Block 1

BBCCT-109
METABOLISM OF
Indira Gandhi
National Open University
CARBOHYDRATES AND
School of Sciences LIPIDS
BLOCK 1
CARBOHYDRATE MATABOLISM I 5
BLOCK 2
CARBOHYDRATE MATABOLISM II 67
BLOCK 3
LIPID METABOLISM I 134
BLOCK 4
LIPID METABOLISM II 180
Programme and Course Design Committee
Prof. Bechan Sharma Prof. K. Vali Pasha Faculty Members
Dept of Biochemistry Dept. of Biochemistry (IGNOU)
University of Allahabad Yogi Vemana University
Andhra Pradesh Dr. Parvesh Bubber
Prof. Ranjit K. Mishra Biochemistry, SOS
Dept. of Biochemistry Prof. Seemi Farhat Basir
University of Lucknow Dept. of Bio Sciences Dr. M. Abdul Kareem
Jamia Milia Islamia Biochemistry, SOS
Prof. Reena Gupta
Dept. of Biotechnology Dr. Sunita Joshi Dr. Arvind Kumar
H.P. University, Shimla Dept. of Biochemistry Shakya
Daulat Ram College Biochemistry, SOS
Prof. D.V. Devaraju University of Delhi
Dept. of Biochemistry Dr. Maneesha
Bangalore University Prof. Vijayshri Pandey
Former Director Biochemistry, SOS
Prof. Sanjeev Puri School of Sciences
UIET, Panjab University IGNOU, New Delhi Dr. Seema Kalra
Biochemistry, SOS
Course Preparation Team

Content Editor Content Preparation
Dr. Sunita Joshi (Retd) Dr. Seema Kalra (Unit 1-5, 8- Dr. Ekta Chitkara (Unit 11, &12)
Dept. of Biochemistry 10,14) Associate Professor, Faculty of
Daulat Ram College SOS, IGNOU Applied Sciences
University of Delhi Manav Rachna International
Dr. Maneesha Pandey (Unit 6) Institute of Research and Studies
SOS, IGNOU Faridabad, Haryana
Dr. Niraj Srivastava (Unit 7) Professor R.K. Sharma (Unit 13)

Jr. Consultant, Biochemistry Sr. Consultant, Biochemistry
SOS, IGNOU SOS, IGNOU
Course Coordinator: Dr. Seema Kalra (seemakalra@ignou.ac.in)
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METABOLISM OF CARBOHYDRATES AND
LIPIDS
Metabolism of Carbohydrates and Lipids is the fifth core course of B.Sc. (Honours)
Biochemistry Programme (BSCBCH. It is being offered as the third Semester course under
UGC-CBCS Scheme. The theory course of Metabolism of Carbohydrates and Lipids (BBCCT-
109) is of 4 credits and a separate laboratory course (BBCCL-110) worth 2 credits is offered
along with this course.
While preparing this course, we have kept in mind that students joining our B.Sc. (Honours)
Biochemistry Programme have studied Biology, Chemistry and Physics upto 10+2 level. Study
of metabolism includes two aspects: catabolism- the breakdown of bio molecules and
anabolism- the synthesis of complex bio molecules. Learning about these pathways gives an
insight into: (i) how living organisms derive energy by catabolism, (ii) how they synthesize
molecules required for their growth and carry out other life sustaining activities, (iii) how different
pathways are integrated with each other and regulated in a coordinated fashion for overall
health and well being of an organism and (iv) biochemical basis of different diseases due to
defect or lack of enzymes catalysing one or more reactions involved in these pathways. That’s
why study of metabolism is at the heart of biochemistry.
We have dealt with metabolic aspects of carbohydrates and lipids in this theory course. It
consists of 14 units divided in 4 blocks. Each block contains 3-4 units which are based on a
common theme. The first two blocks i.e. Block I and Block II deal with metabolism of
carbohydrates. Block III and Block IV deal with metabolism of lipids.
Block 1 on Carbohydrate Metabolism I begins with general overview of metabolism and its
salient features which explain simplicity in its complexity. It will also describe significance and
role of different types of energy rich molecules such as ATP and reducing powers in
metabolism. Units 2-3 describe various catabolic pathways of carbohydrates, their importance
and regulation. These mainly explain breakdown of glucose and other monosaccharides.
Block 2 on Carbohydrates Metabolism II discusses anabolism of carbohydrates. It begins with

synthesis of glucose from non carbohydrates sources (Unit 4). It is followed by metabolism of
glycogen (Unit 5) and starch (Unit 6); the energy storing molecules of animals and plants,
respectively. Unit 7 explains how the metabolic pathways described in Units 2-6 are
interconnected and regulated in a coordinated fashion.
Block 3 on Lipid Metabolism I deals with metabolism of fatty acids formed after fat digestion.
First unit of this block (Unit 8) begins with major pathways of fatty acid oxidation. Study of these
pathways will make you understand why fats yield more energy as compared to carbohydrates.
Many minor pathways for oxidation of fatty acids, formation of ketone bodies and their
significance is described in Unit 9. Unit 10 explains the synthesis of fatty acids.
Block 4 on Lipid Metabolism II includes units on synthesis of complex lipids such as triacyl
glycerides, cholesterol (Unit 11), and membrane lipids (Unit 12). How metabolic pathways of
carbohydrates and lipids are integrated and co-ordinately regulated to meet the energy
requirements of an organism is elaborated in Unit 13. Unit 14 briefly explains about different
disease of lipid metabolism.
The structural outline in the beginning of each unit is a road map to the unit. The mentioned
expected learning outcomes reflect the teaching and learning approaches. The running text
describes and illustrates basics and concepts in a concise, learner friendly and interesting
manner. It is supported by suitable figures, and tables to enrich the concept of the unit. The key
features and concepts have been highlighted. A variety of teaching and learning approaches
such as an inbuilt self-assessment exercises and terminal questions along with answers
provided at the end of each unit will support the learners to evaluate and meet the expected
learning outcomes of the given self-learning material.
You are expected to spend a total of about 120 hours for completing this course. This is the
average time which is to be spent by a learner for studying the course material, doing self-
assessment questions, assignments, watching the audio-video programmes and participation in
IRC/teleconferencing sessions related to this course.
Expected Learning Outcomes

After studying this course, you should be able to:
• Explain metabolism and its salient features
• Discuss about different form of energy currency such as ATP, NADP, FAD in living
beings and their significance
• Describe the pathways involved in catabolism and synthesis of carbohydrates and their
regulation
• Describe the pathways of lipid metabolism, their regulation and importance
• Explain the integration of different metabolic pathways
We wish you the very best and hope you enjoy the learning this course.
BBCCT-109
METABOLISM OF
Indira Gandhi
CARBOHYDRATES AND
National Open University LIPIDS
School of Sciences
Block
1
CARBOHYDRATE METABOLISM I
UNIT 1
Introduction to Metabolism 7
UNIT 2
Glycolysis 28
UNIT 3
Tricarboxylic Acid Cycle 47
BLOCK 1: CARBOHYDRATE METABOLISM I
Metabolism is a complex interplay of various reactions. A cursory look at metabolic reactions
indicates its diversity and complexity across the living organisms. However, careful study
reveals simplicity in complexity. This block will help you understand the common themes of
metabolic reactions as well as meaning of basic terms. Generation of energy is one of the
important functions of metabolism and we all know that carbohydrates are energy producing
molecules. How carbohydrates are utilized by living organisms through glycolysis, a nearly
universal pathway to produce energy will be explained in Unit 2. Infact, glycolysis is a very
primitive pathway which has been conserved through evolution. It constitutes the central
pathway of carbohydrate metabolism. Unit 3 describes about TCA cycle, a pathway which
evolved with introduction of oxygen in the environment of earth. This pathway is able to extract
more energy from glucose oxidation and provides many important products which act as
precursors for other pathways such as amino acid synthesis.
After studying this block, you should be able to:
• Define metabolism and explain other related terms;
• Explain role of energy rich compounds such as ATP, NADP and FAD in metabolism
• Draw reactions of glycolysis and write about the enzymes catalyzing these reactions;
• Explain regulation of glycolysis; and
• Describe TCA cycle, its significance and regulation.
We hope you will have an enjoyable learning experience and wish you success in this
endeavour!!
Unit 1 Introduction to Metabolism
UNIT 1
INTRODUCTION TO
METABOLISM
Structure
1.1 Introduction Primary and Secondary
Pathways
Expected Learning
Outcomes Anaplerotic Reactions and
Amphibolic Pathways
1.2 Autotrophs and
Heterotrophs 1.5 The Source of Energy and
Redox Carriers
1.3 Metabolism
ATP as Universal Free
Catabolism and Anabolism Energy Currency
Schematic Representation of Biological Electron Donors
the Stages of Metabolism and Acceptors
Design of Metabolism 1.6 Summary
1.4 General Organisation of 1.7 Terminal Questions
Metabolic Pathways
1.8 Answers
Metabolic Pathways
1.9 Further Readings
1.1 INTRODUCTION
We know that life of any living organism is driven by energy to perform its
activities. Primary source of energy is food that contains chemical energy of
biomolecules such as carbohydrates, lipids and proteins. Major biochemical
task of living cells is to convert this chemical energy to a form that can be used
by the cells to perform their life supporting activities. This conversion is
accomplished by series of chemical reactions which constitute metabolism.
In this unit, you will study about metabolism and its functions.
You would also learn that in spite of the apparent complexity, there are
common reactions and themes followed in almost all living organisms which
form the basic design of metabolism. A brief outline of the organisation of
metabolic pathways, the role of ATP and redox carriers will also be discussed.
In the next unit, you will study about glycolysis, the central pathway of glucose
oxidation. 7
Block 1 Carbohydrate Metabolism I

After studying this unit, you should be able to:
define the term metabolism;
differentiate between catabolism and anabolism;
classify living beings based on metabolic diversity;
differentiate between autotrophs and heterotrophs;
highlight the salient features in the design of metabolism;
describe the significance, formation and utilization of ATP; and
indicate the role of pyridine and flavin nucleotides in metabolism.
1.2 AUTOTROPHS AND HETEROTROPHS

Life on earth is The biomolecules found in living organisms are essentially carbon based. The
carbon based as it other elements incorporated in the carbon backbone create structural and
constitutes the
functional diversity essential for sustaining life. Living organisms are divided
backbone of all
biomolecules. into two large groups based on the chemical form in which they get carbon
from the environment: autotrophs and heterotrophs.
Autotrophs
Autotrophs use CO2 (the most oxidised form of carbon) from the atmosphere
as their sole source of carbon and reduce it to glucose. They are capable of
synthesising all the required carbon containing biomolecules. The autotrophs
are also known as producers as they bring in fixed usable carbon into the
biosphere for themselves and directly or indirectly support life in this planet.
This group includes photosynthetic bacteria and plants.
Heterotrophs
All animals are Heterotrophs cannot fix atmospheric carbon dioxide. Instead they use organic
heterotrophs while molecules such as glucose and other complex carbohydrates as source of
almost all green carbon. Heterotrophs are also known as consumers as these feed on plants
plants are
and other animals and synthesize the required biomolecules by transforming
autotrophs, the
exceptions being the food they consume.
insectivorous
plants. Both autotrophs and heterotrophs are further classified into two groups based
on source of energy:
1. Phototrophs are capture radiant energy from the sun. They are known as
photosynthetic organisms.
2. Chemotrophs use oxidation- reduction reactions to extract energy from

organic molecules like glucose or oxidizable inorganic substances like Fe2+,
NO2-, NH4+ or elemental sulphur. Table 1.1 summarises the four major groups
8 of organisms based on the source of carbon and energy.
Table 1.1: Metabolic classification of living organisms based on source

of carbon and energy.
Classification Source of Source of Examples

carbon energy
PHOTOTROPHS
Photoautotrophs CO2 Light Green plants, algae,

cyanobacteria,
photosynthetic
bacteria.
Photoheterotrophs Organic Light Nitrifying bacteria;

compounds hydrogen, sulphur
and iron bacteria
CHEMOTROPHS
Chemoautotrophs CO2 Redox reactions Non sulphur

involving inorganic purple bacteria
substrates Fe2+,
NO2-, NH4+ or S
as electron donors
Chemoheterotrophs Organic Redox reactions All animals, most
compounds involving organic microorganism,
molecules such as photosynthetic
glucose as electron cells in dark
donors
Another metabolic classification of organisms is based on whether or not they

can use oxygen as a terminal electron acceptor in energy producing pathways.
The obligate aerobes have an absolute dependence on oxygen. Bacteria like

Azotobacter vinelandii and most eukaryotes couple generation of energy with
oxidation of nutrients by oxygen. These oxidations harvest energy in the form
of reducing equivalents NADH and FADH2. The re-oxidation of NADH and
FADH2 via the electron transport chain (ETC) with oxygen as the terminal
receptor is highly exergonic. There is another class of microbes that also have
an ETC but the terminal electron acceptor is other than oxygen (may be
nitrate, sulphate, etc). This is called anaerobic respiration. It is less efficient
than aerobic respiration.
The extreme group is of obligate anaerobes that are represented by many

prokaryotes belonging to archaea and eubacteria. They cannot tolerate
oxygen and survive in specialised niches. In fact oxygen is a poison for them.
Clostridium botulinum is an example of an obligate anaerobe. Between these
two extremes are some aerobic organisms that can adapt to anaerobic
condition by shifting from respiration to fermentation. The alternative mode is
no doubt inefficient but the organism survives. These organisms are called
facultative anaerobes, for example, Escherichia coli, yeast and human
skeletal muscles. 9
SAQ 1
Differentiate between the following pairs:
i) Facultative and obligate anaerobes
ii) Chemoautotrophs and chemoheterotrophs
1.3 METABOLISM
The word Metabolism is the sum of all enzyme catalyzed chemical reactions taking place
metabolism is in an organism (Fig. 1.1). It is also called intermediary metabolism as a
derived from the substrate goes through multiple intermediary steps to form an end product.
Greek word The outcome of these interconnected pathways is to support the followings:
“metabole”
meaning change. 1) The breakdown of complex biomolecules obtained from the environment
into simpler usable compounds.
2) The efficient extraction of chemical energy of biomolecules into ATP and

reducing equivalents for biosynthesis and other cellular activities.
3) Synthesis of complex biomolecules from simple precursors in

accordance with the changing needs of the organism.
4) Synthesis and storage of long and short term energy reserves in

conditions of excess.
Fig. 1.1: Metabolic map of intermediary reactions operating in human beings.

Dots indicate intermediates in different metabolic pathways and the lines
indicate the enzymes catalyzing the reactions. Many of the pathways are
10 interconnected through common intermediates.
1.3.1 Catabolism and Anabolism

All metabolic reactions participate either in catabolism, anabolism or both. The
overall free energy change of a metabolic pathway is negative.
Catabolism (Greek cata, down and ballein, to throw) refers to reactions

involved in the breakdown of complex biomolecules such as carbohydrates,
lipids and proteins into simple molecules such as CO2, NH3 (ammonia) and
H2O. These reactions are energy generating and oxidative in nature. Some
part of the energy released is utilised for the synthesis of ATP or reduced
energy carriers (NADH, NADPH and FADH2) and the rest is lost as heat. A
less apparent role of catabolism is to provide a variety of anabolic precursors.
Finally, catabolic sequences are convergent in nature. The TCA cycle is a
convergent cycle for the complete oxidation of carbon.
Anabolism (Greek ana, up and ballein, to throw), on the other hand, is

involved in step by step biosynthesis of simple and complex biomolecules,
starting from simple precursors. Anabolic pathways are generally divergent
and depend on a source of energy that is made available in the form of
activated precursors or ATP. They also have one or more steps requiring a
source of reductant. An anabolic process increases the order of a system.
1.3.2 Schematic Representation of the Stages

of Metabolism
Most biological reactions are organised into metabolic sequences and broadly
the process may be subdivided into three stages (Fig. 1.2). During catabolism
we progress from stage one to three whereas anabolic sequences go from
bottom to top. In stage 1 of catabolism, complex biomolecules are broken
down into monomers/ building blocks. This stage does not release energy;
rather, it may need energy for activation. The next two stages generate energy
that is trapped as ATP and reducing power. The biosynthetic pathways on the
other hand use simple precursors to assemble complex molecules and require
both ATP and reducing equivalents.
Fig. 1.2: Stages in the breakdown and synthesis of biomolecules. 11

1.3.3 Design of Metabolism

Large numbers of reactions that occur in all organisms are overwhelming.
Even the simplest unicellular organism is capable of catalyzing a few thousand
reactions. In spite of the complexity, all organisms have a lot in common (unity
in biochemistry). This section highlights the basic design of metabolism.
The central metabolic pathways such as glycolysis are few in number

and have been conserved through evolution; pointing to their importance
and common origin.
Both anabolic and catabolic pathways are essentially irreversible and

this is accomplished by having at least one unique reaction that is
thermodynamically favoured in one direction. Such a reaction derives the
pathway only in one direction.
The number of biochemical reactions is very large but the kinds of

reactions are much less. They predominantly include redox changes
(oxidation-reduction), group transfer, hydrolysis, isomerisation, ATP
dependent ligation and addition to a double bond or removal of
functional groups to form double bonds (lyases).
All reactions are catalysed by enzymes that are either proteins or RNA
(ribozymes). An enzyme allows a thermodynamically feasible reaction to
proceed at a faster rate by reducing the activation energy barrier.
Gluconeogenesis is the
synthesis of glucose from Generally, there are separate pathways for synthesis and degradation.
non- carbohydrate This allows independent and finer control. By having a separate set of
compounds such as
enzymes, the biological system can operate the two pathways
pyruvate.
independently depending on needs and it is not dictated by the law of
mass action. In situations, where two pathways operate in opposite
direction, at least one step is unique to both pathways and that is subject
to stringent control. The pathways of glycolysis and gluconeogenesis, for
example, share seven out of the ten steps; while remaining three are
unique.
The enzymes for synthesis and degradation are often

compartmentalised in eukaryotes. For instance, the enzymes for fatty
acid synthesis are present in cytosol but the enzymes of β-oxidation are
in mitochondria and / peroxisomes. Such a scenario helps in maintaining
different concentration of intermediates, enzymes and above all
regulators.
Many enzymes have additional requirements as coenzymes and / metal

cofactors. Most coenzymes are derived from water soluble B group
vitamins like NAD (P)+ is derived from niacin and coenzyme A is the
active version of pantothenic acid.
In all present day organisms, ribonucleotides play a central role in

metabolism. They function as the energy currency (ATP); structural
components of coenzymes (FAD, NAD+); signal transduction (cAMP);
enzyme regulation; activated donors (CDP-choline, ADP-Glc) in
biosynthesis and molecular switches (G proteins). The range of roles
undertaken by ribonucleotides reflects their ancient origin and is
12 generally taken as strong evidence in favour of the RNA world.
A unique attribute of life is its ability to regulate metabolic processes.

This provides both flexibility and economy which means that an
organism can adapt to changing needs and expresses the
appropriate/desired enzymes as and when they are required. Generally
the first step is the committed step of the pathway; it is irreversible
(exergonic) and stringently regulated.
The metabolic pathways are generally regulated at multiple levels that

include both short and long term measures. Some of them are by
controlling the activity or amount of enzymes. The anabolic and catabolic
sequences are invariably subject to reciprocal regulation that prevents
wasteful outcome. In multicellular organism hormonal signals play a
pivotal role in establishing interdependence between organs to regulate
metabolism.
SAQ 2
Give an example of:
i) A coenzyme derived from a B group vitamin.
ii) An activated donor in biosynthesis.
iii) A modified nucleotide employed for transducing signals.
Synthase is an enzyme
that joins two
1.4 GENERAL ORGANISATION OF substrates without
METABOLIC PATHWAYS direct participation of
ATP or other
A metabolic pathway is a sequence of reactions that produce an end product. nucleoside
triphosphates.
It is the sequence that serves the function. These sequences are organised in
different ways such as linear, branched, cyclic and spiral pathways. Even the
enzymes of a metabolic sequence are generally organised into multi enzyme
complexes; multifunctional enzymes or as membrane bound complexes. Such
organised units of metabolism are called ‘metabolon’ Let us look at examples
of each of these pathways.
1.4.1 Metabolic Pathways

(a) Linear pathway
This type of representation is used when a precursor or substrate is converted

into a product by a series of reactions. The product of one reaction is a
substrate of next reaction until the final product is formed (Fig. 1.3). The partial
breakdown of glucose to pyruvate by glycolysis is an example of a linear
pathway.
Fig.1.3: A linear metabolic pathway. 13

(b) Spiral pathway
A spiral pathway can be viewed as a variation of a linear pathway. In this case

the substrate will be processed through multiple rounds of the same sequence
of reactions. Each time there would be either an incremental decrease or
increase in the length of the spiral (Fig. 1.4). This process will continue until
the desired product is obtained. The β-oxidation of fatty acids is a spiral
pathway that results in the release of acetyl CoA after each round and
decrease in the length of the spiral by two carbon units.
(c) Branched pathway
A branched pathway begins with shared reactions and subsequently an

Fig. 1.4: Schematic intermediate can be diverted to more than one route. The divergent pathway
representation of a spiral
will produce more than one end product. This type of divergent theme is
pathway.
characteristic of anabolic sequences (Fig.1.5). The biosynthesis of three
aromatic amino acids in plants and bacteria is one such example.
Fig. 1.5: The biosynthesis of aromatic amino acids starting from a common
precursor representing branched pathway.
(d) Cyclic pathway
In a cyclic pathway, generally two substrates are involved; one of them is

regenerated through series of reactions and the other is converted to product
(s) (Fig.1.6). Tricarboxylic acid (TCA) cycle is a cyclic pathway in which
oxaloacetate reacts with acetyl CoA and is regenerated with release of two
molecules of CO2.
SAQ 3
Explain the following terms with examples:
Fig.1.6: Schematic
representation of a i) Linear pathway
cyclic pathway.
ii) Cyclic pathway
iii) Branched pathway

14
1.4.2 Primary and Secondary Pathways

Metabolic pathways are broadly classified into two types- primary and
secondary, based on the relative importance of a pathway in fulfilling the basic Secondary
metabolism is also
requirements of an organism.
called special
Primary pathways are indispensable for growth and reproduction of an metabolism.
organism as these govern basic physiological processes. They are meant for
generation of energy. For example, glycolysis is a primary pathway for the
initial breakdown of glucose.
Secondary pathways are not indispensable for completing the life cycle of an
organism. They are responsible for the production of a variety of secondary
metabolites that confer adaptive advantages. . They are produced by most
organisms. Some of these substances include antibiotics, deterrents; attract
pollinators, pigments and even allow an organism to withstand abiotic
stresses. These are synthesised from primary metabolites. The role of most
secondary metabolites is not known and their classification as secondary may
reflect our ignorance.
So far we have learnt that all cells have different types of reactions which are
organized in highly integrated and interconnected metabolic pathways. These
metabolic pathways are tightly regulated. They help in trapping metabolic
energy (ATP) efficiently and utilise it for various cellular activities such as
muscle contraction, nerve transmission, transport of ions and nutrients and
biosynthesis of complex molecules.
1.4.3 Anaplerotic Reactions and Amphibolic

The word amphibolic
Pathways comes from a Greek
word: amphi meaning
Anaplerotic (Greek: ana = up; plerotikos = ‘filling up”) reactions replenish ‘both sides’. In this
intermediates of pathways that are diverted to anabolic routes. The central case it means both in
pathways like glycolysis, TCA cycle and pentose pathway participate in both anabolism and
anabolism and catabolism. Such pathways are generally called amphibolic. catabolism. This term
was proposed by B.
Almost all intermediates of TCA cycle are starting material for the synthesis of Davis in 1961.
divergent anabolic products. For instance, the intermediates oxaloacetate and
α-ketoglutarate serve as precursors for the synthesis of aspartate and
glutamate, respectively which in turn are required for the synthesis of other
non-essential amino acids, purines and pyrimidines.
You will learn in unit 3 that TCA cycle is the major convergent cycle for the
complete breakdown of carbon. Therefore, it is important that these
intermediates are continuously replenished as their shortage may adversely
affect the production of ATP.
The most important anaplerotic reaction is catalysed by biotin dependent

pyruvate carboxylase (Fig.1.7). The enzyme converts pyruvate to
oxaloacetate (OAA) in a two step ATP requiring reaction. The enzyme is
activated by acetyl CoA that signals low level of OAA. Both acetyl CoA and
OAA condense in the first reaction of TCA cycle. 15
ATP ADP + Pi
2+
Mg
Two high energy bonds Adenine NH2 Pyruvate + CO2 Oxaloacetate
(Phosphoanhydride bonds) Biotin
N
N
Fig. 1.7: The carboxylation of pyruvate to oxaloacetate.

O O O N N
−
O P O P O P O There are also cycles like purine nucleotide cycle and glyoxylate cycle that
O
−
O
−
O
−
generate TCA cycle intermediates in specialised tissues. A detailed account
O
H H
of other anaplerotic reactions will be given after discussion on TCA cycle
H H (Unit 3).
OH OH
Ribose
1.5 THE SOURCE OF ENERGY AND
Fig. 1.8: Structure of ATP REDOX CARRIERS
showing the two energy rich
phosphoanhydride bonds. In this section we will discuss the universal role of ATP as the immediate
donor of free energy in biological processes. The major structural features of
ATP that account for its role will be elaborated. In addition, you will be
introduced to two biological redox carriers- nicotinamide nucleotide
coenzymes and riboflavin derived coenzymes / prosthetic groups.
DNA has dATP which
contains 2-
1.5.1 ATP as Universal Free Energy Currency
hydroxyribose as sugar
instead of ribose.
You have learnt in unit 13 of BBCCT-101 that adenosine triphosphate (ATP) is
an activated building block of RNA. It is made up of the base adenine, sugar
ribose and a triphosphate unit (Fig.1.8). A careful look at the structure reveals
Did you know that a that two out of the three phosphates are linked by energy rich
resting human turns phosphoanhydride bonds. The energy is conserved in this triphosphate unit.
over as much as 40 Kg The active form of ATP is complexed with divalent ions (Mg+2 or Mn+2).
of ATP in 24 hours but
the body stores only 5-6 The central role of ATP in biological systems was recognised by F. Lipmann
grams of ATP? and H. Kalckar in 1941. It is the immediate donor of free energy. The turnover
of ATP is high, so it is continuously synthesised. It supports directly or
indirectly energy requiring processes such as biosynthesis, locomotion,
maintenance of membrane gradients, active transport and mechanical
Helicases are activities. In most processes, the energy donation by ATP involves group
enzymes that unwind
transfer of orthophosphate (Pi), pyrophosphate (PPi) or adenylate (AMP) from
double stranded DNA
during DNA ATP to activate the substrate. This is the common way of coupling a
replication, repair thermodynamically unfavourable reaction (endergonic) to an energetically
transcription, etc. favourable reaction (exergonic).
In few instances, however, simple hydrolysis of ATP also occurs like during
muscle contraction; conformational changes in G proteins; reactions catalysed
by helicases and heat generation. ATP is hydrolyzed by ATPase to ADP
(adenosine diphosphate) and ortho phosphate (Pi), accompanied by release of
energy (Fig. 1.9).
16 Fig. 1.9: Hydrolysis of ATP by ATPase.

You may ask what makes ATP a high energy compound or why does ATP has
high phosphoryl group transfer potential?
It is the hydrolysis of ATP that releases lot of energy and accounts for its high
phosphoryl group transfer potential. The structure of ATP and its hydrolysis
products provide an explanation for the greater stability of the products
compared to ATP. The energy
released as water
Let us now elaborate one by one the factors that account for the high molecules surround
phosphoryl group transfer potential of ATP. the ions is called
hydration energy.
(i) Electrostatic Repulsion
At physiological pH, the three phosphate groups present in ATP carry four
negative charges. The closely spaced negative charges repel each other. The
hydrolysis of ATP releases negatively charged terminal phosphate and
relieves some of the electrostatic repulsion.
(ii) Resonance stability of products

The phosphoryl group
The hydrolysis of ATP produces ADP and Pi. Both products are more stable transfer potential of ATP
than the reactant due to resonance and therefore, less reactive. The inorganic is intermediate among
phosphate (Pi) is known to have a number of resonance forms. On the other the phosphorylated
hand, the γ-phosphoryl group of ATP has few resonance forms. compounds. It is an ideal
situation as it can both
(iii) Stabilization due to hydration accept and donate
phosphate groups.
Water molecules bind more to ADP and Pi than they bind ATP stabilising the
products more than ATP due to release of hydration energy.
In biological systems ATP is only one of the high energy compounds. The
standard free energy of hydrolysis of high energy compounds is used to
compare their phosphoryl group transfer potential. The standard free energy of
hydrolysis of some compounds is given in Table 1.2. What is most striking is
that ATP occupies an intermediate position; it can receive phosphate from
compounds such as PEP to regenerate ATP and transfer to those that have
lower transfer potential.
The next question is how cells synthesise ATP. The synthesis of ATP from
ADP and Pi is called phosphorylation. There are three ways to
phosphorylate ADP - substrate level, oxidative and photophosphorylation.
The process of formation of ATP by phosphoryl group transfer from a

metabolic intermediate to ADP is known as substrate level phosphorylation.
Here the energy of oxidation is initially trapped in a compound with a higher
phosphoryl group transfer potential (PEP or 1, 3 BPG) than ATP.
Phosphocreatine in muscles helps in the immediate replenishment of ATP by
a similar mechanism. You would learn about these reactions in subsequent
units. Substrate level phosphorylation is the major source of ATP under
anaerobic conditions.
In aerobic organisms oxidative phosphorylation accounts for most of ATP

generation. It is the synthesis of ATP which is coupled to the oxidation of
NADH or FADH2 via the electron transport chain (ETC) and finally to oxygen.
In eukaryotes, ETC is localised in the inner mitochondrial membrane. The
proton gradient across the membrane drives the synthesis of ATP. 17
Table 1.2: Standard free energy of hydrolysis of some common

phosphorylated intermediates
Redox reactions refer Standard free energy of

to oxidation -reduction hydrolysis (∆G°) KJ/mol
reactions which Phosphorylated compounds
involve both loss and
gain of electrons. Phosphoenol pyruvate (PEP) -14.8
1, 3- bisphosphoglycerate (1,3-BPG) -11.8

(
3-phosphoglycerate + Pi)
Creatine phosphate -10.3
ATP (
ADP + Pi) -7.3
ATP (
AMP + PPi) -7.7
Glucose-1- phosphate -5.0
Fructose-6- phosphate -3.8
Glucose- 6- phosphate -3.3
Glycerol-1- phosphate -2.2
Photophosphorylation as the name suggests, is restricted to photosynthetic

organisms. This is the mechanism of trapping light energy as ATP. A pH
gradient across the thylakoid membrane in chloroplast drives ATP synthesis. It
is the way of bringing a net increase in usable energy into the biosphere.
Let us understand how do we extract energy (ATP) from food? You know that
our food contains biomolecules which are carbon based. These are in reduced
form to varying extent; the fats being the most reduced among these. What do
you think is the relation between reduced state and energy produced? The
more reduced a compound; more will be the energy extracted upon complete
oxidation. The complete oxidation of carbon ends with the production of CO2.
This happens only in the presence of oxygen.
It is important to emphasize that oxidation and reduction go simultaneously

and involve transfer of electrons. The reaction below depicts the outcome of
complete oxidation glucose by oxygen.
C6H12O6 + 6O2 6CO2 + 6H2O
(Reduced) (Oxidized) (Oxidized) (Reduced)
The reaction shows that glucose is oxidized to CO2 by losing electrons and
protons and at the same time the terminal electron acceptor oxygen is reduced
to water. However, unlike direct oxidation of glucose by oxygen, most
biological oxidations go through a series of coupled reduction- oxidation
(redox) reactions in which the reduction of intermediate electron carriers, such
as NAD+ and FAD takes place before electrons are finally transferred to
18 oxygen. The multistep process results in slower release of energy and efficient
trapping in the form of ATP by oxidative phosphorylation. You will read about
the electron transport chain (ETC) and ATP synthesis in later units of this
course.
So far we learnt that phosphoryl transfer potential of ATP is important in

energy transfer during metabolic reactions. Now we shall discuss two redox
carriers that are derived from B group vitamins; niacin and riboflavin. Not only
is it essential to know of the reactions that are dependent on them but it is
equally important to understand how the system regenerates them.
1.5.2 Biological Electron Donors and Acceptors

(a) Nicotinamide derived electron carriers
Nicotinamide adenine dinucleotide (NAD+) and NADP+ are biologically active

coenzymes derived from the vitamin niacin. Both dinucleotides are made up of
a true nucleotide (AMP) and a pseudo nucleotide (nicotinamide
mononucleotide, NMN). There is an additional phosphate esterified to the
ribose of AMP in NADP+. The two nucleotides are linked by a 5’-5’
phosphoanhydride linkage.
Reactive
Site H O
+ +
C NAD and NADP were
NH 2 NMN
O earlier named DPN
−
O P O CH 2 O N
+ (diphosphopyridine
nucleotide) / coenzyme I
H H H H and TPN (triphospho-
pyridine nucleotide)/
OH OH coenzyme II, respectively.
NH2
N
N
N N AMP
−
O P O CH 2 O
O
H H H H
OH OR
Fig. 1.10: Structure of Nicotinamide adenine dinucleotide [NAD (P) +]: R= H in

+ 2- +
NAD and PO3 in NADP .
The oxidised form of NAD (P)+ have absorption maxima at 260nm but the
reduced form has an additional peak at 340nm. This is a useful property that is
extensively used to monitor progress of pyridine nucleotide dependent
reactions.
Please note in Fig. 1.10 that the reactive part of coenzyme is the nicotinamide
ring shown by arrow which carries positive charge in oxidized state. During
oxidation of a substrate, this ring accepts hydride ion (a hydrogen anion; H-)
at position 4 and becomes reduced to NADH + H+. In general, the coenzymes
are loosely associated with the apoenzyme. The enzymes are specific not only
for their substrate but also for the coenzyme. 19
The nicotinamide nucleotide NAD+/NADH is a coenzyme for many

dehydrogenases (DH). Fig. 1.11 gives examples of two redox reactions
dependent on NAD+/NADH + H+. These reactions are generally readily
reversible. In the first reaction lactic acid is oxidized to pyruvic acid and NAD+
is reduced to NADH and in the second reaction acetaldehyde is reduced to
ethanol and NADH is reoxidised to NAD+.
NADPH has
been selected
for reductive CH3 NAD+ NADH2 CH3 CH3 NADH2 NAD+ CH3
biosynthesis.
H C OH C O C H H C H
Lactate dehydrogenase
COOH COOH O OH
Lactic acid Pyruvic acid Acetaldehyde Ethanol
+
Fig. 1.11: Redox reactions catalysed by NAD dependent dehydrogenases.
The biosynthesis of biomolecules is accompanied by reduction of the

intermediates. The source of reductant in most reactions is NADPH. Two
examples of reductive biosynthesis dependent on NADPH are reduction of
carbon dioxide during photosynthesis and the synthesis of fatty acids from
acetyl CoA.
The nicotinamide nucleotide coenzymes are reused and so they need to be

regenerated. NAD+ is regenerated by ETC or fermentation. One mole of
NADH also generates 2.5 moles of ATP by oxidative phosphorylation as
electrons flow via the electron transport chain. NADP+ is converted back to
NADPH by the light reactions of photosynthesis in plants and oxidative
pentose pathway in both plants and animals.
(b) Riboflavin derived coenzymes
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are

coenzymes derived from the vitamin riboflavin. The structures of riboflavin,
FMN and FAD are given in Fig. 1.12. The mononucleotide is simply a
phosphorylated version of riboflavin while the dinucleotide has an additional
AMP residue attached to FMN. The two nucleotides are linked by a 5’-5’
phosphoanhydride linkage. The sugar in FMN is a sugar alcohol (ribitol).
The flavin prosthetic group is firmly associated with the protein and hence they
are called flavoproteins. In their oxidised form the flavins are yellow colored
and become colourless upon reduction. These are capable of performing
reactions involving transfer of one and two electrons, therefore, mediate large
number of reactions. Some of the reactions dependent on flavin prosthetic
group are those catalysed by certain dehydrogenases (succinate
dehydrogenase), oxidases (glucose oxidase), monooxygenases and multi
enzyme complexes (pyruvate dehydrogenase complex). Let us learn about
these reactions. In case of dehydrogenase catalysed reactions the substrate is
dehydrogenated and fully reduced flavin accepts two electron and two protons
20 that is reoxidised ultimately by oxygen.
O
O
H3 C N H3C N
NH
Flavin NH
H3C N N O
H3C N N O
CH 2
CH 2
H C OH
H C OH
Ribose H C OH
H C OH
H C OH
H C OH O
CH 2OH
CH 2O P OH
Riboflavin OH
Flavin mononucleotide (FMN)

O
H3C N
NH
H3C N N O
CH 2
H C OH NH2
N
H C OH N
H C OH O O
N N
CH 2O P O P O H2 C O
OH OH
H
OH OH
FMN Adenylic acid
Flavin adenine dinucleotide (FAD)
Fig. 1.12: Structures of riboflavin, FMN and FAD
The flavin dependent oxidases, on the other hand, reoxidise the flavin
prosthetic group directly by oxygen. Finally, the monooxygenases require
reduced flavin and oxygen to hydroxylate the substrate and reduce oxygen to
water. Now let us consider some specific examples of flavin requiring
reactions to demonstrate their versatility.
(a) The oxidation of succinate to fumarate by succinate dehydrogenase

(Fig.1.13).
COOH COOH
FAD FADH2
CH 2 CH
CH 2 Succinate dehydrogenase HC
COOH COOH
Succinate Fumarate
Fig. 1.13: The reaction catalysed by succinate dehydrogenase.
In this reaction FAD is regenerated by transferring electrons to terminal

oxygen to form water through series of electron carriers in ETC. The re
oxidation of FADH2 by ETC also generates 1.5 ATP by oxidative
phosphorylation
(b) The oxidative deamination of amino acids by L-amino acid oxidase

(Fig.1.14). Unlike dehydrogenases the reduced flavin prosthetic group of
oxidases is directly reoxidised by oxygen and oxygen is reduced to H2O2. In an
21
oxidase catalysed reaction molecular oxygen is the electron acceptor and

oxygen atoms do not appear in the oxidised product.
H2O2 O2
H FAD FADH2 COOH COOH

+
H 2N C COOH R C R C + NH4
L-amino acid NH O
H oxidase
L-amino acid α-imino acid α-keto acid
Fig 1.14: Oxidation of L- amino acid to alpha keto acid.
(c) Some monooxygenases are flavin dependent. They catalyze reactions in

During which the main substrate is hydroxylated by one of the two atoms of molecular
conversion of
phenol to
oxygen and other oxygen is reduced to water by reduced flavin nucleotide.
catechol, FADH2 These enzymes are also called hydroxylases as they hydroxylate the
associated with substrate. Fig. 1.15 shows the hydroxylation of phenol to catechol by phenol
the enzyme monooxygenase.
carries dioxygen
and forms flavin OH OH
peroxide OH
intermediate, Phenol monooxygenase TCA cycle
which transfers intermediates
one of the
oxygen to phenol Phenol FADH2 FAD+ Catechol
to form final
product catechol.
It itself is
converted to
flavin hydroxide NADH + H+ NAD+ + H2O
which forms FAD
+ O2
by removal of
water. FADH2 is
regenerated by Fig. 1.15: Hydroxylation of phenol by phenol monooxygenase.
NADH. So flavin
You must keep in mind that although ATP, NADH, NADPH and FADH2 are
participates in
teh reaction but high energy compounds, yet these are highly stable even in the absence of
is regenerated. enzymes. However, in the absence of enzymes NADH, NADPH and FADH2
react very slowly with oxygen and even ATP is also hydrolyzed very slowly.
Such compounds are said to be kinetically stable. The stability of these
energy rich compounds in the absence of specific catalysts allows cells to
control the flow of energy. There are other high energy molecules in the cell;
we shall consider them as we come across in specific metabolic reactions.
SAQ 4
A) Arrange the following compounds in order of decreasing phosphoryl
group transfer potential?
Glycerol 1-phosphate; Phosphoenol pyruvate (PEP); Adenosine

triphosphate (ATP); Creatine phosphate; 1, 3 bisphosphoglycerate (1, 3
22 BPG)
B) Match the items in column I with their role in column II
Column 1 Column II
i) NAD+ a) Energy currency
ii) ATP b) Reductive biosynthesis
iii) FAD+/FADH2 c) Oxidative catabolism
iv) NADPH d) Accepts two hydrogen atoms
1.6 SUMMARY
• Metabolism is defined as sum of chemical reactions that occur in the
cells of living organisms. These reactions are needed for growth,
reproduction, maintenance and our ability to respond to environmental
cues.
• The functions of metabolism include breakdown of complex

biomolecules present in food to simpler compounds; efficient extraction
of energy into high energy compounds; synthesis of complex
biomolecules from simple precursors and storage of energy reserves in
conditions of excess.
• All living organisms are broadly classified into two groups (autotrophs
and heterotrophs) based on the form in which they obtain carbon from
the environment. The former group includes photosynthetic organisms
(producers) that fix carbon dioxide and are responsible for directly or
indirectly supporting life on earth. The latter group uses organic carbon
compounds from the environment and transform them into specific
biomolecules.
• Both autotrophs and heterotrophs are further classified into phototrophs

and chemotrophs depending on the source of energy.
• All organisms have enzymes that catalyse thousands of reactions.

Inspite of the complexity they all use a common conserved design. Most
metabolic reactions are organised into sequences that participate either
in catabolism, anabolism or both. These pathways may be organised in
different ways such as linear, spiral, cyclic or branched.
• Catabolic pathways are involved in the breakdown of complex

biomolecules into simpler substances. They are oxidative, energy
releasing and convergent pathways. Anabolism on the other hand refers
to pathways that synthesise complex molecules starting from simple
precursors. Anabolic pathways are energy requiring, divergent and
reductive in nature.
• A unique attribute of life is its ability to regulate metabolic processes.

The metabolic pathways are generally regulated at multiple levels that
include both short and long term measures. 23
• Metabolic pathways are broadly classified into primary and secondary,

based on the relative importance of a pathway in fulfilling the basic
requirements of an organism. Metabolic reactions which are responsible
for generation of energy are called primary pathways. Secondary
pathways are not indispensable for completing the life cycle of an
organism. They produce a variety of secondary metabolites that confer
adaptive advantages
• The central pathways like glycolysis, TCA cycle and pentose pathway
participate in both anabolism and catabolism. Such pathways are
generally called amphibolic. The intermediates diverted to anabolic
routes are replenished by anaplerotic reactions.
• Metabolic reactions generate energy by oxidation of reduced

biomolecules. This energy is released in step wise manner during
catabolism and harvested in the form of high energy phosphoryl group of
ATP or as reducing equivalents in NADH, NADPH or FADH2.
• ATP is the universal free energy currency in biological systems. ATP

generally activates a substrate by group transfer of either
orthophosphate (Pi), pyrophosphate (PPi) or adenylate (AMP) making
thermodynamically unfavourable reaction feasible under physiological
conditions.
• NADH and FADH2 are reoxidised either by fermentation or by

transferring their reducing equivalents ultimately to oxygen through the
electron transport chain that is coupled to ATP synthesis.
1.7 TERMINAL QUESTIONS

1. Define metabolism. What are its major functions?
2. What are the differences between heterotrophs and autotrophs?
3. Explain the following terms:
i) Ambhibolic pathway
ii) Anabolism
iii) Catabolism
iv) Anaplerotic reactions
4. Why ATP is a high energy compound and what is the possible reason for
selecting it as the energy currency in biological processes?
5. Compare the structure and function NAD+ and NADP+.
6. Indicate the major features of metabolic design.
1.8 ANSWERS
Self-Assessment Questions
1. i) Many prokaryotes belonging to archaea and eubacteria cannot
24 tolerate oxygen and survive in specialised niches. These
organisms are called obligate anaerobe, for example, Clostridium

botulinum. Organisms that can survive under both aerobic and
anaerobic conditions are facultative anaerobes for example,
Escherichia coli, yeast and even human skeletal muscles. It is an
adaptation to oxygen stress.
ii) Chemoautotrophs Chemoheterotrophs
1. Source of energy Redox reactions involving organic

from redox molecules such as glucose as
reactions involving electron donors
inorganic
substrates such as
Fe2+ or S as
electron donors
2. They can fix They depend on readymade organic

atmospheric CO2. compounds from other organisms as
source of carbon.
3. Examples: Non All animals, most microorganisms,

sulphur purple
bacteria
2. i) NAD(P+)
ii) CDP-choline
iii) cAMP
3. i) In a linear pathway a precursor or substrate is converted into a

product by a series of reactions, for example, glycolysis.
ii) In cyclic pathways, generally two substrates are involved; one of

the substrate is regenerated through series of reactions and the
other is converted to the product. TCA cycle is an example of
cyclic pathway.
iii) A branched pathway begins with shared reactions and

subsequently an intermediate can be diverted to more than one
route resulting in more than one end product. Anabolic pathways
are generally divergent.
4. A) PEP>1,3 BPG>Creatine phosphate> ATP>Glycerol-1-phosphate
B) i) Oxidative catabolism
ii) Energy currency
iii) Accepts two hydrogen atoms
iv) Reductive biosynthesis 25

Terminal Questions
1. Metabolism is the sum of all enzyme catalyzed chemical reactions taking
place in an organism. The role of metabolism includes:
i) Breakdown of complex biomolecules into simpler usable form.
ii) To convert the chemical energy of biomolecules into ATP and

reducing equivalents for biosynthesis and other cellular activities.
iii) Synthesis of complex biomolecules from simple precursors.
iv) Synthesis and storage of long and short term energy reserves.
2. Heterotrophs do not fix atmospheric carbon dioxide. Instead they obtain

readymade organic compounds by feeding on plants and other animals
to synthesize carbon based specific biomolecules. Heterotrophs are also
known as consumers.
Autotrophs use CO2 (the most oxidised form of carbon) from the
atmosphere as their sole source of carbon and reduce it to glucose.
They are capable in synthesising all the required carbon containing
biomolecules. The autotrophs are also known as producers, for example,
photosynthetic bacteria and plants.
3. i) An amphibolic pathway participates in both anabolism and

catabolism such as glycolysis, TCA cycle.
ii) Anabolism is involved in the step by step biosynthesis of simple

and complex biomolecules, starting from simple precursors.
Anabolic pathways are generally divergent, reductive and energy
requiring.
iii) Catabolism refers to reactions involved in the breakdown of

complex biomolecules such as carbohydrates, lipids and proteins
into simple molecules such as CO2, NH3 (ammonia) and H2O.
These reactions are energy generating and oxidative in nature.
iv) Anaplerotic reactions replenish intermediates of amphibolic

pathways, diverted to anabolic routes.
4. The structure of ATP and its hydrolysis products provide an explanation

for the greater stability of the products compared to ATP. It is the
hydrolysis of ATP that releases lot of energy. The energy is conserved in
triphosphate unit. The factors that account for the high phosphoryl
group transfer potential of ATP are electrostatic repulsion, resonance
stability of products and stabilisation due to hydration. ATP occupies an
intermediate position among the high energy compounds in biological
systems. That allows it to function as an efficient carrier of phosphoryl
groups. It can receive phosphate from compounds such as PEP to
regenerate ATP and transfer to those that have lower transfer potential.
This is a possible reason for its selection as the universal carrier of free
26 energy.
5. Differences:
i) Structural: NADP + has an additional phosphate esterified to the

ribose of AMP.
ii) Functional: NAD+ is a coenzyme for DH in catabolic pathways

whereas NADPH is required in reductive biosynthetic pathways.
6. Basic design of metabolism represents unity in biochemistry. Although

there are large number of reactions, yet these metabolic pathways share
many common features. Refer to section 1.3.3 for more details.
27
UNIT 2
GLYCOLYSIS
Structure
2.1 Introduction 2.6 Feeder Pathways for
Glycolysis
2.7 Fates of Pyruvate
2.2 The Road to Glycolysis
2.8 Regulation of Glycolysis
2.3 Glycolysis or Embden-
Meyerhof - Parnas (EMP) 2.9 Summary
Pathway
2.10 Terminal Questions
2.4 Fermentation
2.11 Answers
2.5 Cori Cycle
2.12 Further Readings
2.1 INTRODUCTION
You have been introduced to general terms, concepts and role of metabolism
in unit 1. We also discussed its common features, types of reactions, energy
currency and redox carriers. In this unit, we shall begin carbohydrate
metabolism with glycolysis, an almost universal pathway of sugar catabolism
Glycolysis is the central and primitive pathway of glucose catabolism. It is the

initial route of oxidative catabolism in both anaerobic and aerobic systems. It
also acts as a source of energy and metabolites for anabolism.
In this unit you will learn about the elucidation of this multistep pathway. We
shall discuss the reactions involved in glycolysis and how other sugars enter
into this pathway. You would also study how this pathway yields different
products under different conditions as well as in different tissues. Finally we
shall also explain how this pathway is regulated.

explain the glycolytic pathway and its outcome;
write the structure and point out the step (s) where oxidation and ATP
28 synthesis occurs;
Unit 2 Glycolysis
indicate the fates of pyruvate under different conditions;
describe the feeder pathways for glycolysis and their relevance;
explain the Cori cycle and state its important under anaerobic conditions;
and
describe how the key reactions of glycolysis are regulated.
2.2 THE ROAD TO GLYCOLYSIS

Before we go into the details of the glycolytic pathway, lets us look at some
important leads which were instrumental in the elucidation of the pathway. In
1897 the German brothers, Hans Buchner and Eduard Buchner accidently
Hans and Eduard
found that addition of sucrose to yeast extract led to evolution of bubbles from
Buchner
the solution. The addition of sucrose was meant to preserve yeast extract.
Eduard Buchner concluded that fermentation, a process described by Pasteur
was occurring. He isolated the enzyme from yeast extract and called it
‘zymase’. It was demonstrated for the first time that fermentation could take
place outside the cell and discounted the existing idea of a vital force to carry
out life processes. This work allowed chemists to identify individual steps and
characterise them under controlled conditions. Above all it opened the era of
enzymatic theory of metabolism. Eduard Buchner was rewarded with the
Nobel Prize in 1907.
In 1906, Arthur Harden and William John Young made two very important Harden and Young
observations. They found that inorganic phosphate was required for
fermentation and is incorporated into fructose 1,6 bisphosphate (Harden and
Young ester). They also elaborated Buchner’s work and showed that a cell
free extract can be separated by dialysis into two fractions. One of them was
non dialysable heat labile fraction or zymase and the other is heat stable and
dialysable or cozymase. Both of them are necessary for fermentation. They
also discovered NAD+. Today we know that each of these fractions includes a
mix of enzymes and coenzymes / other low molecular weight substances.
Later studies on muscle extracts showed that many reactions of lactic acid
fermentation were same as those of alcoholic fermentation. The complete
glycolytic pathway was elucidated in 1940 by pioneers in the field including
Gustav Embden, Otto Meyerhof, Carl Neuberg, Robert Robison, Jacob
Parnas, Otto Warburg, Gerty Cori and Carl Cori.
The discovery by Otto Meyerhof and his students that some phosphorylated Otto Fritz Meyerhof
compounds are rich in energy revolutionised our concepts and significance of (1884-1951)
cellular metabolism. One of his associates, K. Lohmann was the first to
discover ATP.
Meyerhof and his colleagues not only discovered the intermediates of the
cycle but played a key role in piecing together the complex puzzle of
glycolysis. He had the gift of integrating a variety of phenomenon. Glycolysis is
also known as Embden- Meyerhof –Parnas pathway. Meyerhof was awarded,
together with the English physiologist A.V. Hill, the Nobel Prize for Physiology
or Medicine in1922. 29
2.3 GLYCOLYSIS OR EMBDEN-MEYERH

OF-PARNAS (EMP) PATHWAY
Glycolysis is the Glycolysis (glykos- sweet; lysis- splitting) is a sequence of reactions which
initial stage of converts glucose and related hexoses into two molecules of pyruvate with net
glucose production of two ATP molecules. It is the most important pathway in energy
metabolism. It metabolism, present in both aerobic and anaerobic organisms. The cycle
occurs in the
completes in ten steps and the enzymes are present in the cytoplasm. None of
cytosol. It does not
involve oxygen. It
the reactions are oxygen dependent. In evolutionary terms it is regarded as a
produces 2 ATP for primitive pathway.
each glucose
oxidised. Its end
Let us see what makes glycolysis an almost universal pathway. Since all these
product is pyruvate. reactions can take place in the absence of oxygen therefore, it is an important
pathway for extraction of energy from nutrients in anaerobic organisms. Even
aerobes begin glucose metabolism with glycolysis and then enter the citric
acid cycle for complete breakdown. In addition, it becomes the major source of
energy in cells lacking mitochondria such as red blood cells and cornea of the
It is important to eye or in rapidly contracting skeletal muscles experiencing transient anaerobic
note that the division conditions.
into phases is for the
ease of The overall pathway of glycolysis is energetically favourable and
understanding. In unidirectional. It can be represented by the following equation:
fact, the product of
one reaction serves Glucose + 2 ADP + 2Pi + 2 NAD+ 2 Pyruvate + 2 ATP + 2NADH+
as the substrate for 2H++ 2H2O
the next reaction in
the pathway.
Let us proceed to learn about the reactions of glycolysis. You would notice that
all the intermediates of the pathway are phosphorylated. The purpose of
phosphorylation is two- fold. It activates the intermediate and polarises it,
thereby preventing it from leaving the cell. Generally, the plasma membrane
lacks transporters for phosphorylated sugars.
The glycolytic pathway is divided conventionally into two phases. They are
called preparatory or energy investment phase and energy yielding / pay off
phase (Fig. 2.1). We shall discuss these phases one by one.
A. Preparatory or energy investment phase
This phase has two reactions which require input of energy in the form of ATP.
The situation is similar to day to day life situations where we invest small
amounts of money to get better returns later. The phase ends with the splitting
of activated fructose1,6- bisphosphate to two sugars.
Step 1: Phosphorylation of glucose
The first step of glycolysis is catalysed by a ubiquitous enzyme, hexokinase.

It is relatively a non specific enzyme as it also phosphorylates mannose,
fructose, glucosamine and 2-deoxyglucose in addition to glucose. It catalyses
the phosphoryl group transfer from ATP to the hydroxyl group at C-6 of
glucose in presence of Mg2+. The reaction is highly exergonic as the
phosphorylated product, glucose-6- phosphate is a low energy ester and the
30 reaction is essentially irreversible under in vivo conditions. In some tissues,
Unit 2 Glycolysis
specific kinases also exist like glucokinase that is specific only for glucose.
Glucokinase has restricted distribution and low affinity for glucose. This first
step of glycolysis is not subject to stringent regulation because the product
formed has multiple fates.
Step 2: Isomerisation of glucose-6 phosphate
The next reaction is the isomerisation of glucose-6-phosphate to fructose-6- Enediol is an organic

phosphate by phosphohexose isomerase (phospho-glucose isomerase). compound in which
The reaction proceeds readily in both directions and the aldoses-ketose two hydroxyl groups
are attached; one
isomerisation involves the formation of an enzyme bound enediol intermediate. each to carbon atoms
of a double bond
Step 3: Phosphorylation of fructose-6 phosphate (>C(OH)=C(OH)<
You learnt that first irreversible reaction is not unique to glycolysis as glucose-
6-phosphate is an intermediate for other metabolic pathways also.
Phosphorylation of fructose-6 phosphate is the first committed reaction to
glycolysis, which means once this reaction occurs; glycolysis will proceed till
the last reaction. This first unique step of glycolysis is catalysed by Mg2+ When two phosphate
groups are present on
dependent phosphofructokinase-I (PFK-I). It catalyses the irreversible
two different carbons of
phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. In this the same molecule, it is
reaction, one molecule of ATP is consumed. In many organisms including named bisphosphate
plants, some bacteria and protists pyrophosphate (PPi) is the phosphoryl as in fructose-1,6-
donor in place of ATP. Due to its uniqueness, this reaction is important in bisphosphate. When
regulation of glycolysis. both phosphates are
present on the same
The next two reactions first split the six carbon bisphosphate intermediate to 2 carbon, it is notated as
three carbon sugars and then triose phosphate isomerase interconvert the two diphosphate as in ADP.
split sugars so that both products can be utilised for oxidation and generation
of ATP in the pay off phase.
Step 4: Cleavage of fructose 1, 6-bisphosphate
The enzyme aldolase splits fructose 1,6-bisphosphate to 3-

phosphoglyceraldehde (an aldose) and dihydroxyacetone phosphate (a
ketose). This reaction has a positive standard free energy change but at lower
concentration of reactant, the reaction is reversible. The enzyme is capable of
splitting a number of ketose mono- and bis-phosphates. This reaction also
operates in gluconeogenesis and Calvin cycle in the reverse direction.
Step 5: Inter conversion of triose phosphates (aldose-ketose)
The production of two sugars by aldolase completes the preparatory phase of

glycolysis. The energy generation phase begins with the oxidation of
glyceraldehyde 3-phosphate. Therefore, dihydroxyacetone phosphate (DHAP)
is isomerised to glyceraldehyde 3-phosphate by triose phosphate isomerase
so that both sugars are degraded by this pathway. The enzyme is extremely
active and is generally dubbed as a perfect enzyme which means that the
product is formed as soon as the enzyme and substrate collide.
So far, we learnt that The cleavage of fructose 1,6 bisphosphate results in two
sugar derivatives; DHAP comes from C-1 to C-3 and glyceraldehyde is derived
from C-4 to C-6. But following the isomerase reaction C-1, C-2 and C-3 are 31
indistinguishable from C-6, C-5 and C-4, respectively. This is because in

glyceraldehyde 3-phosphate, the carbonyl group is C-1. You must always
keep this in mind while tracing the fate of C-14 labelled glucose.
2
1 3
Hexokinase 2- Phosphoglucose
CH 2OPO 3 Phosphofructokinase
6
CH2OH isomerase CH OPO 2- 2-
2 3 CH2OPO 3
5 OH OH O
H H H H CH 2OH O CH 2OPO 3
2-
∆G''= -4.0 ∆G''= +4.0 ∆G''= -3.4
4 1
OH OH H OH OH OH H OH H H
HO OH HO OH
H H
3 2 ATP ADP ATP ADP
H OH OH H
H OH OH H
E n e r g y In v e s t m e n t P h a s e
Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate
4
Aldolase
O O O 6 H ∆G''= +5.7
- - 7 2-
C O 8 C O C OPO 3 ∆ +1.5
∆G''= 4 5
Phosphoglycerate C O
Glyceraldehyde phos- Triose phosphate
2- Phosphoglyceromutase kinase
HCOPO 3 phate dehydrogenase 5 isomerase 1 2-
HCOH HCOH
E n e rg y G e n e ra tio n P h a s e
∆G''= + 1.1
HCOH CH2OPO 3
2- ∆G''= +1.8
CH 2OH ATP ADP 2- Pi
CH 2OPO 3 CH2OPO 3 + +6 2- 2
NADH +H NAD CH 2 OPO 3 C O
∆G''= -4.5
Glyceraldehyde 3
2-Phospho- 3-phosphate CH2OH
3-Phospho- 1,3-Biphospho-
glycerate glycerate glycerate Dihydroxyacetone
phosphate
O O
9
- 10 -
Enolase C O Pyruvate kinase C O
H2O ∆G''= + 0.4 2- ∆G''= - 7.5
C O PO3 C O
CH2 ADP ATP
CH 3
Phosphoenolpyruvate Pyruvate
Fig. 2.1: The Glycolysis Pathway.
B. Energy generation phase
The last five reactions of glycolysis include a single step of oxidation and two
reactions in which ATP is synthesised by substrate level phosphorylation.
Since two molecules of glyceraldehyde 3-phosphate produced in the
preparatory phase enter the energy generation phase, therefore you must
double the final outcome of each reaction in the pay off phase.
Step 6: Oxidation of glyceraldehyde 3-phosphate
The oxidation of glyceraldehyde-3-phoshate is catalysed by a NAD+

dependent glyceraldehyde-3-phosphate dehydrogenase. It has tightly
bound NAD+ which is an exception to the generalisation discussed in unit 1.
The substrate is oxidised to 1,3 bisphosphoglycerate (1,3 BPG) with
concomitant production of NADH and H+ . Formation of 1,3 BPG, a mixed
anhydride (acyl phosphate) conserves the energy released during oxidation of
the carbonyl group to an acid. During the reaction, the substrate is covalently
bound to the -SH group of cysteine in enzyme. Therefore, heavy metals like
32 Hg2+ which react with cysteine can irreversibly inhibit it. Even arsenate, an
Unit 2 Glycolysis
analog of phosphate can replace it and forms an unstable intermediate that

can be non- enzymatically hydrolysed back to 3- phosphoglyceric acid (3
PGA).
You will see that in the next reaction that phosphate group of 1,3 BPG is
essential for ATP synthesis, therefore, in presence of heavy metals, glycolysis
proceeds without generation of ATP.
Step 7: Substrate level phosphorylation by 1, 3 BPG
In this step, ATP is synthesised by phosphoryl group transfer from 1,3BPG to

ADP. The reaction is catalysed by phosphoglycerate kinase generating 3PGA.
The two steps (6 and 7) represent an example of how energy from oxidation of
a substrate is coupled to ATP synthesis.
The next two reactions of glycolysis help to generate another energy rich
intermediate (PEP) starting from an energy poor 3-PGA.
Step 8: Isomerisation of 3-phosphoglyceric acid (3-PGA)
The inter conversion between 3-PGA and 2-PGA is catalysed by

phosphoglycerate mutase. The reaction goes through 2,3 bisphosphoglycerate
(2,3 BPG). The product 2-PGA is obtained by transferring the phosphate at C-
3 to the active site. A small amount of 2, 3 BPG is needed as a cofactor to
initiate the cyclic process.
3-PGA [2, 3 BPG] 2-PGA
Step 9: Dehydration of 2-PGA
The dehydration of 2 PGA by the enzyme enolase yields an energy rich enolic
phosphate, phosphoenol pyruvate (PEP). This reaction requires Mg2+ or Mn2+
and it is inhibited by fluoride.
Step 10: Substrate level phosphorylation by PEP
The enzyme pyruvate kinase catalyses irreversible phosphoryl group transfer

from PEP to ADP. The enol form of pyruvate non- enzymatically tautomerises
to the more stable keto form. The reaction is irreversible under physiological
conditions.
Thus each molecule of 3-phosphoglyceraldehyde is processed in the second

phase to produce 1NADH + H+ and 2 ATP. We will double this to account for
both molecules of 3-phosphoglyceraldehyde. In the preparatory phase two
ATP were consumed and so the net yield of ATP is only two (4 - 2).
Many of the intermediates of this pathway also provide anabolic precursors;

DHAP is the precursor for the glycerol backbone of glycerolipids, 3-PGA can
be converted to serine and other amino acid and PEP is one of the precursors
for the synthesis of aromatic amino acids.
We learnt that 2 NAD+ are consumed in one cycle of glycolysis. This NAD+
must be regenerated in all organisms to allow oxidative catabolism. There is
more than one way to do it depending on the presence or absence of oxygen.
In the following section, we will cover fermentation and demonstrate its role in
regeneration of NAD+ by taking suitable examples. 33
SAQ 1
Match the enzymes in column A to substrate /product pair in column B.
Column A Column B
i) Hexokinase a) 3-Phosphoglycerate(3-PGA)/2-
PGA
ii) Glyceraldehyde-3 b) 2-phosphoglycerate / phosphoenol

phosphate pyruvate
dehydrogenase
iii) Phosphoglycerate c) fructose-1,6-bisphosphate/
mutase glyceraldehyde 3-phosphate &
DHAP
iv) Phosphofructokinase-I d) glyceraldehyde-3-phoshate/1,3-

bisphosphoglycerate (BPG)
v) Enolase e) Fructose-6-phosphate / Fructose-

1,6-biphospahate
vi) Aldolase f) Glucose / glucose-6-phosphate
2.4 FERMENTATION
Breakdown of glucose You may know that earliest organisms lived in an atmosphere which was
into pyruvic acid is
devoid of oxygen; therefore they had to develop strategies to derive energy
called glycolysis and
further processing of from fuel molecules to survive under anaerobic conditions. Most of them
pyruvic acid in depended on glycolysis for the breakdown of carbon. It is a near universal
anaerobes is called pathway as it is equally relevant in aerobic organisms. With the emergence of
fermentation. Its main oxygen there has been shift towards a more efficient mode of energy
purpose is to
generation involving complete breakdown of carbon to CO2 and ATP
regenerate NAD+ so
that ATP production by generation by oxidative phosphorylation. Yet most organisms / tissues have
glycolysis can continue. retained the ability to ferment under low oxygen such as skeletal muscles
during strenuous muscular activity, solid tumours, and cornea of the eye. An
extreme example is of erythrocytes that lack mitochondria and hence, ferment
glucose even in the presence of oxygen. In many niches anaerobic organisms
still survive by utilising primitive catabolic pathways. The process of
generation of energy (ATP) by substrate level phosphorylation from
incomplete oxidation of fuels like glucose under anaerobic conditions is
known as fermentation.
It is our general understanding that microorganisms are employed for

fermentation of sugar to produce alcohol in brewing industry. In the microbial
world we encounter enormous variation in the end product of fermentation. In
some microbes more than one end product is produced. The type of
fermentation is named on the end products such as homo lactate
34 fermentation, mixed acid fermentation, etc. Some of these organic end
Unit 2 Glycolysis
products for instance, citric acid, propionic acid, butanol, acetone and ethanol
are produced commercially
Fermentation is an inefficient mode of energy generation as the end products

are organic compounds rather than CO2 that still conserve lot of energy. Let us
study homo lactate and alcohol fermentation reactions. In both examples you
will note that there is no net change in the oxidation state of carbon which
means that H: C ratio is same for glucose and the end product.
Lactic acid fermentation
Lactic acid fermentation is carried out by bacteria (Lactobacillus species),

some fungi and rapidly contracting skeletal muscle. A single reaction catalysed
by lactate dehydrogenase (LDH) reduces pyruvate to lactate (Fig. 2.2). It is
not specific for pyruvate and reduces a number of other keto acids, including
phenylpyruvic acid. If the end product is only lactate it is homo lactate
fermentation. The NADH utilized in this step is obtained from the reaction
catalyzed by glyceraldehyde-3-phosphate dehydrogenase in glycolysis. The
formation of lactate allows the regeneration of NAD+ which can be reused in
glycolysis. In this case, glycolysis results in net formation of 2 ATP for each
molecule of glucose and no net production of NAD+/NADH.
− −
O O NADH + H+ NAD+ O O
C C
C O HC OH
LDH
CH 3 CH 3 Km is defined as the
Lactate substrate concentration
Pyruvate
at which half of the
maximum velocity of a
Fig. 2.2: Reduction of pyruvate to lactate: Pyruvate formed in glycolysis is reaction is attained. Its
reduced to lactate under anaerobic conditions to regenerate NAD+. value for an enzyme is
inversely proportional
In vertebrates multiple isozymes of LDH exist that are encoded by two genes. to the affinity of the
The active form of this enzyme is a homo tetramer and the isozymes differ in enzyme for the
ratio of the two polypeptides (M or H chain). The skeletal muscles have substrate.
predominantly LDH5 that four M (muscle) chains while the major variant in the
heart is LDH1 with four H (heart) chains. The other tissues have a mix of H and
M polypeptides. Isozymes have different amino acid composition, kinetic and
immunological properties. The heart enzyme has a low Km (high affinity for the
CO2 released during
substrate) while the muscle enzyme works best at higher concentration of
alcohol fermentation
pyruvate (high Km). These properties are consistent with their roles. gives the
characteristic
Alcohol fermentation
carbonation of
In some bacteria and fungi (e.g. yeast), pyruvate is fermented to ethyl alcohol champagne in
brewing and allows
in two steps. In the first step, pyruvate is decarboxylated irreversibly to
dough to rise in
acetaldehyde by a thiamine pyrophosphate (TPP) dependent pyruvate baking.
decarboxylase. It also requires Mg2+. This enzyme is absent in animal tissues.
In the second step, acetaldehyde is reduced to ethyl alcohol by alcohol

dehydrogenase (Fig. 2.3). Ethyl alcohol is excreted by microorganisms as 35
accumulation of alcohol beyond a certain limit can kill them. In many

organisms including humans, the enzyme metabolises ethyl alcohol.
Alcohol fermentation is very important from industrial point of view and has
been extensively exploited for beer and wine production and baking industry.
−
O O
+ NAD+
C CO2 H O NADH + H H
C H C OH
C O Pyruvate Alcohol
Decarboxylase CH 3 Dehydrogenase
CH 3
CH 3 TPP
Pyruvate Acetaldehyde Ethanol
Fig. 2.3: The conversion of pyruvate to ethanol reoxidizes NADH.
SAQ 2
Fill in the blanks:
i) The general term used for the anaerobic degradation of glucose to

obtain energy is _______________.
ii) The net yield of ATP during lactate fermentation is ---------------
iii) ____________ is the example of human tissue that can ferment glucose
to __________.
iv) The end products of alcohol fermentation are _____ and ______.
v) In both lactate and alcohol fermentation the purpose of going beyond

glycolysis is to _______.
2.5 CORI CYCLE

In section 2.3, we discussed that lactic acid is formed in skeletal muscles
Carl and Gerty Cori
during strenuous exercise. It is a temporary measure taken by the muscles to
cope up with the energy requirements under hypoxic (low oxygen) conditions.
As far as the muscle is concerned it is a dead end. Lactate must be converted
back to pyruvate. The muscles use the inefficient mode to buy time and then
shift their burden to the liver for gluconeogenesis (synthesis of carbohydrates
from non carbohydrate sources). Liver, in turn, makes glucose again available
to the muscles to generate energy or storage. Some tissues like liver, kidney
and intestine can hydrolytically cleave glucose 6-phosphate to glucose that
leaves the cells with ease. We shall discuss about gluconeogenesis in unit 5,
however, you may note in Fig. 2.4 that gluconeogenesis is an expensive
process and uses 6 ATP to get back glucose while lactate fermentation in
muscles results in net synthesis of 2 ATP molecules.
The synthesis of lactate in the skeletal muscles and its conversion back to
36 glucose by the liver for use largely by the muscles constitutes the Cori cycle
Unit 2 Glycolysis
(Fig. 2.4). This cyclic route was worked out by the husband and wife team of
Carl Ferdinand Cori and Gerty Theresa Cori. Their contributions in glycogen
metabolism were recognised with the 1947 Nobel Prize in Physiology or
Medicine along with Bernardo Houssay.
Fig. 2.4: The Cori cycle illustrates metabolic interdependence between the
muscle and liver.
In situations, where strenuous activity continues for a long time, the formation
of lactate exceeds the capacity of the liver to regenerate glucose, resulting in
rise of lactic acid concentration in blood. The mildly acidic lactate will lower
blood pH (lactic acidosis) leading to tissue damage and symptoms associated
with panic, such as hyperventilation, abdominal cramps, vomiting, etc. All
these symptoms are a part of the body's natural defence mechanisms
designed to slow down rigorous activity, so that permanent damage can be
avoided.
So far we have discussed metabolism of glucose through glycolysis and the

fate of lactate in the skeletal muscle. In the next section, we shall learn how
carbohydrates other than glucose are processed into intermediates of the
glycolysis. They may be either obtained from diet or synthesised
endogenously. The additional reactions that allow their entry into glycolysis
constitute the feeder pathways.
2.6 FEEDER PATHWAYS FOR

GLYCOLYSIS
Glycolysis is the central metabolic pathway for generation of energy. We also
know that whenever our body needs energy, glycogen stores are mobilized. In
addition, polysaccharides, oligosaccharides and disaccharides are also
present in our diet. The complex dietary carbohydrates are hydrolyzed into
their constituent monosaccharides prior to absorption and assimilation. When
there is a need to degrade carbohydrates; the first step is to break them into
monosaccharides and process sugars other than glucose to an intermediate in
glycosis. Fig. 2.5 gives an overview of the feeder pathways of glycolysis. 37
Trehalose CH 2 OH
Lactose
Trehalase
Lactase HO H
O H
D-Galactose
CH 2OH Glycogen; starch H OH H OH
H H
O H Pi
H OH
HO OH H OH
UDP-galactose
H OH
Su c ra se
ATP Glucose-
Sucrose D-Glucose UDP-glucose
Hexokinase 1-phosphate
Phosphogluco
HOCH2 O CH 2 OH
mutase CH 2 OH
H H
O H
Glucose-
H H HO OH 6-phosphate
ATP
HO OH HO OH
OH H Hexokinase
D-Fructose H H
Fructose- D-Mannose
ATP Fructokinase 6-phosphate ATP
Hexokinase
Fructose-1-phosphate Mannose-6-phosphate
Fructose-1- Phosphomannose
phosphate isomerase
aldolase Fructose-1,6-
bisphosphate
Glyceraldehyde + Dihydroxyacetone
phosphate
Triose phosphate
ATP Triose isomerase
kinase Glyceraldehyde-
3-phosphate
Fig. 2.5: Feeder pathways for glycolysis.
Fig. 2.5 shows the breakdown of storage polysaccharides (glycogen and

starch), disaccharides (lactose, maltose, trehalose and sucrose) and
monosaccharides (fructose, galactose and mannose) to glycolytic
intermediates.
Phosphorolysis of
glycogen releases The dietary polysaccharides, oligosaccharides and disaccharides are
glucose-1-phosphate hydrolysed to monosaccharides by a combination of digestive enzymes that
which enters glycolysis act step by step, as the food moves down the digestive tract. On the other
at fructose-6-phospahte, hand, the endogenous glycogen undergo phosphorolytic cleavage from the
thereby, bypassing the
first step in which ATP is
non reducing end, releasing glucose 1-phosphate one by one until they reach
consumed. This is a branch point. The debranching enzyme takes over to remove branches. The
advantageous as it details of this process will be dealt in glycogen metabolism. Finally, glucose-1
increases the yield of phosphate is isomerised to the glycolytic intermediate glucose-6 phosphate by
ATP to three per
a phosphoglucomutase (PGM).
glucose from glycolysis
instead of two.
The disaccharides like sucrose, lactose, maltose and trehalose are hydrolysed
to monosaccharides (Fig.2.5). The entry of fructose, mannose and galactose
to the glycolytic pathway is explained in detail below.
Metabolism of Fructose
Fructose is a ketohexose that is released from the hydrolysis of sucrose (table

sugar) and is also present in free form in many fruits and honey. It is either
38 directly phosphorylated by hexokinase (muscles and kidney) or goes through
Unit 2 Glycolysis
the fructose 1-phosphate pathway (liver). The latter pathway is a three

step conversion of fructose to glyceraldehyde 3-phosphate, initiated by
fructokinase (Fig.2.5):
1. The liver fructokinase transfers the phosphoryl group from ATP to C-1 of
fructose.
2. Fructose-1-phosphate is split by fructose-1-phosphate aldolase into

Many people do not
glyceraldehyde and DHAP. You already know how DHAP enters produce enough lactase;
glycolysis. therefore, cannot digest
milk or milk products.
3. Glyceraldehyde is converted into glyceraldehyde-3-phosphate by triose As a result, lactose is
kinase. fermented by gut
bacteria leading to
Metabolism of Galactose symptoms like gastric
discomfort, diarrhoea,
D-Galactose is an aldohexose that is obtained by the hydrolysis of milk sugar, bloating, nausea and
lactose. Lactose is a disaccharide that is hydrolysed by lactase to glucose gas.
and galactose by β-galactosidase / lactase. The conversion of galactose to
glucose 1-phosphate goes through UDP-linked sugar derivative and uses
NAD+ for both oxidation and reduction (Fig. 2.6).
1. Galactose is phosphorylated to galactose-1-phosphate by galactokinase.
Galactose + ATP Galactose-1-phosphate + ADP + Pi
2. Galactose-1-phosphate is converted into UDP-Galactose by UDP- glucose:

galactose-1-phosphate uridylyl transferase. In this reaction UDP-glucose is the
donor of uridine monophosphate (UMP) and itself is converted to glucose-1-
phosphate. Glucose-1-phosphate is isomerised to glucose-6-phosphate by
PGM.
3. UDP-Galactose is converted to UDP-glucose by UDP galactose-4-

epimerase. In this reaction C-4 of galactose is first oxidised and then reduced
by NAD+ that also results in the inversion of its configuration. UDP-glucose
can participate in another round.
Fig. 2.6: Feeder pathway of galactose. 39

Individuals with a defect in any of three enzymes of galactose catabolism have

galactosemia. The most severe condition results from the deficiency of
uridylyl transferase in which children have mental retardation, poor growth and
liver damage that may lead to death even when they are fed on galactose free
diet.
The survivors also suffer from cataract due to deposition of galactitol in lens of
the eye. A deficiency of the other two enzymes is relatively less severe
especially when dietary control is rigidly followed.
Finally, D-mannose is converted to mannose-6- phosphate by hexokinase and

then isomerised to fructose-6- phosphate by phosphomannose isomerase
(Fig. 2.5).
SAQ 3
Complete the feeder pathway of galactose with names of missing
intermediates and the enzymes involved.
E1 E2 E3
Galactose ___ ____ Glucose-1- phosphate _______ glycolysis.
2.7 FATES OF PYRUVATE

We studied in the previous sections that fate of pyruvate depends on whether
it is catabolised under aerobic or anaerobic conditions. We learnt that under
anaerobic conditions it is fermented to a variety of end products like lactic acid
Pyruvate, the end or ethanol.
product of glycolysis
can form lactate, In this section we shall discuss what happens to pyruvate under aerobic
ethanol or acetyl condition.
CoA under different
conditions. Under aerobic conditions pyruvate is completely degraded to CO2 and water
by the combined action of pyruvate dehydrogenase complex (PDH), citric acid
cycle and electron transport chain. In eukaryotes, all this takes place in the
mitochondria.
Most of the ATP is generated by oxidative phosphorylation. In this respect

Louis Pasteur made a very significant observation while studying fermentation
in yeast. He discovered that glucose consumption dramatically falls if yeast
(facultative anaerobe) is shifted from anaerobic to aerobic conditions.
The basis of ‘Pasteur effect ‘can now be explained by the stringent regulation
of glycolysis at PKF-I catalysed step. The details of catabolism beyond
pyruvate under aerobic conditions are discussed in the next unit.
In addition to the catabolic fate, pyruvate also serves as starting material in

anabolism in all organisms. Fig. 2.7 shows diverse fates of pyruvate under
40 different conditions.
Unit 2 Glycolysis
Gluconeogenesis
Phosphoenolpyruvate
Lactate
Alanine CO2 + Acetaldehyde

Glycolysis LDH
ADH
Pyruvate dehydrooxylase
Transamination Ethanol
Oxaloacetate Pyruvate Acetyl CoA + CO2

PDH
Pyruvate carboxylase complex
Citrate synthase
Citrate + CoA
Fig. 2.7: Fates of pyruvate.
2.8 REGULATION OF GLYCOLYSIS

Glycolysis is the first step towards generation of energy from glucose. In The regulation of
carbohydrate
additon, it also serves as primary pathway for metabolism of other
breakdown occurs at
monosaccahrides. In general, the regulation of metabolic pathways is best the level of
achieved at steps that are far from equilibrium and are essentially irreversible. glycolysis, citric acid
The glycolysis pathway is therefore regulated at steps catalysed by cycle and
glycogenolysis.
hexokinase, phosphofructokinase-I and pyruvate kinase. In this section,
allosteric regulation of the three enzymes will be explained. We will come back
to this again after gluconeogenesis to understand how the two pathways are
reciprocally regulated.
Hexokinase
The enzyme hexokinase is inhibited by its product, glucose-6-phosphate. In

many organisms including humans, isozymes of hexokinase exist and they
may or may not be inhibited by glucose-6-phosphate; for instance the human
liver enzyme is not subject to product inhibition. This reaction is not subject to
stringent control so that glucose-6-phosphate can be fed to other pathways
such as glycogen synthesis and pentose phosphate pathway.
Phosphofructokinase-I (PFK-I)
The reaction catalysed by PFK-I is the first unique step of glycolysis and the
product is only fed to glycolysis. The enzyme is regulated by multiple allosteric
activators and inhibitors, although the regulation varies between organisms. 41
Allosteric inhibitors include ATP, citrate and H+ ion concentration (low pH).
ATP inhibits the enzyme by decreasing its affinity (high Km) for fructose 6-
phosphate. A high concentration of citrate intensifies the inhibitory effect of
ATP and it favours the dissociation of PFK-I from an active tetramer to an
inactive dimer.
Allosteric activators are fructose 2,6-bisphosphate, ADP, AMP and Pi.

Among them fructose 2,6-bisphosphate is the most important regulator of
PFK-I. It activates PFK-I at very low concentration as compared to other
Insulin is effectors by increasing the affinity of the enzyme for the substrate and
released when simultaneously decreasing the affinity of ATP and citrate.
blood glucose
levels are high Fructose 2,6-bisphosphate (F26BP) is formed from fructose-6-phosphate by a
and glucagon is bifunctional enzyme that has both kinase (PFK-2) and phosphatase activity
released when (Fig. 2.8). The activities of PFK-2 and fructose 2,6-bisphosphatase are
blood glucose controlled by reversible covalent modification (phosphorylation/
levels drop.
dephosphorylation) that is dependent on hormonal (insulin/glucagon)
response. Glucagon released in response to low blood glucose levels results
in phosphorylation of enzyme which converts fructose- 2, 6- bisphosphate
back to fructose- 6- phosphate which slows down the glycolysis. Insulin has
opposite effect.
Fig. 2.8: Regulation of PFK –I by fructose-2,6- bisphosphate.
Pyruvate kinase is regulated by allosteric effectors. Some of its isozymes are

also regulated by reversible covalent modification. The enzyme is allosterically
inhibited by high concentrations of ATP, acetyl CoA and long chain fatty acids.
The liver isozyme is regulated by phosphorylation and is active as
dephosphoenzyme. The hormone glucagon released in response to low
glucose slows down hepatic glycolysis by activating cAMP dependent protein
kinase that phosphorylates pyruvate kinase. As conditions change a
42 phosphatase removes the phosphate and makes it active. Fig. 2.9 summarizes
Unit 2 Glycolysis
the regulation of glycolysis. In muscles an increase in cAMP stimulates

glycolysis.
Fig. 2.9: Regulation of glycolysis- An overview.
SAQ 4
Choose the most appropriate glycolytic enzyme:
i) Depletion of cell’s ATP supply activates ---------- (Hexokinase / Pyruvate

kinase / Glucokinase / Phosphofructokinase-1).
ii) High concentration of glucose-6-phasphate inhibits --------------

(Hexokinase /Pyruvate kinase / Glucokinase /PFK -1)
iii) The committed step of glycolysis is catalyzed by -------------------

(Hexokinase/Pyruvate kinase/ Glucokinase / Phosphofructokinase-1).
2.9 SUMMARY
• Glycolysis is a near universal pathway of ten reactions in which glucose
(or related hexoses) is converted to two molecules of pyruvate with net
production of two ATP.
• It is divided into two phases: Energy investment / preparatory phase

and energy generation / pay off phase. 43
• The preparatory phase has two reactions which require input of energy
in the form of ATP to synthesise activated phosphorylated intermediates.
It ends with the splitting of fructose1,6- bisphosphate to two 3 carbon
sugars.
• The pay off phase includes one oxidation and two steps of substrate
level phosphorylation from each three carbon sugar. It ends with the
generation of two molecules of pyruvate, 2 NADH + H+ and 4 ATP. The
net ATP yield of glycolysis is therefore only 2.
• Under anaerobic conditions, pyruvate is converted to either lactate or

ethanol. This process is known as fermentation and its purpose is to
regenerate NAD+. It is an inefficient process.
• The glycolytic pathway is regulated at steps catalysed by hexokinase,

phosphofructokinase-I and pyruvate kinase. These steps are essentially
irreversible under physiological conditions.
• The synthesis of lactate in the skeletal muscles and its conversion back
to glucose by the liver for use largely by the muscles constitutes the Cori
cycle. It is a temporary measure taken by the muscles to cope up with
the energy requirements under hypoxic (low oxygen) conditions.
• Monosaccharides other than glucose such as fructose, mannose and

galactose can be transformed into glycolytic intermediates and
catabolised.
• Under aerobic conditions pyruvate is completely degraded to CO2 and

water by the combined action of pyruvate dehydrogenase complex
(PDH), citric acid cycle and electron transport chain.

1. What are the two phases of glycolysis? What is the net outcome of each
phase?
2. What is substrate level phosphorylation? Give an example of glycolytic

reaction that synthesises ATP by substrate level phosphorylation.
3. Indicate the significance of the Cori cycle.
4. Briefly explain the regulation of glycolysis. Why is step 3 and not step 1
of glycolysis the major control point?
5. What is the role of feeder pathways in carbohydrate metabolism?

Support your answer with suitable examples.
6. If C-1 of glucose is 14C labelled, which carbon(s) of ethanol will be

labelled?
7. Differentiate between PFK-1 and PFK-2
8. What would be the net yield of ATP if glucose is fermented to lactate in

44 the presence of arsenate?
Unit 2 Glycolysis
2.11 ANSWERS
1. i) f) ii) d) iii) a) iv) e) v) b) vi) c)
2. i) Fermentation
ii) Two
iii) Skeletal muscle/ cornea of eye;
iv) Ethanol and CO2
v) Regenerate NAD+
3. E1- galactokinase; E2- galactose-1-phosphate uridylyl transferase; E3-

phosphoglucomutase
Intermediates: galactose-1-phosphate; glucose-6- phosphate
4. i) Pyruvate kinase and Phosphofructokinase-1
ii) Hexokinase
iii) Phosphofructokinase-1
Terminal Questions
1. Investment (preparatory) and pay off phase. The investment phase
consumes two molecules of ATP where as the pay off phase results in
formation of 4 ATP molecules by substrate level phosphorylation and 2
molecules of NADH. Refer to section 2.3 for more details.
2. When ATP is synthesised by phosphoryl group transfer from activated

phosphorylated substrate to ADP is known as substrate level
phosphorylation. For example the conversion of 1,3 BPG to 3- PGA..
3. The synthesis of lactate in the skeletal muscles and its conversion back
to glucose by the liver for use largely by the muscles constitutes the Cori
cycle. Lactic acid formation is temporary measure taken by the muscles
to cope up with the energy requirements under hypoxic (low oxygen)
conditions.
4. The glycolytic pathway is regulated at the steps catalysed by

hexokinase, phosphofructokinase-I and pyruvate kinase. Step 1 of
glycolysis is not subject to stringent control because glucose-6-
phosphate also fed to pathways such as glycogen synthesis and
pentose phosphate pathway. Reaction 3 catalysed by PFK-I is the first
unique step of glycolysis and the product is only directed to glycolysis.
Therefore, it is more important for regulation of glycolysis. Refer to
Fig.2.11 for an overview of regulation of glycolysis.
5. Feeder pathways allow use of sugars other than glucose, either obtained
from diet or synthesised endogenously to be processed by glycolysis.
Refer to section 2.5 for more details. 45
6. None; labelled C-1 will be removed as CO2 because following the

isomerase reaction C-1, C-2 and C-3 are indistinguishable from C-6, C-5
and C-4.
7. PFK-1 catalyzes conversion of fructose-6- phosphate to fructose-1,6-

phosphate and is the stringently regulated step of glycolysis. PFK-2 is
part of a bifunctional enzyme and converts fructose-6- phosphate to
fructose-2,6- phosphate that functions as a positive allosteric regulator of
PFK-1 activity. PFK-1 is affected by ATP concentration but PFK-2 is not.
8. Arsenate resembles phosphate and may replace phosphate in

glyceraldehyde-3-phosphate dehydrogenase reaction. As a result, ATP
is not generated by substrate level phosphorylation, although glycolysis
will proceed. Therefore, there would be no net yield of ATP.
46
Unit 3 Tricarboxylic Acid Cycle
UNIT 3
TRICARBOXYLIC ACID CYCLE
Structure
3.1 Introduction 3.6 Glyoxylate Pathways
Expected Learning 3.7 Coordinated Regulation of

Outcomes TCA and Glyoxylate
Pathways
3.2 Unravelling the
Tricarboxylic Acid (TCA) 3.8 Pentose Phosphate
Cycle Pathway (PPP)
3.3 Synthesis of Acetyl CoA 3.9 Summary
3.4 TCA cycle 3.10 Terminal Questions
Amphibolic Role 3.11 Answers
Anaplerotic Reactions 3.12 Further Readings
3.5 Regulation of TCA Cycle
3.1 INTRODUCTION
In unit 2, you studied about glycolysis and how organisms extract energy
under anaerobic conditions. It is a primitive and almost universal pathway of
glucose catabolism. With the appearance of oxygen, the major change is in
our ability to completely breakdown carbon and to trap energy largely by
oxidative phosphorylation. Yet the initial catabolism is by glycolysis.
In this unit we shall discuss complete breakdown of pyruvate under aerobic

conditions by first converting it to acetyl CoA followed by entry to the TCA
cycle. We shall describe the amphibolic nature of this cycle and its anabolic
variant present in some organisms, the glyoxylate cycle. A brief description of
how to calculate the ATP yield from the complete breakdown of glucose will be
explained. Finally, the pentose phosphate pathway (PPP) and its role will be
explained.

elaborate the role of pyruvate dehydrogenase (PDH) complex in linking

glycolysis and TCA cycle and its regulation; 47
write about the intermediates of TCA cycle and the net outcome;
calculate the ATP yield of aerobic respiration;
indicate the amphibolic nature of TCA cycle;
highlight the relevance of anaplerotic reactions;
explain glyoxylate cycle in specialised tissues;
describe the regulation of TCA cycle and the underlying logic behind the
choice of allosteric modulators; and
explain the two phases and role of pentose phosphate pathway.
Sir Hans Adolf Krebs

(1900-1981) was a German 3.2 UNRAVELLING THE TRICABOXYLIC
born British biochemist.
His research work had
ACID (TCA) CYCLE
been mainly concerned
with various aspects of The elucidation of TCA cycle is the result of concerted effort of many
intermediary metabolism. investigators. Other commonly used names for TCA cycle are Kreb’s cycle or
He is best known for citric acid cycle. This work began in earnest from early 1930’s when it was
elucidating the pathways of observed that stimulated muscle exposed to air (oxygen) ceased to
both the urea cycle (1932) accumulate lactate, indicating further catabolism. The addition of dicarboxylic
in liver and the TCA cycle acids such as succinate, fumarate and malate was known to increase the rate
(1937) in pigeon breast of oxygen uptake in muscle. By 1920s Thunberg had added many more
muscles. The urea cycle
compounds to this list.
incidentally is the first
metabolic cycle worked In 1935, Albert von Szent-Györgyi showed in minced pigeon breast muscle
out. Interestingly, Nature that the complete oxidation of pyruvate occurs if catalytic quantities of
rejected Krebs’s paper on
dicarboxylic acids were added. He proposed that certain pairs of these acids
TCA cycle in 1937.
are interconnected by dehydrogenases and play role in respiration.
Krebs was elected Fellow
of the Royal Society of Carl Martius and F. Knoop (1936) demonstrated that citric acid is oxidised to
London in 1947. He was α-ketoglutarate (α-KG) by way of isocitrate. It was already known that α-KG
knighted in 1958. He forms succinate.
shared 1953 Nobel Prize in
Physiology or Medicine The single most important contributor was the German-British biochemist Sir
with F. Lipmann for the Adolf Hans Krebs. He and Johnson in 1937 showed that citrate was derived
discovery of citric acid from pyruvate and oxaloacetate (OAA). They found that citrate was not only
cycle. rapidly broken down in muscle; but is also formed readily if oxaloacetate is
(From Nobel Lectures, Physiology added. Krebs finally placed the intermediates in a cyclic pathway. In 1950,
or Medicine 1942-1962, Elsevier Fritz Lipmann discovered that acetyl CoA is derived from pyruvate and S.
Publishing Company, Amsterdam, Ochoa and F. Lynen showed that it condenses with OAA, thereby slightly
1964) modifying the original cycle.
E.P Kennedy and A.L. Lehninger in 1948 found that rat liver mitochondria
could catalyse the oxidation of pyruvate and all the intermediates of the TCA
cycle by molecular oxygen.
3.3 SYNTHESIS OF ACETYL COA

We already discussed in the previous unit that under aerobic conditions,
pyruvate undergoes oxidative decarboxylation to acetyl CoA. Let us see how
this reaction proceeds. Pyruvate is largely a product of glycolytic breakdown of
48 carbohydrates in the cytosol from where it is transported for further breakdown
into mitochondria by the pyruvate carrier. The conversion to acetyl CoA (2-C)
is catalysed in eukaryotes by pyruvate dehydrogenase (PDH) multienzyme
complex localised in the mitochondria. The reaction is irreversible under
physiological conditions. It is a bridge between glycolysis and TCA cycle. The
net outcome of the reaction is:
2 Pyruvate + 2NAD+ + 2HSCoA 2AcetylCoA + 2CO2 + 2NADH +

2H+
The enzyme complex has three different kinds of catalytic units, each present
in multiple copies. The number of copies of each enzyme varies from one
organism to another. The mammalian enzyme also has additional regulatory
units associated with the enzyme complex. In all organisms, it is a large
complex of variable molecular weight (4 to 10 million Daltons) that can be
observed under electron microscope.
Three enzymes (E1, E2 and E3) and their coenzyme / prosthetic groups of PDH
are:
• E1 : pyruvate dehydrogenase (TPP)
• E2: dihydrolipoyl transacetylase (lipoamide, HSCoA)
• E3: dihydrolipoyl dehydrogenase (FAD, NAD+)
The two coenzymes (HSCoA and NAD+) and three prosthetic groups (thiamine
pyrophosphate, FAD, and lipoamide) required are derived from B-group
vitamins. During the multistep reaction, there is net reduction of NAD+ and
synthesis of ‘active acetate’ (acetyl CoA) by linking acetate to coenzyme A
while all others factors are regenerated. Later NADH enters the electron
transport chain (ETC) for its re-oxidation and production of ATP by oxidative
phosphorylation. The recycling of cofactors is economic functioning and
especially relevant for vitamin derived organic cofactors.
The reaction catalyzed by PDH complex is summarized in Fig. 3.1.The

reaction is initiated by E1 enzyme. It catalyses the C-C bond cleavage of α-
keto acid (bond between carbons of ketone and carboxylic acid groups),
releasing CO2. The intermediate product of decarboxylation is resonance
stabilised and protonated to yield hydroxy ethyl-TPP. Next the hydroxy ethyl
group is oxidised to acetyl and transferred by E1 to lipoamide bound to E2. The
oxidation is accompanied by the reduction of disulphide linkage and the
intermediate is acetyl dihydrolipoamide. The first enzyme has accomplished
oxidative decarboxylation. The subsequent reactions have the job of
appropriate segregation of acetyl units and reducing equivalents. Lipoamide is
unique as it couples both electron and acyl group transfer processes. The
acetyl group is transferred to HSCoA generating acetyl CoA and reducing
equivalents are transferred to the FAD prosthetic group of E3 by E2, reoxidising
the –SH groups to disulphide. Acetyl CoA has an energy rich thioester linkage
and is released. You might have noticed that the flexible arm of lipoamide
interacts with all three active sites. Finally the enzyme bound reduced FADH2
is reoxidised by NAD+. In this reaction HSCoA and NAD+ are stoichiometric
cofactors while others are needed in catalytic amounts. 49
FAD
NAD+
Dihydrolipoyl 5
dehydrogenase
S (E3) NADH +H+
4
OH S FADH2
CO2 R
CH 3 CH TPP
Hydroxyethyl- Lipoamide HS
TPP Dihydrolipoyl
Pyruvate transacetylase HS
1 2
dehydrogenase (E2) R
(E1)
O O O 3 O
CH 3 C C TPP CH 3 C S CH 3 C S CoA
- CoA
O Acetyl-CoA
Pyruvate HS
R
Acetyl-dihydrolipoamide
Fig. 3.1: Formation of acetyl CoA from pyruvate by PDH complex.
It is important to mention here that TPP, one of the prosthetic groups of PDH
is derived from thiamine (vitamin B1). Deficiency of thiamine causes Beriberi,
which is primarily a neurological and cardiovascular disorder. Degeneration of
nervous system is prominent resulting in symptoms such as weakness of
muscles leading to pain in the limbs. This is because brain depends only on
glucose as fuel to get energy by TCA cycle whereas other tissues can use fats
as fuel. The activity of PDH is crucial for pyruvate to enter the TCA cycle
Two other enzymes whose activity is also affected in thiamine deficiency are
α-ketoglutarate dehydrogenase and transketolase. As a result, the levels of
pyruvate and α-ketoglutarate in the blood are elevated.
In unit 2, we learnt that glucose and other carbohydrates are broken down to
pyruvate by glycolysis. These reactions extract only a small fraction of energy
from glucose. In aerobic organisms, pyruvate is first converted to acetyl CoA.
One of the fates of acetyl CoA is to enter the TCA cycle for complete
oxidation. Acetyl CoA (or intermediates of the cycle) is also generated from the
partial breakdown of other biomolecules especially fatty acids and few amino
acids. It is a convergent pathway for the complete oxidation of carbon to
CO2.
The citric acid cycle / Krebs cycle is a sequence of eight reactions in which
(OAA) is regenerated. All but one enzyme (succinate dehydrogenase is
membrane bound) are present in the mitochondrial matrix of eukaryotes and
cytosol of aerobic prokaryotes. The intermediates of the cycle are also
anabolic precursors. The pathway includes two decarboxylation steps, four
oxidations (three hydride ions to NAD+ and one pairs of hydrogen atoms to
FAD) and one substrate level phosphorylation. The purpose of TCA cycle is to
harvest high energy electrons from breakdown of carbon fuels. The cyclic
50 pathway neither consumes oxygen nor directly produces enough ATP.
The TCA cycle operates in collaboration with the mitochondrial electron

Synthase is an enzyme
transport chain (ETC). The latter serves a dual purpose of re-oxidation of that joins two
reducing equivalents (NADH and FADH2) and ATP production by oxidative substrates without
phosphorylation. This pathway accounts for most of the ATP in aerobic direct participation of
heterotrophs and non green parts of plants. Let us now learn the reactions of ATP or other
TCA cycle. nucleoside
triphosphates.
SAQ 1
Match the enzymes of PDH complex with their prosthetic groups:
i). Pyruvate dehydrogenase a) FAD
ii). Dihydrolipoyl transacetylase b) TPP
iii). Dihydrolipoyl dehydrogenase c) Lipoamide
3.4 TCA CYCLE

The first condensation product of the cycle is citric acid so it is also called citric
acid cycle. Citric acid and the next two intermediates are tricarboxylic acids; TCA cycle is a
therefore it is tricarboxylic acid cycle (TCA cycle). It is also known as Krebs convergent pathway for
the complete oxidation
cycle as he was the single most important contributor in elucidating the cycle.
of carbon to CO2. Each
Let us look at Fig. 3.2 for an overview of the cycle and then study step wise round of the cycle
produces two molecules
the key events taking place.
of CO2, one GTP and
1. In the first reaction, citrate synthase catalyses aldol condensation of reducing equivalents in
the form of NADH and
acetyl CoA (2-C) with oxaloacetate (4-C) to form citrate. The reaction
FADH2. The re-oxidation
proceeds via citryl CoA as hydrolysis of this thioester drives the reaction of NADH and FADH2 by
towards citrate synthesis. ETC is coupled to ATP
synthesis (oxidative
2. Citrate is isomerised to isocitrate in a two step reaction of dehydration phosphorylation).
followed by hydration. This reaction is catalyzed by covalently bound
non heme iron (NHI) activated enzyme, aconitase. The product
isocitrate is formed through an intermediate cis - aconitate (not shown).
This isomerisation is a prerequisite for subsequent oxidative
decarboxylation. The reaction is inhibited by fluoroacetate, after its
conversion to fluorocitrate.
3. NAD+ dependent isocitrate dehydrogenase converts isocitrate (6-C)

to α-ketoglutarate (5-C) by oxidative decarboxylation.
4. The reaction occurs in two steps with formation of the enzyme bound
intermediate; oxalosuccinate. It is the first reaction of the cycle that
produces NADH.
5. Another oxidative decarboxylation is catalysed by α-ketoglutarate

dehydrogenase complex.
6. It is a multienzyme complex like PDH and catalyses α-ketoacid

decarboxylation by a similar mechanism. 51
7. The E3 enzyme is identical in both. In this reaction, α-ketoglutarate (5-C)

is converted to succinyl CoA accompanied by the reduction of NAD+ to
NADH.
8. Two CO2 molecules released by oxidative decarboxylations are

derived from the original OAA molecule because aconitase
distinguishes the two arms of citrate.
9. Succinate thiokinase catalyses substrate level phosphorylation of GDP

(ADP) to GTP (ATP).
10. The reaction proceeds via the displacement of HSCoA by

orthophosphate to an energy rich succinyl phosphate that transfers
phosphate group ultimately to GDP and generates succinate. GTP can
be converted to ATP.
11. The last three reactions regenerate OAA and provide additional reducing
equivalents that can be diverted to the ETC for re-oxidation and ATP
production.
12. Succinate is oxidized to fumarate by succinate dehydrogenase (SDH).

The enzyme has covalently bound FAD and multiple Fe-S clusters. FAD
is reduced to FADH2 during the reaction.
13. It is the only integral membrane enzyme of the TCA cycle and is also a
component of the succinate-ubiquinone reductase complex of ETC.
14. SDH is competitively inhibited by malonate. You must note that

succinate is a symmetrical molecule unlike succinyl CoA.
15. Fumarate is converted to L-malate by stereospecific addition of water by

fumarase.
16. An oxidation reaction catalysed by NAD+ dependent malate

dehydrogenase converts malate into oxaloacetate. This reaction is
driven to completion by removal of the products - oxaloacetate by citrate
synthase and NADH by ETC.
Let us calculate how much energy (ATP) is extracted from glucose breakdown
under aerobic conditions:
• The degradation of glucose to 2 pyruvate by glycolysis yields 2 ATP and 2

NADH+ 2H+.
• The oxidative decarboxylation of 2 pyruvate to 2 acetyl CoA generates 2

NADH+ 2H+.
• Oxidation of each acetyl CoA via citric acid cycle produces 3 NADH, 1
FADH2 and 1GTP. Therefore two acetylCoA will produce double the
number of reducing equivalents and GTP.
• Oxidation of NADH and FADH2 by ETC is coupled to oxidative

phosphorylation. Each NADH produces 2.5 ATP and FADH2 produces 1.5
ATP.
52 • The final count of ATP is : 10 x 2.5 + 2 x 1.5 + 4 = 32

O O
From glycolysis H3C C C
−
O
Pyruvate
NAD+ CoASH
Pyruvate dehydrogenase
NADH + H+ CO2
O
H3C C S CoA From β oxidation of fatty acids
Malate Acetyl-CoA
dehydrogenase
O CoASH
−
8 C COO
+
−
HO NAD H2C COO 1 Citrate
+
NADH + H oxaloacetate Synthase
− HO 2 -
H C COO H2C COO
− −
H2C COO HO C COO
H2O
Malate −
H2C COO
Citrate
7 Fumarase
2 Aconitase
H COO
−
Fumarate C
C -
− H H2C COO
OOC
FADH2 CH COO
−
−
HC COO
Succinate
6 dehydrogenase OH
Isociitrate
FAD
NAD+
−
H2C COO
− NADH + H+ 3 Isocitrate
H2C COO
- dehydrogenase
Succinate - NAD+ H2C COO
H2C COO
Succinate H2 C CO2
+
thiokinase H2C NADH + H 4
−
C COO
CoASH C SCoA
5 O a-ketoglutarate
O CO2 CoASH
GTP GDP + Pi
Succinyl-CoA a-ketoglutarate
dehydrogenase
ADP ATP
Nucleoside
diphosphate
kinase
Fig. 3.2: Tricarboxylic acid (TCA) cycle.
Net outcome of TCA cycle:

Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 +
3NADH + 3H+ + FADH2 + GTP + HSCoA 53
The number of ATP produced varies from 30 – 32, depending on the shuttle
operative in a given cell. Remember two NADH are produced in the cytosol
and eukaryotes do not interchange the pool of NAD+ / NADH between different
compartments. The movement of reducing equivalents as reduced organic
compounds depends on the shuttle systems.
You have seen that many enzymes such as PDH and KDH are multi enzyme
complexes. Such large complexes have some advantages over individual
enzymes.
i) These enzyme complexes allow product of one reaction to diffuse from

one active site to another without releasing intermediates into the
cytosol. This increases the overall efficiency of the reaction.
ii) The side reactions are minimized.
iii) It prevents exposure of cytosol to some of the intermediates which may

be highly reactive and toxic to the cell.
iv) It allows co-regulation of the enzymes in the complex.
3.4.1 Amphibolic Role

You may wonder why evolution selected use such an elaborate cyclic pathway
just to convert two carbons of acetyl CoA to CO2. TCA cycle intermediates
served as an important source of biosynthetic precursors and not energy in
early anaerobes. Some present day anaerobes have an incomplete TCA cycle
to provide anabolic precursors. In aerobic organisms, TCA cycle plays a dual
role; it is a convergent cycle for oxidative catabolism as well as a source of
anabolic precursors. We discussed in unit 1 that such pathways are more
appropriately called ambhibolic pathways. The anabolic fates of TCA cycle
intermediates are given in Fig. 3.3. You will study the synthesis of fatty acids
as we progress through this course. The metabolism of nitrogen containing
amino acids, nucleotides and compounds derived from them is the subject
matter of a separate course where again TCA cycle provides the starting
material for synthesis.
Fig. 3.3: Biosynthetic role of TCA cycle: The intermediates are drawn off for
54 biosynthesis of biomolecules for other pathways.
3.4.2 Anaplerotic Reactions

We discussed in the preceding section that TCA cycle is the primary source of
key biosynthetic precursors. In order to maintain the level of intermediates,
they are replenished by anaplerotic (‘filling up’) reactions, a term proposed by
H. L. Kornberg. Let us look at Fig. 3.4 for some anaplerotic reactions.
Fig. 3.4: Anaplerotic reactions: The reactions labelled 1 to 6 are some of the
ways to replenish TCA cycle intermediates. Out of these reactions 1 and 3 are
the major anaplerotic reactions.
There are a number of ways to replenish TCA cycle intermediates. One

important reaction is the conversion of pyruvate (1) or phosphoenol pyruvate
(6) to OAA or malate. The carboxylation of pyruvate is catalysed by biotin
dependent pyruvate carboxylase. This enzyme is activated allosterically by
acetyl CoA. It makes sense to synthesise more OAA when acetyl CoA levels
are high for efficient catabolism by TCA cycle.
Amino acids like aspartate and glutamate can either by transamination or

deamination generate OAA and α- ketoglutarate, respectively (2 and 5). The
partial breakdown of some amino acids (valine and isoleucine) or odd chain
fatty acids yields propionyl CoA that can be converted in three steps to
succinyl CoA (3). Some amino acids (Phe and Tyr) enter at the level of
fumarate (4).
In addition to reactions shown in Fig. 3.4, phosphoenol pyruvate also enters

TCA cycle at the level of oxaloacetate (6). Some cycles like the glyoxylate
cycle and purine nucleotide cycle operate in specialised tissues and can
replenish TCA cycle intermediates. 55
SAQ 2
A)
i) Individuals with thiamine deficiency have high levels of pyruvate in their

blood, why?
ii) Why thiamine deficiency is accompanied by neurological symptoms?
B)
i) The TCA cycle enzymes that participate in oxidative decarboxylations

are ___________ and ____________
ii) The only membrane bound enzyme of the TCA cycle is -------------
iii) The conversion of α-ketoglutarate to malate by TCA cycle and

reoxidation of reducing equivalents by ETC will generate ------- ATP
molecules.
iv) Pyruvate carboxylase is allosterically activated by ----------------
3.5 REGULATION OF TCA CYCLE

You have studied different mechanisms of enzyme regulation like feedback
regulation, reversible covalent modifications and by control of substrate
availability. Similar mechanisms are used by cells to tightly regulate the supply
of acetyl CoA and three enzymes of TCA cycle so as to ensure need based
catabolism (Fig. 3.5). The regulated steps are essentially irreversible under
physiological conditions. Now we will describe the regulation at each of these
High adenylate steps.
charge means high
[ATP/ADP] ratio, The activity of PDH complex regulates the supply of acetyl CoA derived from
indicating cell has carbohydrate sources. It is activated when the energy demands go up. In
enough energy to mammals two regulatory strategies are employed.
support itself.
PDH is activated by low concentration of fatty acids or low ratio of
[ATP]/[ADP], and [NADH]/[NAD+]. The products of the reaction inhibit the
enzyme by feedback inhibition as high ratio of [NADH/ NAD+] and [acetyl CoA
/HSCoA] maintain E2 in the acetylated form, incapable of accepting
hydroxyethyl group from TPP.
In addition to allosteric regulation, PDH is also regulated by reversible covalent

modification. High adenylate charge and interaction with the acetylated form of
E2 activates PDH kinase that phosphorylates a specific serine residue of E1,
inhibiting the enzyme. As the situation changes and the cell need more ATP, a
phosphatase hydrolyses the phosphate from phosphoserine thereby activating
the enzyme.
Finally, an increase in intracellular Ca2+ concentration activates the

phosphatase leading to PDH activation as during muscle contraction.
The rate controlling enzymes of the TCA cycle are citrate synthase, isocitrate
56 dehydrogenase and α- ketoglutarate dehydrogenase complex. The activity of
these enzymes is controlled by substrate availability (acetyl CoA and OAA),

product inhibition and feedback inhibition by other intermediates further along
the cycle. Both OAA and acetyl CoA are present at concentrations that do not
saturate the enzyme.
• Citrate synthase which catalyzes the synthesis of citrate is inhibited by

NADH, ATP, citrate (product inhibition) and succinyl CoA (competitive
feedback inhibition).
• The rate of citrate removal is dependent on the activity of isocitrate DH. It is

inhibited by NADH and ATP and activated by ADP, NAD+ and Ca+2. In
organisms that express the enzymes of glyoxylate cycle, isocitrate is a
branch point intermediate which helps in coordinated regulation of TCA and
glyoxalate cycle as explained in the next section.
• α- ketoglutarate dehydrogenase complex is strongly inhibited by its

products (succinyl CoA and NADH) and activated by Ca+2.
Fig. 3.5: Regulation of TCA cycle: The pathway is regulated at the level of PDH,
citrate synthase, isocitrate dehydrogenase and α- ketoglutarate dehydrogenase
complex by allosteric and covalent modification mechanisms primarily
determined by the energy status of the cell.
In general, the pathway is inhibited by high[ NADH/ NAD+ ] and high adenylate
charge where as high level of ADP, AMP, NAD+ and Ca2+ activate the
enzymes as they are indicate increase in energy demands.
SAQ 3
Name two enzymes of TCA cycle that are allosterically inhibited by ATP.
Earlier a reference to an incomplete TCA cycle operative in anaerobic

microorganisms was made in subsection 3.4.1. Glyoxylate cycle is another
variation of this cycle that has both anabolic and anaplerotic role. Let us learn
about this pathway in more detail. 57
3.6 GLYOXYLATE PATHWAYS

Glyoxylate cycle is an anabolic variant of citric acid cycle that synthesises
carbohydrates from acetylCoA / acetate derived from fatty acid metabolism. It
derives its name from the production of an intermediate glyoxylate in the first
unique step of the pathway.
The cycle was elucidated by Hans Kornberg and Neil Madsen. Essentially, it
results in the net synthesis of succinate (a gluconeogenic substrate) from two
molecules of acetyl CoA by bypassing the decarboxylation reactions of TCA
Glyoxylate cycle plays
cycle and including two additional enzymes. It is present in some
important role in the
microorganisms such as fungi, bacteria and protists and transiently expressed
metabolism of pathogenic
in germinating seedlings of oil seeds.
species including fungi
and bacteria. The The pathway is important during germination till plants become competent to
enzymes of the cycle are
fix CO2. Vertebrates lack the glyoxylate cycle so they cannot convert fatty
important drug targets in
acids or acetyl CoA to carbohydrates.
treatment of diseases
such as tuberculosis. The net reaction of glyoxylate cycle is:
2 Acetyl CoA + 2NAD+ +2H2O Succinate + 2HSCoA + 2NADH +2H+
In plants, the enzymes of the cycle are compartmentalised in glyoxysomes,

special kind of peroxisome. It shares three out of the five enzymes with TCA
cycle.
The isoenzymes of citrate synthase, aconitase and malate dehydrogenase are

present both in mitochondria and the glyoxysomes. The two unique enzymes
of this cycle are isocitrate lyase and malate synthase. Glyoxysomes also have
enzymes for the breakdown of fatty acids.
The first two reactions are identical to citric acid cycle. The difference is in the
processing of isocitrate; it is cleaved by isocitrate lyase into succinate and
glyoxylate. Glyoxylate combines with another molecule of acetyl CoA to form
malate in the presence of malate synthase which completes the cycle,
regenerating OAA by malate dehydrogenase. Fig. 3.6 shows the complete
glyoxylate cycle.
58 Fig. 3.6: Glyoxylate cycle.

Glyoxylate cycle results in synthesis of a succinate molecule from acetyl CoA,

derived from fat breakdown. Let us see how this cycle helps in conversion of
fats reserves to carbohydrates. Succinate is transported to the mitochondria
where the TCA cycle enzymes convert it to malate which leaves the
mitochondria. In the cytosol malate is oxidised to OAA by cytosolic malate DH
and then converted to glucose by gluconeogenesis. The entire process is
completed by participation of enzymes in glyoxysomes, mitochondria and
cytosol (Fig. 3.7). The cycle can also serve an anaplerotic role as succinate is
a TCA cycle intermediate.
Fig. 3.7: The synthesis of carbohydrates from fat reserves: Note the
compartmentalisation of enzymes and the movement of intermediates between
glyoxysomes, mitochondria and cytosol to complete the entire process.
SAQ 4
Which reaction of the glyoxylate cycle is analogous to the reaction catalysed
by citrate synthase in Krebs cycle?
3.7 COORDINATED REGULATION OF TCA

AND GLYOXYLATE PATHWAYS
You know that TCA and glyoxylate cycles branch before the first
decarboxylation step of TCA cycle. At this point, the two cycles are co-
ordinately regulated (Fig. 3.8). The branch point intermediate of these two
pathways is isocitrate and the regulation of isocitrate DH by reversible
covalent modification determines its fate. When it is phosphorylated by a 59
specific protein kinase, the enzyme is inactive and isocitrate is diverted to the
glyoxylate cycle. On the other hand, the removal of phosphate by
phoshoprotein phosphatase activates this enzyme and isocitrate is fed to the
TCA cycle for degradation. The two regulatory enzymes are present in a single
polypeptide (bifunctional enzyme).
The intermediates of TCA cycle and glycolysis that activate isocitrate DH by

stimulating the phospho protein phosphatase are allosteric inhibitors of
isocitrate lyase. These modulators sense the energy needs of the cells and if
they are fulfilled; the glyoxylate cycle is favoured and isocitrate DH is
phosphorylated.
Fig. 3.8: Coordinated regulation of TCA and glyoxylate cycles: Energy status of
the cell is the primary determinant of the fate of isocitrate (degradation Vs
Otto Warburg in 1931 synthesis).
discovered the first
enzyme of the 3.8 PENTOSE PHOSPHATE PATHWAY
pathway that oxidises
glucose 6-phosphate (PPP)
to 6-phoshogluconic
acid & its coenzyme The pentose phosphate pathway is an alternative to glycolysis for glucose
NADP+. The complete metabolism. It is also known as hexose monophosphate shunt (HMP shunt) or
pathway was
phosphogluconate pathway. The existence of an alternate route was based on
subsequently
elucidated by F. the initial observations that in some tissues, the classical inhibitors of
Lipmann, F. Dickens, glycolysis had no effect on the utilisation of glucose. It was also shown that
B. Horecker and E. glucose labelled in C-1 is more readily oxidised to CO2 than that is labelled at
Racker. C-6.
The pentose pathway operates in the cytosol. The cycle is primarily

60 responsible for the generation of NADPH for reductive biosynthesis and a
variety of sugars such as ribose 5-phosphate that may be utilised for

biosynthesis or provide a link to the glycolytic cycle. The activity of the
pathway is directly correlated with reductive biosynthesis; for instance it is very
low in skeletal muscles and high in adipose tissue, liver, adrenal glands.
Ribose-5-phosphate is required to synthesise nucleic acid building blocks and

biomolecules containing nucleotides like NAD+, FAD and HSCoA. Similarly,
erythrose-4-phosphate produced in the non- oxidative branch is one of the
starting materials for aromatic amino acid synthesis. This pathway also
assumes significance in RBC where a large amount of NADPH produced is
used for the reduction of glutathione by GSH reductase. This reduced
glutathione in turn reduces organic peroxides and H2O2 and protects RBCs
from oxidative damage. The inability to maintain reduced glutathione in RBCs
Pentose phosphate
leads to increased accumulation of peroxides, weakening of cell membrane pathway is an alternate
and haemolysis. pathway of glucose
metabolism in which
The pentose pathway is divided into two distinct phases, namely oxidative interconversions of
(irreversible) phase and non oxidative (reversible) phase. sugars occurs. It results
in the formation of
A. Oxidative branch tetroses and pentoses
which are used for the
i) The oxidative branch starts with the oxidation of glucose 6-phosphate synthesis of aromatic
(glycolytic intermediate) to 6-phosphogluconolactone by a NADP+ amino acids and nucleic
dependent glucose-6-phosphate dehydrogenase. acids respectively. It
also yields NADPH
ii) Next the lactone (an internal ester) is hydrolysed by lactonase to 6- required for reductive
biosynthesis and
phosphogluconic acid.
maintenance of
iii) The sugar acid is oxidatively decarboxylated by NADP+ dependent 6- reduced forms of
glutathione and other
phosphogluconate dehydrogenase to ribulose 5-phosphate.
biomolecules.
iv) The 5C ketose sugar is then isomerised to ribose 5-phosphate by
phosphopentose isomerase.
The net outcome of glucose 6-phosphate oxidation is:
Glucose-6 phosphate + 2NADP+ +H2O Ribose 5-

phosphate + 2NADPH + 2H+ + CO2
You will note that every glucose 6-phosphate oxidised produces one ribose 5-
phosphate and two NADPH + H+. The cellular requirements are not always in
the same proportion for both products of the oxidative branch. Many cells need
more NADPH than ribose 5-phosphate. The non oxidative branch converts the
excess sugar to glycolytic intermediates.
B. Non oxidative branch
The non oxidative branch involves the inter conversion of sugars by

transketolase and transaldolase. In both these reactions the sugar that
donates 2C or 3C unit is always a ketose and the acceptor is an aldose. The
net result is the formation of two hexoses and one triose from three pentoses.
To begin with ribulose 5 phosphate is converted to xylulose 5-phosphate by an
epimerase. The three interconversions are:
61
i) A two carbon moiety is transferred from xylulose-5-phosphate to ribose-

5-phosphate by transketolase resulting in the synthesis of
sedoheptulose-7-phosphate (7-C) and glyceraldehydes-3-phosphate (3-
C). Transketolase is a TPP dependent enzyme.
ii) Transaldolase catalyses the transfer of a three carbon moiety from

sedoheptulose-7-phosphate to glyceraldehydes-3-phosphate to form
fructose-6-phosphate (6-C) and erythrose-4-phosphate (4-C).
iii) Finally, transketolase transfers a 2 carbon ketol group from xlulose-5-

phosphate to erythrose-4-phosphate to produce fructose-6-phosphate
and glyceraldehydes-3-phosphate.
Fructose-6-phosphate and glyceraldehyde-3-phosphate can resynthesise five

molecules of glucose-6-phosphate by gluconeogenesis. This allows the
recycling of ribose 5-phosphate produced by the oxidative branch of PPP to
glucose 6-phosphate.Therefore the end result is the complete oxidation of a
molecule of glucose 6-phosphate to 6 molecules of CO2 and 12 molecules of
NADPH.
The net outcome PPP and gluconeogenesis is:
Glucose 6-phosphate + 12 NADP+ + 7H2O 6CO2 + 12 NADPH +12 H+ +

Pi
Fig. 3.9: Pentose phosphate pathway: The pathway is depicted in two parts
representing the oxidative and non- oxidative branches.
The first reaction of the oxidative branch catalysed by glucose 6-phosphate

DH is the rate limiting step that is regulated by NADP+ level. This ensures that
NADPH generation is tightly coupled to its utilisation in reductive biosynthesis.
The non oxidative branch on the other hand is controlled by the availability of
62 substrates.
SAQ 5
Fill in the blanks:
i) The end products of the oxidative branch of PPP are ________and

_________.
ii) In plants the enzymes of glyoxylate cycle are present in-------------
iii) A PPP enzyme whose activity is impaired by thiamine deficiency is -------
3.9 SUMMARY
• In aerobic organisms, pyruvate is oxidatively decarboxylated to acetyl
CoA before it enters the TCA cycle for complete oxidation. The reaction
is catalyzed by a multienzyme pyruvate dehydrogenase complex,
present in mitochondria. It is irreversible under physiological conditions
and is a bridge between glycolysis and TCA cycle.
• The citric acid cycle / Krebs cycle is a sequence of eight reactions in

which oxaloacetate is regenerated. All but one enzyme (succinate
dehydrogenase is membrane bound) are present in the mitochondrial
matrix of eukaryotes and cytosol of aerobic prokaryotes
• TCA cycle is a convergent pathway for the catabolism of carbohydrates,

lipids and proteins. Each acetyl CoA produces two molecules of CO2,
one molecule of GTP and reducing equivalents in the form of NADH and
FADH2 which enter ETC for reoxidation and ATP production. The
complete oxidation of one glucose molecule through glycolysis, TCA
cycle and ETC produces 30-32 ATP.
• In aerobic organisms TCA cycle plays a dual role; that of oxidative

catabolism and a source of anabolic precursors. It is more appropriately
called an ambhibolic pathway. As intermediates of TCA cycle are also
used for biosynthesis they are replenished by anaplerotic reactions.
• TCA cycle is regulated at steps catalysed by PDH, citrate synthase,

isocitrate dehydrogenase and α- ketoglutarate dehydrogenase complex.
Regulation is exerted by allosteric modulators and /or covalent
modification. In general, the pathway is activated in response to
indicators of cell activity.
• Anabolic variants of TCA cycle exist in nature, where the purpose is to

provide intermediates for biosynthesis. One such pathway is glyoxylate
cycle which is present in plants (germinating seeds) and many
microorganisms such as fungi, bacteria and protists. It is involved in the
synthesis of carbohydrates from fats. This pathway is absent in
vertebrates.
• TCA and glyoxylate cycles are co-ordinately regulated. The branch point
intermediate of the two pathways is isocitrate and the regulation of
isocitrate DH by reversible covalent modification determines its fate. 63
When cell needs energy, isocitrate dehydrogenase is activated by

covalent modification and isocitrate lyase is allosterically inhibited by
intermediates of TCA cycle.
• An alternative pathway of glucose oxidation is known as pentose

phosphate pathway or hexose monophosphate (HMP) shunt. The cycle
is primarily responsible for generating NADPH for reductive biosynthesis
and a variety of sugars such as ribose 5-phosphate that may be utilised
for biosynthesis or provide a link to the glycolytic cycle. The activity of
the pathway is directly correlated with reductive biosynthesis; for
instance it is very low in skeletal muscles and high in adipose tissue,
liver, adrenal glands. In RBCs, NADPH is used to reduce glutathione
and protect the cells from oxidative stress and haemolysis.

1. Indicate the role of each coenzyme / prosthetic group involved in the
oxidative decarboxylation of pyruvate by PDH.
2. Highlight two advantages of multienzyme complexes as compared to

isolated enzymes.
3. a) How many molecules of CO2, NADH and FADH2 are produced in

one round of citric acid cycle?
b) Calculate the ATP yield from complete oxidation of glucose under

aerobic conditions.
4. Describe the role of Pentose Phosphate Pathway.
5. How does the glyoxylate cycle help in the synthesis of carbohydrates

from fats?
6. Explain the regulation of TCA cycle.
7. TCA cycle operates only under aerobic conditions although oxygen is

not a direct participant. Explain.
3.11 ANSWERS
1. i) b) ii) c) iii) a)
2. A) i) Individuals with thiamine deficiency have low activity of PDH. They

have limited ability to catalyse TPP dependent α-ketoacid
decarboxylation of pyruvate. This leads to an increase in pyruvate
levels in blood.
ii) Thiamine deficiency is accompanied by neurological disorder as brain

relies on glucose to get energy via TCA cycle. Its deficiency depletes
brain of its energy causing neurological damage and symptoms such
64
as limbs weakness and abnormal skin sensation.
B) i) Isocitrate dehydrogenase and α- ketoglutarate dehydrogenase
ii) SDH; iii) Five; iv) acetyl CoA;
3. i) Citrate synthase ii) isocitrate dehydrogenase
4. Malate synthase
5. i) Ribose 5-phosphate and NADPH ii) Glyoxysomes iii) Transketolase
Terminal Questions
1. Refer to section 3.3 for more details.
Enzyme of Prosthetic Role

PDH complex group/Cofactor
E1: Pyruvate thiamine Decarboxylation of pyruvate and

dehydrogenase pyrophosphate carries remaining molecules as
(TPP) hydroxyl ethyl
E2: lipoamide and Lipoamide oxidizes

Dihydrolipoyl coenzyme A hydroxyethyl- to acetyl- and
transacetylase (HSCoA) then transfers acetyl unit to
HSCoA
E3: flavin adenine Regeneration of lipoamide and

Dihydrolipoyl dinucleotide (FAD) FAD
dehydrogenase and nicotinamide
adenine
dinucleotide
(NAD+)
2. Advantages of multienzyme complexes:
i) The product of one reaction diffuses from one active site to

another without releasing intermediates into the cytosol. This
increases the overall efficiency of the reaction.
ii) The side reactions are minimized.
iii) It prevents exposure of cytosol to some of the intermediates which

may be highly reactive and toxic to the cell.
iv) It allows co-regulation of the enzymes in the complex.
3. a) One round of TCA cycle produces 2 CO2, 3NADH and one FADH2.
b) Total number of ATP from complete aerobic oxidation of glucose

range from 30 to 32. Refer to section 3.4 for detailed calculations.
4. Pentose phosphate pathway operates in the cytosol and is primarily

responsible for the generating of NADPH for reductive biosynthesis. It
also yields a variety of sugars such as ribose 5-phosphate that may be
utilised for biosynthesis or provide a link to the glycolysis. 65
5. Acetyl CoA from fatty acid oxidation is converted to succinate that enters
glyoxylate cycle. Succinate is transported to the mitochondria where
TCA cycle enzymes convert it to malate which then leaves the
mitochondria. In the cytosol, malate is oxidised to OAA by cytosolic
malate DH and then converted to glucose by gluconeogenesis. Refer
section 3.6 for more details and Fig.3.7.
6. TCA cycle is regulated at the level of PDH complex, citrate synthase,

isocitrate dehydrogenase and α- ketoglutarate dehydrogenase complex
by various mechanisms, Refer section 3.5 for details.
7. TCA cycle operates in collaboration with the mitochondrial electron

transport chain (ETC). ETC reoxidizes reducing equivalents (NADH and
FADH2) formed in TCA cycle and produces ATP production by oxidative
phosphorylation. The terminal acceptor of ETC is oxygen therefore both
these processes occur only in the presence of oxygen.
66

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BBCCT-109

Course Preparation Team

Dr. Niraj Srivastava (Unit 7) Professor R.K. Sharma (Unit 13)

Course Coordinator: Dr. Seema Kalra (seemakalra@ignou.ac.in)

Print Production Team

Block 2 on Carbohydrates Metabolism II discusses anabolism of carbohydrates. It begins with

Expected Learning Outcomes

Expected Learning Outcomes

After studying this block, you should be able to:

• Define metabolism and explain other related terms;

• Explain regulation of glycolysis; and

• Describe TCA cycle, its significance and regulation.

Expected Learning Outcomes

define the term metabolism;

differentiate between catabolism and anabolism;

classify living beings based on metabolic diversity;

differentiate between autotrophs and heterotrophs;

highlight the salient features in the design of metabolism;

describe the significance, formation and utilization of ATP; and

indicate the role of pyridine and flavin nucleotides in metabolism.

1.2 AUTOTROPHS AND HETEROTROPHS

2. Chemotrophs use oxidation- reduction reactions to extract energy from

Table 1.1: Metabolic classification of living organisms based on source

Classification Source of Source of Examples

Photoautotrophs CO2 Light Green plants, algae,

Photoheterotrophs Organic Light Nitrifying bacteria;

Chemoautotrophs CO2 Redox reactions Non sulphur

Another metabolic classification of organisms is based on whether or not they

The obligate aerobes have an absolute dependence on oxygen. Bacteria like

The extreme group is of obligate anaerobes that are represented by many

i) Facultative and obligate anaerobes

ii) Chemoautotrophs and chemoheterotrophs

2) The efficient extraction of chemical energy of biomolecules into ATP and

3) Synthesis of complex biomolecules from simple precursors in

4) Synthesis and storage of long and short term energy reserves in

Fig. 1.1: Metabolic map of intermediary reactions operating in human beings.

1.3.1 Catabolism and Anabolism

Catabolism (Greek cata, down and ballein, to throw) refers to reactions

Anabolism (Greek ana, up and ballein, to throw), on the other hand, is

1.3.2 Schematic Representation of the Stages

Fig. 1.2: Stages in the breakdown and synthesis of biomolecules. 11

1.3.3 Design of Metabolism

The central metabolic pathways such as glycolysis are few in number

Both anabolic and catabolic pathways are essentially irreversible and

The number of biochemical reactions is very large but the kinds of

The enzymes for synthesis and degradation are often

Many enzymes have additional requirements as coenzymes and / metal

In all present day organisms, ribonucleotides play a central role in

A unique attribute of life is its ability to regulate metabolic processes.

The metabolic pathways are generally regulated at multiple levels that

i) A coenzyme derived from a B group vitamin.

ii) An activated donor in biosynthesis.

iii) A modified nucleotide employed for transducing signals.

1.4.1 Metabolic Pathways

This type of representation is used when a precursor or substrate is converted

Fig.1.3: A linear metabolic pathway. 13

(b) Spiral pathway

A spiral pathway can be viewed as a variation of a linear pathway. In this case