 mole of GTP during each turn of the cycle.
• The citrate cycle is a "metabolic engine" in
  which all eight of the cycle intermediates are
  continually replenished to maintain a
  smooth-running energy conversion process. The
  fuel for this metabolic engine is acetyl-CoA, the
  exhaust is two molecules of CO2, and the energy
  output is redox energy used in the electron
  transport system for ATP synthesis.
• Oxaloacetate is both the product of reaction 8,
  and the reactant for reaction 1, which means that
  flux through the pathway is continuously
  monitored by resetting the level of available
  substrate after each turn of the cycle. Two
  other regulatory mechanisms at play in the
  citrate cycle are product inhibition and feedback
  control of key enzymes.

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• The citrate cycle is considered the "hub" of
  cellular metabolism because it not only links the
  oxidation of metabolic fuels (carbohydrate, fatty
  acids and proteins) to ATP synthesis, but it also
  provides shared metabolites for numerous other
  metabolic pathways.

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Pathway OverviewThe eight reactions of the
citrate cycle oxidize acetyl-CoA to generate 2
CO2, and in the process, reduce 3 NAD and 1 FAD.
In addition, 1 GTP is produced by substrate
level phosphorylation which is converted to ATP
by nucleotide kinase.
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• All of the enzymes in the citrate cycle, electron
  transport chain and oxidative phosphorylation
  reside in the mitochondrial matrix where pyruvate
  is converted to acetyl-CoA by the enzyme pyruvate
  dehydrogenase.

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The "currency exchange" for redox energy and ATP
synthesis in the mitochondria electron transport
chain is 2.5 ATP/ NADH. Oxidation of 2 FADH2
molecules by the electron transport chain results
in only 3 molecules of ATP (1.5 ATP/FADH2)
because of differences in where these two
coenzymes enter the electron transport chain.
Based on this ATP currency exchange ratio, and
the one substrate level phosphorylation reaction,
each turn of the cycle produces 10 ATP for every
acetyl-CoA that is oxidized.
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Hans Krebs Elucidated the Citrate Cycle

• Hans Krebs, a biochemist who fled Nazi Germany
  for England in 1933, first described the citrate
  cycle in 1937.
• The citrate cycle is sometimes called the Krebs
  cycle, the citric acid cycle, or the
  tricarboxylic acid cycle, although we will refer
  to it as the citrate cycle because citrate is the
  first product of the pathway.
• The unprotonated form of citric acid is citrate
  which is the predominant species at physiological
  pH (the pKa values of the three carboxylate
  groups are 3.1, 4.7 and 6.4).

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Pathway Questions

• What does the citrate cycle accomplish for the
  cell?
• Transfers 8 electrons from acetyl-CoA to the
  coenzymes NAD and FAD to form 3 NADH and 1 FADH2
  which are then re-oxidized by the electron
  transport chain to produce ATP by the process of
  oxidative phosphorylation.
• Generates 2 CO2 as waste products and uses
  substrate level phosphorylation to generate 1 GTP
  which is converted to ATP by nucleoside
  diphosphate kinase.
• Supplies metabolic intermediates for amino acid
  and porphyrin biosynthesis.

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Pathway Questions

• What is the overall net reaction of citrate
  cycle?
• Acetyl-CoA 3 NAD FAD GDP Pi 2 H2O ?
• CoA 2 CO2 3 NADH 2 H FADH2 GTP
• ?Gº -57.3 kJ/mol

Is the citrate cycle a favorable reaction? Of
course it is, you are alive arent you!
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Pathway Questions

• What are the key regulated enzymes in citrate
  cycle?
• Pyruvate dehydrogenase not a citrate cycle
  enzyme but it is critical to flux of acetyl-CoA
  through the cycle this multisubunit enzyme
  complex requires five coenzymes, is activated by
  NAD, CoA and Ca2 (in muscle cells), and
  inhibited by acetyl-CoA, ATP and NADH.
• Citrate synthase catalyzes the first reaction
  in the pathway and can be inhibited by citrate,
  succinyl-CoA, NADH and ATP inhibition by ATP is
  reversed by ADP.

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Pathway Questions

• What are the key regulated enzymes in citrate
  cycle?
• Isocitrate dehydrogenase - catalyzes the
  oxidative decarboxylation of isocitrate by
  transferring two electrons to NAD to form NADH,
  and in the process, releasing CO2, it is
  activated by ADP and Ca2 and inhibited by NADH
  and ATP.
• a-ketoglutarate dehydrogenase - functionally
  similar to pyruvate dehydrogenase in that it is a
  multisubunit complex, requires the same five
  coenzymes and catalyzes an oxidative
  decarboxylation reaction that produces CO2, NADH
  and succinyl-CoA it is activated by Ca2 and AMP
  and it is inhibited by NADH, succinyl-CoA and
  ATP.

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Pathway Questions

• What are examples of citrate cycle in real life?
• Citrate is produced commercially by fermentation
  methods using the microorganism Aspergillus
  niger. Every year almost a half of million tonnes
  (5 x 108 kg) of citrate are produced worldwide by
  exploiting the citrate synthase reaction.

Purified citrate is a food additive
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The complete oxidation of glucose to CO2 and H2O
is summarized by the reaction

• Glucose (C6H12O6) 6O2 ? 6CO2 6H2O
• ?Gº -2,840 kJ/mol?G -2,937 kJ/mol
• Four of the CO2 molecules are produced in
• the Citrate Cycle, but what reaction
• generates the other two CO2?

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Eight Reactions of the Citrate Cycle
In the first half of the cycle, the two carbon
acetate group of acetyl-CoA is linked to the four
carbon oxaloacetate substrate to form a six
carbon citrate molecule.
Citrate is then converted to isocitrate to set up
two decarboxylation reactions yielding two NADH
and the high energy four carbon cycle
intermediate succinyl-CoA. In the second half
of the cycle, oxaloacetate is regenerated from
succinyl-CoA by four successive reactions that
lead to the formation of one GTP (ATP), one
FADH2, and one NADH.
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Reaction 1 Condensation of oxaloacetate and
acetyl-CoA by citrate synthase to form citrate
This reaction commits the acetate unit of
acetyl-CoA to oxidative decarboxylation Reaction
follows an ordered mechanism Oxaloacetate binds,
inducing a conformational change in the enzyme
that facilitates - acetyl-CoA binding -
formation of the transient intermediate,
citryl-CoA - rapid hydrolysis that releases
CoA-SH and citrate
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Reaction 2 Isomerization of citrate by aconitase
to form isocitrate
This is a reversible two step isomerization
reaction. The intermediate, cis-aconitate, is
formed by a dehydration reaction that requires
the participation of an iron-sulfur cluster
(4Fe-4S) in the enzyme active site. H2O is added
back to convert the double bond in cis-aconitate,
to a single bond with a hydroxyl group, on the
terminal carbon.
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Aconitase is one of the targets of fluorocitrate

• Fluorocitrate is derived from fluoroacetate.
  Fluoroacetate-containing plants, such as acacia
  found in parts of Australia and Africa, are so
  deadly that Australian sheep herders have
  reported finding sheep with their heads still in
  the bush they were feeding on when they died.
• Fluoracetate is the active ingredient in the
  poison compound 1080 used to kill rodents and
  livestock predators. Sometimes, the poison is
  used indiscriminately, causing animal deaths.

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Reaction 3 Oxidative decarboxylation of
isocitrate by isocitrate dehydrogenase to form
a-ketoglutarate, CO2 and NADH
First of two decarboxylation steps in the citrate
cycle First reaction to generate NADH used for
energy conversion reactions in the electron
transport system Catalyzes an oxidation reaction
that generates the transient intermediate
oxalosuccinate In the presence of the divalent
cations Mg2 or Mn2, oxalosuccinate is
decarboxylated to form a-ketoglutarate
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Reaction 4 Oxidative decarboxylation of by
a-ketoglutarate dehydrogenase to form
succinyl-CoA, CO2 and NADH
Second oxidative decarboxylation reaction and
also produces NADH. a-Ketoglutarate dehydrogenase
complex utilizes essentially the same catalytic
mechanism we have already described for the
pyruvate dehydrogenase reaction. Includes the
binding of substrate to an E1 subunit
(a-ketoglutarate dehydrogenase), followed by
decarboxylation and formation of a TPP-linked
intermediate.
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Reaction 5 Conversion of succinyl-CoA to
succinate by succinyl-CoA synthetase in a
substrate level phosphorylation reaction that
generates GTP
The available free energy in the thioester bond
of succinyl-CoA (?Gº' -32.6 kJ/mol) is used in
the succinyl-CoA synthetase reaction to carry out
a phosphoryl transfer reaction (?Gº' 30.5
kJ/mol), in this case, a substrate level
phosphorylation reaction, that produces GTP (or
ATP).
Nucleoside diphosphate kinase interconverts GTP
and ATP by a readily reversible phosphoryl
transfer reaction GTP ADP ? GDP ATP (?Gº'
0 kJ/mol).
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Reaction 6 Oxidation of succinate by succinate
dehydrogenase to form fumarate
This coupled redox reaction directly links the
citrate cycle to the electron transport system
through the redox conjugate pair FAD/FADH2 which
is covalently linked to the enzyme succinate
dehydrogenase, an inner mitochondrial membrane
protein. Oxidation of succinate results in the
transfer of 2 e- to the FAD moiety, which in
turn, passes the two electrons to the electron
carrier coenzyme Q in complex II of the electron
transport system.
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Reaction 6 Oxidation of succinate by succinate
dehydrogenase to form fumarate
Compounds that that donate electrons are called
reductants, and compounds that accept electrons
are called oxidants.?
Is FAD oxidized or reduced in this redox
reaction? Is succinate the reductant or the
oxidant in this reaction?
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Reaction 7 Hydration of fumarate by fumarase to
form malate
Fumarase the reversible hydration of the CC
double bond in fumarate to generate the L-isomer
of malate.
Fumarate and malate are citrate cycle
intermediates that enter and exit the cycle from
several different interconnected pathways.
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Reaction 8 Oxidation of malate by malate
dehydrogenase to form oxaloacetate
Oxidation of the hydroxyl group of malate to form
oxaloacetate in a coupled redox reaction
involving NAD/NADH. The change in standard free
energy for this reaction is unfavorable (?Gº'
29.7 kJ/mol), but the actual ?G for this
reaction is favorable.
In order for this unfavorable ?Gº to allow for a
favorable ?G, the metabolite concentrations need
to be far from equilibrium. Based on what you
know about the citrate cycle, what do you think
explains the favorable ?G in terms of
metabolite in this case?
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Bioenergetics of the citrate cycle
??
Glycolysis pyruvate dehydrogenase reaction
citrate cycle net reactionGlucose 2 H2O
10 NAD 2 FAD 4 ADP 4 Pi ?6 CO2 10 NADH
6 H 2 FADH2 4 ATP
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The reducing power of NADH and FADH2 can be
converted to ATP equivalents using the currency
exchange ratio. 2.5 ATP/NADH 1.5 ATP/FADH2
This yields 28 ATP based on 3 NADH and 1 FADH2
Anoter 4 ATP are synthesized by substrate
phosphorylation, generates a maximum of 32 ATP.
The complete oxidation of glucose by the
pyruvate dehydrogenase complex and the citrate
cycle leads to the production of 6 CO2 molecules
as waste.
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Radioactive 14C-acetyl CoA

• In the first turn of the cycle, both carbons are
  incorporated into oxaloacetate.
• It isn't until the second turn of the cycle that
  the first carbon is released as CO2 by the
  isocitrate dehydrogenase reaction.
• The second carbon is not lost as CO2 until the
  a-ketoglutarate reaction in the fourth turn of
  the cycle.
• Intermediates must be continually regenerated in
  a cyclic pathway to maintain metabolic flux.

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• Three main control points in the cycle
• Citrate synthase
• Isocitrate
• dehydrogenase
• a-ketoglutarate
• dehydrogenase

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• Pyruvate dehydrogenase is activated by CoA-SH to
  stimulate acetyl-CoA production.
• Pyruvate carboxylase in turn, is stimulated by
  acetyl-CoA to maintain OAA for citrate synthesis.
  If carbohydrate is limiting, (low pyruvate), then
  acetyl-CoA is converted to ketone bodies
  (ketogenesis) or citrate is shuttled to the
  cytosol.