Download Lecture 28 - Citrate Cycle

Citrate Cycle:
Energy 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!
Compound 1080 contains
fluoracetate, a poison that blocks
the activity of aconitase
If the citrate cycle is an engine,
what is the fuel, the energy
output, and the exhaust?
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 acetylCoA, 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.
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.
Pathway
Overview
The 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.
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.
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.
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).
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 reoxidized 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.
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
ΔGº’ = -57.3 kJ/mol
Is the citrate cycle a favorable reaction?
Of course it is, you are alive aren’t you!
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.
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.
α-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.
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
The complete oxidation of glucose to CO2 and H2O
is summarized by the reaction:
Glucose (C6H12O6) + 6O2 → 6CO2 + 6H2O
ΔGº’ = -2,840 kJ/mol
ΔG = -2,937 kJ/mol
Four of the CO2 molecules are produced in
the Citrate Cycle, but what reaction
generates the other two CO2?
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.
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
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.
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.
Reaction 3: Oxidative decarboxylation of isocitrate by
isocitrate dehydrogenase to form α-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 α-ketoglutarate
Reaction 4: Oxidative decarboxylation of by
α-ketoglutarate dehydrogenase to form succinyl-CoA,
CO2 and NADH
Second oxidative decarboxylation reaction and also produces NADH.
α-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 (α-ketoglutarate
dehydrogenase), followed by decarboxylation and formation of a
TPP-linked intermediate.
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 (ΔGº' =
-32.6 kJ/mol) is used in the succinyl-CoA synthetase reaction to carry
out a phosphoryl transfer reaction (ΔGº' = +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 (ΔGº' = 0 kJ/mol).
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.
Reaction 6: Oxidation of succinate by succinate
dehydrogenase to form fumarate
Is FAD oxidized or reduced in this redox reaction?
Is succinate the reductant or the oxidant in this reaction?
Reaction 7: Hydration of fumarate by fumarase to form
malate
Fumarase the reversible hydration of the C=C 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.
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
(ΔGº' = +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?
Bioenergetics of the citrate cycle
??
Glycolysis + pyruvate dehydrogenase reaction + citrate cycle = net reaction:
Glucose + 2 H2O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi →
6 CO2 + 10 NADH + 6 H+ + 2 FADH2 + 4 ATP
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”.
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 α-ketoglutarate reaction
in the fourth turn of the cycle.
Intermediates must be continually
regenerated in a cyclic pathway to
maintain metabolic flux.
Three main
control points in
the cycle:
• Citrate synthase
• Isocitrate
dehydrogenase
• α-ketoglutarate
dehydrogenase
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.