Transcript Bioenergetics and Metabolism

‫چرخه کربس مرکز‬
‫سوخت و ساز سلولي‬
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‫سوخت و سوزي‬
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‫چرخه است‬
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.
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
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.
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 succinylCoA.
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.
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