UNIVERSITY OF LUSAKA

School of Medicine and Health Sciences

Department of Basic Sciences

Biochemistry II

BMPS 111

LECTURE 2

The Citric acid cycle (Krebs Cycle/Tricarboxylic acid

cycle (TCA))
 Learning Outcomes
• Describe the reactions of the citric acid cycle.
• Describe the function of the citric acid cycle and identify
the products produced
• Understand the importance of acetyl CoA as a metabolic
intermediary
• Understand the regulation of the TCA (ATP demands,
ADP levels.)
Catabolism of proteins, fats, and carbohydrates in the three
stages of cellular respiration
 The Citric acid cycle
• The citric acid cycle (Krebs cycle or tricarboxylic acid—TCA
cycle) is the most important metabolic pathway for the energy
supply to the body.
• About 65-70% of the ATP is synthesized in Krebs cycle.
• Citric acid cycle essentially involves the oxidation of acetyl
CoA to CO2 and H2O.
• This cycle utilizes about two thirds of total oxygen consumed
by the body.
• The name TCA cycle is used, since, at the outset of the cycle,
tricarboxylic acids (citrate, cis-aconitate and isocitrate)
participate.
 TCA cycle—the central metabolic
pathway
• The citric acid cycle is the common oxidative pathway for
carbohydrates, fats and amino acids.
• This cycle not only supplies energy but also provides
many
• intermediates required for the synthesis of amino acids,
glucose, heme etc.
• Krebs cycle is the most important central pathway
connecting almost all the individual metabolic pathways
(either directly or indirectly).
• Thus it acts as an amphibole - acts both catabolically and
anabolically.
 Overview
• Krebs cycle basically involves the combination of a two
carbon acetyl CoA with a four carbon oxaloacetate to produce
a six carbon tricarboxylic acid, citrate.
• In the reactions that follow, the two carbons are oxidized to
CO2 and oxaloacetate is regenerated and recycled.
• Oxaloacetate is considered to play a catalytic role in citric
acid cycle. An overview of Krebs cycle is depicted below.
 Location of the TCA
• The enzymes of TCA cycle are located in mitochondrial matrix, in
close proximity to the electron transport chain.
• This enables the synthesis of ATP by oxidative phosphorylation
without any hindrance
• Substrates have to flow across the outer and inner parts of the
mitochondria
An overview of intermediary metabolism

• Once pyruvate is formed

during glycolysis, it then
travels to the matrix of
the mitochondria to
undergo the first
decarboxylation to form
acetyl Co-A.
• The pyruvate moves
across the membrane of
the mitochondria via
transport proteins known
as pyruvate translocases.
 Co-A acts as a carrier of acetyl groups
• Acetyl-CoA is a “high energy” compound: The G' for the
hydrolysis of its thioester is -31.5 kJ• mol -1 making it greater than the
hydrolysis of ATP .
• The enzyme Pyruvate dehydrogenase(PDH) complex converts
pyruvate to acetyl-CoA and CO2
Conversion of pyruvate to acetyl CoA- Oxidative
Decarboxylation
• Pyruvate is converted to acetyl CoA by oxidative
decarboxylation.
• This is an irreversible reaction, catalyzed by a multi-
enzyme complex, known as pyruvate dehydrogenase
complex (PDH), which is found only in the mitochondria.
• High activities of PDH are found in cardiac muscle and
kidney.
• The enzyme PDH requires five cofactors (coenzymes),
namely—TPP, lipoamide, FAD, coenzyme A and NAD+
(lipoamide contains lipoic acid linked to ε-amino group of
lysine).
Oxidative Decarboxylation of Pyruvate

Allosteric
Regulation
 Pyruvate dehydrogenase
• Multi-enzyme complexes are groups of non-
covalently associated enzymes that catalyze two or
more sequential steps in a metabolic pathway.
 Reactions of PDH complex
• Step 1: Thiamine pyrophosphate of E1 de-carboxylates pyruvate. The acetyl
group of pyruvate is attached to the TPP and a CO2 as a bi-product is lost in
the process.
• Step 2: Lipoyllysine of the E2 enzyme Dihydrolipoyl transacetylase, accepts
the acetyl group from TPP. Forming an acetyl (acyl lipoyllysine) lipoamide.
• Step 3: Acetyl group is transferred to co-enzyme A. Forming acetyl- CoA.
This leaves a reduced lipoyllysine.
• Step 4: The cycle is complete when reduced lipoamide is converted to
oxidized lipoamide by dihydrolipoyl dehydrogenase, transferring the
reducing equivalents to FAD.
• FADH2, in turn, transfers the reducing equivalents to NAD+ to give NADH
+ H+, which can pass through the respiratory chain to give 3 ATP (6 ATP
from 2 moles of pyruvate formed from glucose) by oxidative
phosphorylation.
• The intermediates of PDH catalysed reaction are not free but bound with
enzyme complex.
 Regulation of the PDH complex
• The conversion of pyruvate into Acetyl co-A is a key step in glucose
metabolism. It is an irreversible step that commits the glucose
derivative into one of two pathways (1) the citric acid cycle or (2) lipid
synthesis.
• This step must be closely regulated to meet the needs of the cell. The
regulation of this step at the PDH complex.
• The PDH complex is controlled by phosphorylation.
• When the PDH enzyme complex is phosphorylated, it is inactive,
when it is dephosphorylated, it is active.
1. Resting conditions
•Under such conditions, the energy charge of the cell is high.
•This means that there are high concentrations of Acetyl CoA,
NADH and ATP.
•Because the cell does not require more ATP, these three molecules
promote phosphorylation of the E1 enzyme of the PDH complex
inhibiting its activity.

2. Exercising conditions
•Under these conditions, the energy charge of the cell is low
because the ATP is used up. The ADP and pyruvate will inhibit the
kinase from phosphorylating E1 of the PDH.
•There is also a rise in Ca2+ ions that stimulate phosphatase to
remove phosphates off of PDH there by activating it.
Epinephrine
•When released, this hormone will bind to liver cells and
stimulate a signal transduction mechanism which increases
levels of Ca2+ in the cell thereby stimulating phosphatase.

Insulin
•After a meal, insulin indirectly stimulates phosphatase to
activate PDH in liver and fat cells. This helps transform
pyruvate into acetyl CoA to help form lipids.
 Why such a complex set of enzymes?
1. Enzymatic reactions rates are limited by
diffusion, with shorter distance between
subunits a enzyme can almost direct the
substrate from one subunit (catalytic site) to
another.
2. Channeling metabolic intermediates between
successive enzymes minimizes side reactions
3. The reactions of a multi-enzyme complex can
be coordinately controlled.
The Citric Acid Cycle Has
Eight Steps
Step 1: Condensation reaction
Formation of citrate : Krebs cycle proper starts with the condensation of
acetyl CoA and oxaloacetate to form citrate, catalysed by the enzyme
citrate synthase.
• Step 2: Citrate is isomerized to iso-citrate by the enzyme
aconitase.
• This is achieved in a two stage reaction of dehydration
followed by hydration through the formation of an
intermediate cis-aconitate.
• Step 3: Oxidation of Isocitrate to Ketoglutarate and CO2:
• The enzyme isocitrate dehydrogenase (ICD) 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.
• Step 4: Conversion of α-ketoglutarate to succinyl CoA
occurs through oxidative decarboxylation, catalysed by α-
ketoglutarate dehydrogenase complex.
• This enzyme is dependent on five cofactors—TPP, lipoamide,
NAD+ ,FAD and CoA.
• The mechanism of the reaction is analogous to the conversion of
pyruvate to acetyl CoA
• Step 5: Formation of succinate : Succinyl CoA is converted to
succinate by 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
• Step 6: Oxidation of succinate to fumarate : Succinate is
oxidized by succinate dehydrogenase to fumarate. This reaction
results in the production of FADH2 and not NADH
• Step 7 Hydration of Fumarate to Malate: The enzyme
fumarase catalyses the conversion of fumarate to malate
with the addition of H2O. The transition state
• in this reaction is a carbanion.
• Step 8: Oxidation of Malate to Oxaloacetate: Malate is then
oxidized to oxaloacetate by malate dehydrogenase.
• The third and final synthesis of NADH occurs at this stage.
• The oxaloacetate is regenerated which can combine with another
molecule of acetyl CoA, and continue the cycle.
 Energetics of citric acid cycle
• During the process of oxidation of acetyl CoA via the
citric acid cycle, 4 reducing equivalents (3 as NADH
and one as FADH2) are produced.
• Oxidation of 3 NADH by electron transport chain
coupled with oxidative phosphorylation results in the
synthesis of 9 ATP, whereas FADH2 leads to the
formation of 2 ATP.
• Besides, there is one substrate level phosphorylation.
Thus, a total of twelve ATP (10 as per recent evidence)
are produced from one acetyl CoA.
Products of one turn of the citric acid
cycle • At each turn of the cycle,
three NADH, one
FADH2, one GTP (or
ATP), and two CO2 are
released in oxidative
decarboxylation
reactions.
• Most of the reactions of
the TCA are reversible.
 Regulation of citric acid cycle
• The TCA has Three regulatory points that are used to control its rate.
• The first is at the conversion of pyruvate to Acetly Co-A, the second is
at step 3 the conversion of iso-citrate to α-ketoglutarate and the final is
at step 4, the conversion of α-ketoglutarate to succinyl Co-A
• Step 2 and 3 are allosteric regulation.
1. Iso-citrate dehydrogenase
• Catalyses the first oxidative decarboxylation step of the citric acid cycle.
• Allosteric effectors include ADP, which binds to a regulatory site of the
enzyme when the cell requires energy. This binding increases the
affinity of the enzyme for the isocitrate substrate which will increase the
rate of the TCA cycle.
• High levels of ATP means no need to generate more, thus, ATP will act
as an allosteric inhibitor of Iso-citrate dehydrogenase and decrease its
affinity for the substrate.
• NADH also acts as an inhibitor of the enzyme.
 Regulation of citric acid cycle
• Α-ketoglutarate dehydrogenase catalyses
the second oxidative decarboxylation step.
• When the energy charge of the cell is high,
ATP and succinyl CoA and NADH act as
allosteric inhibitors.
 Regulation of citric acid cycle
• Availability of ADP is very important for the citric acid cycle
to proceed.
• This is due to the fact that unless sufficient levels of ADP are
available, oxidation (coupled with phosphorylation of ADP to
ATP) of NADH and FADH2 through electron transport chain
stops.
• The accumulation of NADH and FADH 2 will lead to
inhibition of the enzymes (as stated above) and also limits the
supply of NAD+ and FAD which are essential for TCA cycle
to proceed.
 Regulation of Oxidative
Decarboxylation and Krebs Cycle
• PDH complex and the TCA cycle are both up-regulated in
response to a decrease in the ratio of :
• ATP:ADP
• NADH:NAD+
• TCA cycle activators are:
• ADP
• Ca2+
• TCA cycle inhibitors are:
• ATP
• NADH
 Krebs Cycle: Energy Yield

Number of ATP molecules produced from the oxidation of one molecule of

acetyl coenzyme A (CoA) using both substrate-level and oxidative
phosphorylation.
 Net ATP Production by
Complete Glucose Oxidation

Aerobic glycolysis: 8 ATP

Oxidative decarboxylation: 2X3= 6 ATP

Krebs cycle: 2 X 12 = 24 ATP

Net: 38 ATP
 Summary
• Pyruvate is oxidatively de-carboxylated by PDH to acetyl CoA
inside the mitochondria.
• For each acetyl-CoA that enters the citric acid cycle;
• 2 molecules of carbon dioxide,
• 3 molecules of NADH,
• 1 molecule of ATP, and
• 1 molecule of FADH2 is produced.