Citric acid cycle

Respiration
• In broader sense respiration refers to animal’s
uptake of oxygen and release of carbon
dioxide.
• In narrower sense, cellular respiration refers
to the utilization of oxygen by cells to produce
carbon dioxide and energy.
 Universal electron carrier
• Cellular oxidations channel electrons to a few universal
electron carriers.
• Energy is stored by the catabolic process of reduction of
electron carriers.
• NAD, NADP, FMN and FAD are water-soluble universal
electron carriers.
• NAD and NADP readily move from one enzyme to another.
• The flavin nucleotides, FAD and FMN, are tightly bound to
enzymes, called flavoproteins, for which they serve as
prosthetic groups.
• Iron-sulfur proteins and cytochromes also serve as electron
carriers
NAD(P) as an electron carrier
 NAD and NADP
• NAD and NADP are composed of two nucleotides joined by
phosphoanhydrite bond.
• The vitamin niacin is the source of nicotinamide.
• NAD and its phosphorylated analogue NADP take up 2
electrons and 1 H+ ions to form NADH or NADPH.
• The second H+ taken up from the substrate is released as H+
in the solution.
• The hydrogen can be added to the front (the A side) and the
back (the B side) of the planar nicotinamide ring.
• Note that these electron carriers are not overall positively
charged. The positive charge on NAD+ denotes the positive
charge on the nicotinamide ring.
 Roles of NAD and NADP
• In most cells concentration of NAD+ is higher than the
concentration of NADH favoring the transfer of hydrite from the
substrate.
• On the other hand, the concentration of NADPH is higher than the
concentration of NADP+ favoring the reverse reaction.
• NAD+ generally functions in oxidation reactions.
• NADPH is the usual coenzyme used in reduction.
• In addition there is spatial separation in the use of these cofactors.
• Oxidative processes such as oxidation of pyruvate, fatty acids and
alpha-keto acids derived from amino acids occurs in mitochondrial
matrix, where the concentration of NAD+ is high
• Reductive biosynthetic processes take place in the cytosol, where
the concentration of NADPH is high.
 Roles of NAD and NADP
• Enzymes that use NAD or NADP are often called
dehydrogenases.
• They carry these cofactors at Rossmann fold, which
generally contains 6 stranded parallel beta sheets and 4
associated alpha helices.
• The enzymes are generally very specific about their use of
NAD or NADP and their preference for A or B side of the
ring.
• These cofactors are synthesized from niacin, which is again
synthesized from tryptophan.
• Populations that use maize as a staple crop suffer from a
disease called pallegra associated with niacin deficiency.
 FAD and FMN as electron carriers
• Flavoprotein catalyze enzymes using either
flavin mononucleotide (FMN) and flavin
adenine dinucleotide(FAD) as coenzymes.
• They are derived from the vitamin riboflavin.
• The isoalloxazine ring undergoes reduction
accepting either 1 or 2 electrons while also
accepting 1 or 2 protons.
• FAD and FMN are involved in more reactions
than NAD and NADP
 FAD and FMN as electron carriers
• They are usually bound tightly to the enzymes.
• They are temporary sinks of electrons.
• When bound to flavoproteins their affinity of electrons
changes substantially.
• They are often complex. Often they have additional
inorganic ions such as iron and molybdenum capable
of participating in electron transfers
• Photolyases that repair DNA are flavoproteins.
• Crytochromes in plants mediate their development
using blue light and in mammals regulate circadian
rhythm
Three stages of cellular respiration
• In stage 1 of cellular respiration Acetyl-CoA is formed from
the organic molecules: amino acids, fatty acids and glucose.
• In stage 2 acetyl-CoA is fed into citric acid cycle. Acetate is
oxidized to carbon dioxide through this cycle, and the
energy released is conserved as reduced electron carriers
NADH and FADH2.
• Citric acid cycle is also called tricarboxylic acid (TCA) cycle or
Kreb’s cycle.
• The reduced molecules transfer protons and electrons to
electron transport chain. The electrons are finally taken up
by oxygen while forming water. During this process energy
from electrons is transferred to ATP.
Pyruvate to acetyl-CoA using pyruvate
dehydrogenase (PDH) complex
 PDH complex
• Pyruvate dehydrogenase (PDH) complex is a classic example
of a multienzyme complex in which intermediates are
bound to the complex.
• 5 cofactors, 4 derived from vitamins, participate in the
reaction.
• Regulation takes place through both covalent modification
and allosteric mechanism.
• This complex possibly shares evolutionary origins with
alpha-ketoglutarate dehydrogenase of the citric acid cycle
and branched chain alpha keto acid dehydrogenase of the
oxidative pathway of several amino acids. The three share
remarkable similarities in protein structure, cofactor
requirements and reaction mechanisms.
Cofactors of PDH complex
Structure of Coenzyme A
Lipoyl group
 PDH complex
PDH complex consists of 3 enzymes:
• 1. Pyruvate dehydrogenase (E1)
• 2. Dihydrolipoyl transacetylase (E2)
• 3. Dihydrolipoyl dehydrogenase (E3)
Size of PDH complex in mammals is about 50 nm.
The bovine enzyme has a diameter of 25 nm, and
consists of 60 identical copies of E2 forming a
pentagonal docecahedron.
E. coli enzyme contains 24 copies of E2
E1 is bound to TPP while E3 is bound to FAD
PDH complex
 PDH complex
• Above shown is a 3-D image showing three
domains of PDH. The image is generated by
combining cryoelectron microscopy images with
crystal images of individual units.
• The core consists of 60 molecules of E2, arranged
in 20 trimers to form a pentagonal dodecahedron.
• Lipoyl domain of E2 (blue) reaches out to touch
the active site of E1.
• By swinging arms E2 can reach E3.
PDH complex
 Lipoyl domains
• E2 has 3 functionally different domains: lipoyl
domain, binding domain and acyltransferase
domain.
• E. coli, mammals and yeast have 3, 2 and 1
lipoyl domains.
5 reactions in PDH complex
 Step 1 and 2
• Step 1: C-1 of pyruvate is released as carbon
dioxide and C-2 of pyruvate that has the oxidation
state of aldehyde is attached to TPP of E1 as a
hydroxyethyl group. This is the slowest and
rate-limiting step of the reaction.
• Step 2: Pyruvate dehydrogenase transfers of
electrons and the acetyl group from TPP to the
oxidized form of the lipoyllysl group of the core
enzyme E2, to form the acetyl thioester of the
reduced lipoyl group.
 Steps 3, 4 and 5
• Step 3: Transesterification, in which the –SH group
of CoA replaces the –SH group of E2 to yield
acetyl-CoA and fully reduced lipoyl group.
• Step 4: Dihydrolipoyl dehydrogenese (E3)
promotes transfer of 2 H atoms from the reduced
lipoyl group to prosthetic group of E2(FAD).
• Step 5: The reduced FADH2 transfers hydrite ions
to NAD+ forming NADH.
 Location of citric acid cycle
• In eukaryotes, the entire citric acid cycle takes
place in mitochondria.
• In most bacteria, citric acid cycle takes place in
the cytosol; the plasma membrane serves a
role analogous to the inner mitochondrial
membrane in ATP synthesis.
• There are a total of eight steps in citric acid
cycle.
 1. Formation of citrate
• In this reaction the methyl group of acetyl is joined to the
carbonyl carbon (C2) of oxaloacetate.
• To drive the reaction forward, the exergonic hydrolysis to
form free CoA and citrate is critical.
• This exergonic reaction is important to counter the slowness
of the reaction arising from a low concentration of
oxaloacetate.
• The CoA is recycled by PDH complex to decarboxylate
another molecule of pyruvate.
• Citrate synthase undergoes induced conformation change
after binding to oxaloacetate to fit acetyl CoA. Another
change of conformation after forming a bond between
oxaloacetate and acetyl-CoA causes hydrolysis of CoA.
1. Formation of citrate
2. Citrate to isocitrate
 2. Citrate to isocitrate
• Aconitase is an enzyme that is capable of adding water
to the cis-Aconitate to form both isocitrate and citrate.
• Although less than 10% isocitrate is formed at pH 7.4
and 25 degree Celsius, the reaction moves forward
when the product, isocitrate, is consumed in later
steps.
• Aconitase contains an iron sulfur center, which is
important in holding the substrate at the active site
and in carrying out catalysis.
• Aconitase acquires a role in regulating iron
homeostasis when iron is scarce. There are number of
such enzymes that serve two functions.
3. Isocitrate to alpha-ketoglutarate
3. Isocitrate to alpha-ketoglutarate
• In this step isocitrate dehydrogenase catalyzes oxidative
decarboxylation of isocitrate to form alpha-ketoglutarate.
• Dehydrogenation takes place with the help of NAD(P)+
before decarboxylation happens.
• Two forms of isocitrate dehydrogenases exist depending on
their preference for NAD+ or NADP+.
• The enzyme that prefers NAD is found in the mitochondrial
matrix and functions in the citric acid cycle.
• The enzyme with a preference for NADP is found both in
mitochondria and cytosol and may have a function of
generating NADPH.
4. Alpha-ketoglutarate to succinyl CoA
4. Alpha-ketoglutarate to succinyl CoA
• This reaction is another example of oxidative
decarboxylation and is carried out by
alpha-ketoglutarate dehydrogenase complex.
• NAD+ is reduced to NADH, while CoA
covalently bonds with succinyl group.
• The energy of oxidation is conserved by the
formation of thioester bond.
5. Succinyl CoA to succinate
 5. Succinyl CoA to succinate
• The breaking of thioester bond is used to drive the
formation of phosphoanhydrite bond.
• A GTP or an ATP is formed from this reaction.
• There are two versions of the enzyme: one that
transfers phosphate group to GDP and another
that transfers it to ADP.
• GTP thus formed can easily be used to form ATP
from ADP using nucleoside diphosphate kinase.
• The enzyme that catalyzes this reversible reaction
is called a succinyl-CoA synthetase or succinic
thiokinase.
6. Succinate to fumarate
 6. Succinate to fumarate
• This reaction is catalyzed by a flavoprotein
succinate dehydrogenase.
• In eukaryotes this enzyme is bound to the
inner membrane of mitochondria, whereas in
bacteria it is found to the plasma membrane.
• Electrons pass from succinate to FAD to iron
sulfur center (3 found in this enzyme) to a
chain of electron carriers in the mitochondrial
inner membrane.
7. Fumarate to malate
 7. Fumarate to malate
• The hydration of fumarate to form malate is
catalyzed by fumarase. The reaction goes
through carbanion transition state.
• The enzyme is very stereospecific. In the
forward direction it is very selective for the
trans isomer (not the cis isomer), and in the
reverse directly it exclusively works with
L-malate (not D-malate).
8. Malate to oxaloacetate
 8. Malate to oxaloacetate
• The equilibrium of this reaction, described by
the delta G value, lies far to the left in this
reaction.
• But a highly negative delta G of the
subsequent reaction drives this reaction
forward.
 Following citric acid cycle
• The citric acid cycle is studied by following
radioactively labeled acetate and pyruvate.
Initially the fate of these molecules was
worked out in vitro. Nowadays, these
compounds are followed in vivo using whole
tissue NMR spectroscopy.
Product of 1 turn of citric acid cycle
• 1 complete turn of citric acid cycle releases
two carbon dioxides one from isocitrate and
another from alpha-ketoglutarate
• It also results in the yield of 3 NADHs, 1 FADH2
and 1 ATP or GTP.
• It should be noted that the two carbon
dioxides released do not come from 2 carbons
of acetyl-CoA.
Stoichiometry of oxidation of glucose
 Stoichiometry
• There is a net yield of 2 ATPs and 2 NADHs from glycolysis.
• From citric acid cycle, we get a net yield of 8 NADHs and 2
FADH2s.
• Additionally 2 ATPs are produced from one cycle of citric
acid cycle.
• Given that 2.5 ATPs are produced per molecule of NADH
and 1.5 ATPs per molecule of FADH2, we get a total of
(2.5*10+1.5*2+2+2 =32) 32 ATPs per molecule of glucose.
• This means a total of 32*30.5 kJ/mol =976 kJ/mol of energy
is conserved per molecule of glucose. That is roughly 34%
(976/2840) efficiency in the conversion of energy from
glucose to ATP. When corrected for actual free energy
required for ATP production within cells this efficiency
number goes up to 65%.
Incomplete citric acid cycle
 Incomplete citric acid cycle
• Components of citric acid cycle probably first evolved
in anaerobic bacteria. In these bacteria, alpha
ketoglutarate and succinyl-CoA serve as biological
precursors for synthesis of biological products such as
amino acids, nucleotides, heme, etc.
• These bacteria have enzymes that can form
alpha-ketoglutarate, but cannot convert it to
succinyl-CoA.
• They also have enzymes that catalyze the reverse
citric acid cycle to give Succinyl-CoA.
• NADH produced by citrate oxidation is recycled to
synthesize succinyl-CoA.
Citric acid
cycle in
anabolism
 Citric acid cycle in anabolism
• Citric acid cycle intermediates are used for various anabolic
processes.
• Anaplerotic reactions replace citric acid cycle intermediates
so that their concentration remains constant. These
reactions are shown by red arrows in the figure.
• When the citric acid cycle intermediates are in low
concentration, pyruvate carboxylase with the help of ATP
forms oxaloacetate from pyruvate.
• Pyruvate carboxylase is positively stimulated by acetyl-CoA
• PEP carboxylase is activated by the accumulation of fructose
1,6-bisphosphate. This happens when citric acid cycle
operates slowly.
 Pyruvate carboxylase
• This enzyme forms oxaloacetate from pyruvate.
• This enzyme requires the vitamin, biotin, for its function.
• Biotin plays a key role in many carboxylation reactions.
• It carries carbon dioxide in these reactions.
• The reaction mechanism consists of 2 major steps:
carboxylation of biotin and transfer of carbon dioxide to
pyruvate.
• These two reactions occur at different sites in the enzyme.
• Biotin provides flexible tether to move carbon dioxide
around.
 Regulation
• The activity of PDH complex and citrate
synthase is heavily regulated.
• Similarly, the activity of isocitrate
dehydrogenase and alpha-ketoglutarate
dehydrogenase complexes are also regulated.
Regulation
 Allosteric control of PDH complex
• The reaction is inhibited by the products of
the reaction: ATP, acetyl-CoA and NADH.
• The accumulation of AMP, CoA, NAD+ signals
little flow of acetate activates PDH complex.
• Availability of fuel signaled by fatty acids also
inhibits the complex.
 Covalent control of PDH complex
• The complex is inhibited by phosphorylation of
a specific Ser residue in one of the two units of
E1.
• Pyruvate dehydrogenase kinase
phosphorylates and inactivates E1. Kinase is
allosterically activated by a high concentration
of ATP.
• A specific phosphatase removes phosphoryl
group and activates PDH.
 Regulation
• There are variations in the regulation of the enzyme in plants, animals and
bacteria.
• 3 factors govern flux through the cycle: substrate availability, inhibition by
accumulating products, and allosteric feedback inhibition
• Three exergonic steps catalyzed by citrate synthase, isocitrate dehydrogenase,
and alpha-ketoglutarate dehydrogenase can become rate limiting under certain
conditions.
• Accumulation of NADH blocks the two dehydrogenases.
• succinyl-CoA inhibits alpha-ketoglutatrate dehydrogenase; citrate blocks citrate
synthase; and ATP blocks both citrate snythase and isocitrate dehydrogenase.
• ADP relieves ATP’s inhibition of citrate synthase.
• Ca2+ that signals contraction and increase in ATP consumption in muscles
activates alpha-keto glutarate dehydrogenase, isocitrate dehydrogenase and PDH
• The concentration of pyruvate, lactate and acetyl-CoA are maintained at steady
levels. Regulation also involves inhibition of phosphofructokinase-1 by citrate.