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 Three stages of catabolism

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 Fate of Pyruvate
Under aerobic conditions, pyruvate is converted to acetyl-CoA in all tissues
containing mitochondria. Both pyruvate molecules are oxidized to two acetyl-
CoA molecules.

Entry of Pyruvate into Mitochondria

The mitochondrial membrane is not permeable to pyruvate, which is formed in
cytosol. A specific carrier present in mitochondrial membrane transports
pyruvate across mitochondrial membrane.

Fate of Pyruvate in Mitochondria

In mitochondria, pyruvate undergoes oxidative decarboxylation and remaining
two carbon fragment is converted to acetyl-CoA. The reaction is irreversible
and multi-step process. This reaction is catalyzed by pyruvate dehydrogenase
(PDG) multi enzyme complex present in inner mitochondrial membrane. This
multi enzyme complex consist of three enzymes.
Pyruvate dehydrogenase (PDH or E1, also called a decarboxylase)
Dihydrolipoamide acetyltransferase [E2]
Dihydrolipoamide dehydrogenase [E3]
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 Medical Importance
1.Pyruvate dehydrogenase serve as a link between aerobic
glycolysis and citric acid cycle.

2. Since the reaction catalyzed by this enzyme is irreversible,

acetyl -CoA can not be converted to pyruvate. For this reason, fat
can not be converted to carbohydrate.

3. Lactic acidemia occurs in some individuals due to deficiency

of pyruvate dehydrogenase.

4. Arsenic compounds inhibits this enzyme by reacting with-SH

of lipoic acid. War gases and pesticides containing arsenic inhibit
this enzyme. Deaths due to arsenic poisoning are well
documented in history.
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 Regulation of pyruvate dehydrogenase (PDH)

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 Regulation of the Pyruvate Dehydrogenase Complex

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 Coenzymes: The pyruvate dehydrogenase complex contains five
coenzymes that act as carriers or oxidants for the intermediates of the
reactions are thiamine pyrophosphate, lipoic acid , coenzyme A, FAD and
NAD+

Pyruvate dehydrogenase deficiency: A deficiency in the pyruvate

dehydrogenase complex is the most common biochemical cause of
congenital lactic acidosis. This enzyme deficiency results in an inability to
convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactic acid
via lactate dehydrogenase .This causes particular problems for the brain,
which relies on the TCA cycle for most of its energy, and is particularly
sensitive to acidosis.

Inhibition of Pyruvate dehydrogenase

Arsenite and mercuric ions react with lipoic acid and inhibit pyruvate
dehydrogenase.

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 The central role that acetyl CoA plays in metabolism
•Acetyl CoA plays a central
role in metabolism. In fact,
most energy generating
metabolic pathways of the cell
eventually produce it.

•It can be formed from

carbohydrate, fat and protein.

•It is also the starting point for

the synthesis of fats, steroids
and Ketone bodies.

•Its oxidation provides energy

for many tissues.

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 Tricarboxylic acid cycle
Citric acid cycle or Krebs cycle
• The citric acid cycle is the final common
pathway for the oxidation of fuel
molecules amino acids, fatty acids, and
carbohydrates. Most fuel molecules enter
the cycle as acetyl coenzyme A.
• A cyclical series of eight reaction that
oxidize one molecule of acetyl CoA
completely to two molecules of CO2,
generating energy, either directly as ATP
or in the form of reducing equivalents
(NADH or FADH2).
• The cycle is aerobic; the absence or
deficiency of oxygen leads to total or
partial inhibition of the cycle.
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  Location: All
mammalian cells that
contain mitochondria
(i.e. not red blood
cells)

 Site: All the enzymes

of TCA cycle are
located in the
mitochondrial
matrix.

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 Function of TCA cycle
1. The TCA cycle provides final common pathway for the
oxidation of carbohydrate, fat and protein.
2. The main function of the cycle is the production of energy,
either, directly as ATP or as the reducing equivalents, NADH
or FADH2, which are oxidized by ETC.
3. The cycle provides substrates for the ETC.
4. The cycle is also a source of biosynthetic precursors, for
example, porphyrin is synthesized from succinyl CoA, and
amino acids are synthesized from oxaloacetate and α-
ketoglutarate.

5. Some of the cycle intermediates also exerts regulatory effects

on other pathways; for example, citrate inhibits PFK-1 in
glycolysis.

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 Reactions of citric acid cycle
 Oxidative decarboxylation of pyruvate to acetyl
CoA by pyruvate dehydrogenase complex.
1. Formation of citrate:
 Krebs cycle proper starts with the condensation
of acetyl CoA and oxaloacetate, catalysed by
the enzyme citrate synthase.
2. Citrate is isomerized to isocitrate:
It is done by enzyme aconitase. It is achieved in a
two stage reaction of dehydration followed by
hydration through the formation of internediate
compound is cis-aconitase.

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 3. Formation of a-ketoglutarate:
 The enzyme isocitrate dehydrogenase (lCD) catalyses
the conversion (oxidative decarboxylation) of isocitrate
to oxalosuccinate and then to 𝛼-ketoglutarate.
 The formation of NADH and the liberation of CO2 occur
at this stage.

4. Conversion of a-ketoglutarate to succinyl CoA:

 It occurs through oxidative decarboxylation and
catalysed by 𝛼 -ketoglutarate dehydrogenase complex.
 The enzymes dependent on cofactors-TPP, lipoamide,
NAD+, FAD and CoA.
 The mechanism of the reaction is analogous to the
conversion of pyruvate to acetyl CoA.
 At this stage, second NADH is produced and the
second CO2 is liberated.

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 5. Formation of succinate:
 Succinyl CoA is converted to succinate by enzymes
succinate thiokinase.
 This reaction is coupled with the phosphorylation of
GDP to GTP.
 This is a substrate level phosphorylation. GTP is
converted to ATP by the enzyme nucleoside
diphosphate kinase.
 GTP + ADP→ATP + GDP

6. Conversion of succinate to fumarate:

by succinate dehydrogenase. This reaction results in
the production of FADH2 and not NADH.

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 7. Formation of malate:
 The enzyme fumarase catalyses the conversion of
fumarate to malate with the addition of H2O.

8. Conversion of malate to oxaloacetate:

 By malate dehydrogenase and third and final synthesis
of NADH occurs in this stage.
 The oxaloacetate is regenerated which can combine
with another molecule of acetyl CoA, and continue the
cycle.

 Summary of this cycle:

 Acetyl CoA + 3 NAD+ + FAD + CDP + Pi + 2H2O →2CO2
+ 3NADH + 3H+ + FADH2 + GTP + CoA.

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 Generalizations on the Regulation of Metabolic
Pathways
1. Regulation of metabolic pathways occurs at rate-limiting
steps, the slowest steps, in the pathway. These are reactions in
which a small change of rate will affect the flux through the
whole pathway.
2. Regulation usually occurs at the first committed step of a
pathway or at metabolic branchpoints. In human cells, most
pathways are interconnected with other pathways and have
regulatory enzymes for every branch point.
3. Regulatory enzymes often catalyze physiologically
irreversible reactions. These are also the steps that differ in
biosynthetic and degradative pathways.

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 4. Many pathways have “feedback” regulation, that is, the end
product of the pathway controls the rate of its own synthesis.
Feedback regulation may involve inhibition of an early step in
the pathway (feedback inhibition) or regulation of gene
transcription.
5. Human cells use compartmentation to control access of
substrate and activators or inhibitors to different enzymes.
6. Hormonal regulation integrates responses in pathways
requiring more than one tissue. Hormones generally regulate
fuel metabolism by:
a. Changing the phosphorylation state of enzymes.
b. Changing the amount of enzyme present by changing its rate
of synthesis (often induction or repression of mRNA synthesis)
or degradation.
c. Changing the concentration of an activator or inhibitor.

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 Regulation of TCA cycle

Three regulatory enzymes-

namely

• Citrate synthase

• Isocitrate dehydrogenase

• α-Ketoglutarate dehydrogenase

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 Regulation of TCA cycle

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 1. Citrate synthase: Citrate synthase, which is the first enzyme of
the TCA cycle, is a simple enzyme that has no allosteric
regulators.
 Citrate is a competitive inhibitor of oxaloacetate for citrate
synthase (product inhibition); the fall in [citrate] caused by
increased isocitrate dehydrogenase activity increases the rate of
citrate formation.
 Succinyl-CoA also competes with acetyl-CoA in the citrate
synthase reaction (competitive feedback inhibition).

2. Isocitrate dehydrogenase is considered one of the ratelimiting

steps of the TCA cycle, and is allosterically activated by ADP and
inhibited by NADH

3. The α -ketoglutarate dehydrogenase complex, although not an

allosteric enzyme, is product-inhibited by NADH and succinyl
CoA, and may also be inhibited by GTP.
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  Thus, both α-ketoglutarate dehydrogenase and
isocitrate dehydrogenase respond directly to changes
in the relative levels of ADP and hence the rate at
which NADH is oxidized by electron transport. Both
of these enzymes are also activated by Ca2+.
 In contracting heart muscle, and possibly other
muscle tissues, the release of Ca2+ from the
sarcoplasmic reticulum during muscle contraction
may provide an additional activation of these
enzymes when ATP is being rapidly hydrolyzed.

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Calcium (Ca2+) as a regulator
 Ca2+ among its many biological functions, is an essential
metabolic regulator.
 It stimulates glycogen breakdown, triggers muscle
contraction,and mediates many hormonal signals as a second
messenger.
 Ca2+ also plays an important role in the regulation of the citric
acid cycle.
 It activates pyruvate dehydrogenase phosphatase and inhibits
pyruvate dehydrogenase kinase, thereby activating the PDC to
produce acetyl-CoA .
 In addition, Ca2+ activates both isocitrate dehydrogenase and α
-ketoglutarate dehydrogenase.
 Thus, the same signal stimulates muscle contraction and the
production of the ATP to fuel it.
 Respiratory control
 This is governed by the activity of the electron transport chain
(which oxidizes NADH and FADH2) and the rate of oxidative
phosphorylation (ATP synthesis)
 The activity of the TCA cycle is dependent on the continuous
supply of NAD+ and FAD, cofactor for dehydrogenases.
 The ETC is responsible for oxidizing any NADH and FADH2
formed during glycolysis and TCA cycle back to their
oxidized forms, i.e. NAD+ and FAD.
 Therefore, anything affecting the supply of substrates, namely
oxygen, ADP, or the source of reducing equivalents, may
inhibit the cycle.

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 Inhibitors of TCA cycle

Fluoroacetate

Arsenite

Malonate

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 Inhibitors of Krebs cycle
Enzyme Inhibitor

Aconitase Fluoroacetate
(non-competitive)
𝛼-Ketoglutarate Arsenite
dehydrogenase (non-competitive)
Succinate Malonate
dehydrogenase (competitive)

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Biosynthetic Roles of the Citric Acid Cycle
• The main synthetic pathway that use TCA
cycle intermediates are:

• Mitochondrial citrate is transported to the

cytosol, where it is cleaved to provide
acetyl CoA for the biosynthesis of fatty
acids and sterols.

• Amino acid synthesis: for example,

aspartate from oxaloacetate and glutamate
from α-ketoglutarate.

• Porphyrin biosynthesis from succinyl CoA.

• Gluconeogenesis: this occurs in the cell

cytosol from oxaloacetate.
 Biosynthetic Pathways Begin with Glycolysis or TCA
Cycle  Intermediates can be
used by other
enzymes as the
starting point in
making amino acids,
nucleotides, lipids
and other small
organic compounds
 Black arrows – one
enzyme reaction
 Red arrows – multi
step reactions

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 Anaplerotic reactions
The reaction

• The intermediates that are

used for synthetic reaction
must be replaced for the TCA
cycle to be able to continue.

• The reaction concerned to fill

up the intermediates of TCA
cycle are called Anaplerotic
reaction.

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 Examples
 Since large entry of Acetyl CoA to TCA cycle depletes the
supply of oxaloacetate, required for synthesis of citrate.
Oxaloacetate has to be replenished. So most important
anaplerotic reaction is formation of oxaloacetate from
pyruvate by the enzyme pyruvate carboxylase.

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  Formation of alpha-ketoglutarate by glutamate
dehydrogenase reaction.
 Formation of fumarate from amino acids
phenylalanine and tyrosine.
 Formation of succinyl CoA from propionyl CoA.
 Formation of oxaloacetate via an enzyme called
malic enzyme, which gives malate and upon
oxidation gives oxaloacetate.

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 What is the maximum yield of high energy ATP in
the aerobic catabolism of glucose?

Glycolysis:
glucose 2pyruvate + 2NADH+2ATP 8 ATPs
Pyruvate Dehydrogenase:
2pyruvate  2acetyl CoA + 2NADH 6 ATPs
TCA cycle:
acetyl CoA2CO2+3NADH+FADH2+GTP
2x12ATPs

OVERALL yield from glucose 38 ATPs

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 Old:
1FADH2= 2 ATP.
1NADH=3 ATP

New:
1FADH2= 1.5 ATP
1 NADH= 2.5 ATP

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 Generation of high-energy
phosphate in the catabolism of glucose

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 Vitamins Play Key
Roles in the Citric Acid Cycle
Four of the B vitamins are essential in the citric acid cycle and
hence energy-yielding metabolism:
(1) Riboflavin, in the form of flavin adenine dinucleotide
(FAD), a cofactor for succinate dehydrogenase;
(2) Niacin, in the form of nicotinamide adenine dinucleotide
(NAD), the electron acceptor for isocitrate dehydrogenase, -
ketoglutarate dehydrogenase, and malate dehydrogenase;
(3) Thiamin (vitamin B1), as thiamin diphosphate, the
coenzyme for decarboxylation in the -ketoglutarate
dehydrogenase reaction; and
(4) Pantothenic acid, as part of coenzyme A, the cofactor
attached to "active" carboxylic acid residues such as acetyl-
CoA and succinyl-CoA.
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 The Citric Acid Cycle Plays a Pivotal
Role in Metabolism
 The citric acid cycle is not only a pathway for oxidation of
two-carbon units, but is also a major pathway for
interconversion of metabolites arising from transamination and
deamination of amino acids, and
 Providing the substrates for amino acid synthesis by
transamination, as well as for gluconeogenesis and fatty acid
synthesis.
 Because it functions in both oxidative and synthetic processes,
it is amphibolic

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 The Citric Acid Cycle
Takes Part in Fatty Acid Synthesis
 Acetyl-CoA, formed from pyruvate by the action
of pyruvate dehydrogenase, is the major substrate
for long-chain fatty acid synthesis.
 Pyruvate dehydrogenase is a mitochondrial
enzyme, and fatty acid synthesis is a cytosolic
pathway; the mitochondrial membrane is
impermeable to acetyl-CoA.
 Acetyl-CoA is made available in the cytosol from
citrate synthesized in the mitochondrion,
transported into the cytosol, and cleaved in a
reaction catalyzed by ATP-citrate lyase.
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  Citrate is only available for transport out of the mitochondrion
when aconitase is saturated with its substrate, and citrate
cannot be channeled directly from citrate synthase onto
aconitase.
 This ensures that citrate is used for fatty acid synthesis only
when there is an adequate amount to ensure continued activity
of the cycle.

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