The citric acid (krebs, tca) cycle
The Citric Acid (Krebs, TCA) Cycle
Step 1: Condensation
In step 1 of the Krebs cycle, the two-carbon compound,
acetyl-S-CoA, participates in a
condensation reaction with the four-carbon compound,
oxaloacetate, to produce
citrate:
• This reacion is moderately
exergonic. Thermodynamically, the equilibrium is in
favor of the products. Thus, this is considered to be the first
committed step of the Krebs cycle
• Being the first committed step, this is a likely step to have some kind of
regulatory control mechanism (which will effectively regulate the entire cycle)
• The Krebs cycle is also known as the
citric acid cycle. Citrate is a tricarboxylic
acid, and the Krebs cycle is also known as the
tricarboxylic acid (or
TCA) cycle
Step 2. Isomerization of Citrate
As we will see later on in the Krebs cycle, there will be a
decarboxylation reaction.
• Such decarboxylation reactions usually involve α- (or β-) keto acids • The hydroxyl group of citrate can be oxidized to yield a keto group, but to form
an α-keto acid it needs to be adjacent to one of the terminal carboxyl groups
Thus, step 2 involves moving the hydroxyl group in the citrate molecule so that we can
later form an α
-keto acid
• This process involves a sequential dehydration and hydration reaction, to form the
D-Isocitrate isomer (with the hydroxyl group now in the desired α- location), with
cis-Aconitase as the intermediate
• A single enzyme,
Aconitase, performs this two-step process:

• This reaction is endergonic, so the equilibrium is in favor of the
reactants and not
the desired product. However, the exergonic character of the
next reaction in the cycle helps shift the equilibrium of
this reaction towards the right.
• There are two asymmetric centers in the D-Isocitrate molecule. Eeach can adopt
either the L- or D- rotamer, thus there are 4 possible isomers of this molecule
•
Aconitase only produces the single form of Isocitrate (D-Isocitrate). Thus,
Aconitase is a
stereospecific enzyme
Note: the stereospecificity of Aconitase was established by introducing carboxyl-
labeled Acetate into the Krebs cycle. The conversion of Acetate into Acetyl-CoA can
subsequently result in the labeling of Citrate. Although Citrate is a symmetric
molecule, the labeled carboxyl-group always ends up on the γ
- carbon group in D-
Isocitrate
Step 3: Generation of CO2 by an NAD+ linked enzyme
• The Krebs cycle contains two oxidative decarboxylation steps; this is the first one • The reaction is catalyzed by the enzyme
Isocitrate dehydrogenase

• The reaction involves dehydrogenation to
Oxalosuccinate, an unstable
intermediate which spontaneously decarboxylates to give α
-Ketoglutarate
• The reaction is
exergonic, with a ΔG0' = -20.9 kJ/mol. This helps drive the
preceding (endergonic) reaction in the cycle
• In addition to decarboxylation, this step produces a reduced nicotinamide adenine
dinucleotide (NADH) cofactor, or a reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor
• If the NAD+ cofactor is
reduced, then the
D-Isocitrate must be
oxidized when
forming α
-Ketoglutarate. Thus, this step is referred to as an
oxidative
decarboxylation step
Step 4: A Second Oxidative Decarboxylation Step
• This step is performed by a multi-enzyme complex, the α
-Ketoglutarate
• The multi-step reaction performed by the α
-Ketoglutarate Dehydration Complex
is analogous to the
Pyruvate Dehydrogenase Complex, i.e. an α-keto acid undergoes oxidative decarboxylation with formation of an acyl-CoA
• Overall, this oxidative decarboxylation step is more exergonic than the first
Summary of Kreb cycle reactions up to this point Two carbons have been added to
Oxaloacetate by the action of
Citrate Synthase (and
Acetyl-CoA)
• Two carbons have been lost as CO2 by oxidative decarboxylation steps • Two oxidized NAD+ cofactors have been reduced to NADH • Due to the stereospecific action of
Aconitase, the two carbons added are
not the
same two carbons lost in the oxidative decarboxylation steps
In the remaining steps of the Krebs cycle, the Succinyl-CoA is converted back into the original substrate for the cycle: Oxaloacetate
Step 5: Substrate-Level Phosphorylation
Succinyl-CoA is a high potential energy molecule. The energy stored in this molecule is used to form a high energy phosphate bond in a Guanine nucleotide diphosphate (GDP) molecule:
• Most of the GTP formed is used in the formation of ATP, by the action of
• In plants and bacteria ATP is formed directly in the
Succinyl-CoA Synthase
catalyzed reaction by phosphorylation of ADP directly. In animals, GDP is the substrate in the reaction with formation of GTP (which is then used to form ATP by
Nucleoside Diphosphokinase)
Step 6: Flavin-Dependent Dehydrogenation
The Succinate produced by
Succinyl CoA-Synthetase in the prior reaction needs to be converted to Oxaloacetate to complete the Krebs cycle.
• Both Succinate and Oxaloacetate are 4-carbon compounds • The first step in the conversion is the dehydrogenation of Succinate to yield
• In this reaction a C-C bond is being oxidized to produce a C=C bond. This
oxidation is energetically more costly than oxidizing a C-O bond.
• The
redox coenzyme for this reaction is therefore FAD, rather than NAD+ (FAD is
a more powerful oxidizing agent compared to NAD+)
• FAD is covalently bound to the
Succinate Dehydrogenase molecule (via a
• The FADH2 has to be oxidized for the enzyme activity to be restored. This
oxidation occurs via interaction with the mitochondrial electron transport system (bound to mitochondrial inner membrane).
•
Succinate Dehydrogenase is tightly bound to the mitochondrial inner membrane
•
Succinate Dehydrogenase is stereo-specific: the
trans- isomer (Fumarate) is
produced and not the
cis- isomer (Maleate)
Step 7: Hydration of a Carbon-Carbon Double Bond
Fumarate undergoes a stereo-specific hydration of the C=C double bond, catalyzed by
Fumarate Hydratase (also known as
Fumarase), to produce L-Malate:

•
Fumarase is a stereo-specific enzyme: it will only hydrate Fumarate, it will not
hydrate Maleate. Furthermore, the enzyme can not use D-Malate as a substrate in the reverse reaction
Step 8: A Dehydrogenation Reaction that will Regenerate Oxaloacetate
L-Malate (Malate) is dehydrogenated to produce Oxaloacetate by the enzyme
Malate Dehydrogenase
• This is a highly endergonic reaction (ΔG0' = +29.7 kJ/mol) and so the equilibrium
strongly favors the
reactants over the products.
• However, the
next step in the Krebs cycle (i.e. the
first step in the process) is the
highly exergonic reaction (ΔG0' = -32.2 kJ/mol) catalyzed by
Citrate Synthase and this keeps the levels of Oxaloacetate low (<10-6 M), thus allowing the above reaction to proceed
• The formation of Oxaloacetate completes the Krebs cycle
Stoichiometry and Energetics of the Citric Acid Cycle
Reaction
(kJ/mol)
Acetyl-CoA + Oxaloacetate + H2O Ö Citrate + CoA-SH + H+
cis-Aconitase + H2O Ù Isocitrate
Isocitrate + NAD+ Ù α-Ketoglutarate + CO2 + NADH
α-Ketoglutarate + NAD+ + CoA-SH Ù Succinyl-CoA + CO2 + NADH α
-Ketoglutarate
Succinyl-CoA + Pi + GDP Ù Succinate + GTP + CoA-SH
L-Malate + NAD+ Ù Oxaloacetate + NADH + H+
NET: -44.8
Acetyl-CoA + Oxaloacetate + H2O Ö Citrate + CoA-SH + H+
cis-Aconitase + H2O Ù Isocitrate
Isocitrate + NAD+ Ù α-Ketoglutarate + CO2 + NADH
α-Ketoglutarate + NAD+ + CoA-SH Ù Succinyl-CoA + CO2 + NADH
Succinyl-CoA + Pi + GDP Ù Succinate + GTP + CoA-SH
L-Malate + NAD+ Ù Oxaloacetate + NADH + H+
Acetyl-CoA + 2H2O + 3NAD+ + Pi + GDP + FAD Ö 2CO2 + 3NADH + GTP + CoA-SH
One turn of the citric acid cycle generates:
• One high-energy phosphate through substrate-level phosphorylation • Three NADH • One FADH2
Catabolism of Glucose through Glycolysis and the Krebs Cycle
• Each molecule of Glucose produces two molecules of Pyruvate
Glucose + 2NAD+ + 2ADP + 2Pi Ö 2Pyruvate + 2NADH + 2H+ + 2H2O +2ATP
• Action of
Pyruvate Dehydrogenase on Pyruvate:
Pyruvate + CoA-SH + NAD+ Ö CO2 + Acetyl-CoA + NADH
• The overall catabolism of Glucose to 2 Pyruvate molecules:
Glucose + 2NAD+ + 2ADP + 2Pi Ö 2Pyruvate + 2NADH + 2H+ + 2H2O +2ATP
2Pyruvate + 2CoA-SH + 2NAD+ Ö 2CO2 + 2Acetyl-CoA + 2NADH
Glucose + 4NAD+ + 2ADP + 2CoA-SH + 2Pi Ö 2CO2 + 2Acetyl-CoA + 4NADH + 2H+
• The GTP formed in the animal
Succinyl-CoA Synthetase reaction in the Krebs
cycle is readily converted to ATP (by
Nucleoside Diphosphokinase)
2Acetyl-CoA + 4H2O + 6NAD+ + 2Pi + 2ADP + 2FAD Ö 4CO2 + 6NADH + 2ATP +
Glucose + 4NAD+ + 2ADP + 2CoA-SH + 2Pi Ö 2CO2 + 2Acetyl-CoA + 4NADH + 2H+
Glucose + 10NAD+ + 4ADP + 2H2O + 4Pi + 2FAD Ö 6CO2 + 10NADH + 4ATP +
Yield of ATP
At this point the yield of ATP is 4 moles per mole of Glucose as it passes through the Krebs cycle
• This is not much more than the 2 moles which would have been produced from
• However, NADH and FADH2 are energy rich molecules • Their oxidation is highly exergonic and is coupled with the production of ATP
• Oxidation of 1 mole NADH produces 3 moles ATP • Oxidation of 1 mole FADH2 produces 2 moles ATP • Thus total ATP yield = (10 x 3) + (2 x 2) + 4 = 38 moles ATP per mole Glucose
Prepared by
Dr Hana M Gashlan
Source: http://hgashlan.kau.edu.sa/Files/0002526/files/20209_citric_acid%5B1%5D.pdf
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