The document discusses various metabolic pathways in plants including primary and secondary metabolites. It describes the shikimic acid pathway and its role in synthesizing aromatic amino acids. The acetate/mevalonate pathway and its role in terpenoid biosynthesis is also covered. Various techniques used to study these pathways are outlined, including the use of radioactive isotopes as tracers to investigate biosynthesis through precursor-product relationships. The summary focuses on the key metabolic pathways and tracer techniques discussed in the document.

2.  Introduction –
 Overview of Basic metabolic pathways
 Role of Enzymes
 Role of Co-enzymes
 Shikmic acid pathway
 Role of shikmic acid pathway
 Acatate pathway
 Amino acid pathway
 Utilization of Radioactive isotopes in the
investigation of biogenetic disease.

3.  Metabolic pathways:
Metabolic pathway comprises of series of
chemical reactions which occurs inside the
cells. The reactants, products, and
intermediates which are formed as a result of
these enzymatic reactions are called as
Metabolites. These enzymes typically need
dietary minerals, vitamins, and different
cofactors to perform.
 There are two types of metabolites-
1. Primary plant metabolites.
2. Secondary plant metabolites.

4. 1. Primary plant metabolites- These are considered as
basic plant constituents. They are simple in structure
and they do not have biological and pharmacological
activity associated with them. Primary metabolites
are utilized by plants for building up their body and
responsible for physiological functioning of the plant
body. eg. Starch, Cellulose, Chlorophyll, Calcium
oxalate crystals etc.
 Degradation of carbohydrates and sugars which
release energy from the organic compounds by
oxidative reactions.
 Oxidation of fatty acid from fats by β- oxidation also
provides energy.
 Proteins taken via diet provide amino acid most
organism can synthesize only proportion of amino
acid they actually required for protein synthesis those
not synthesized they are called as essential amino
acid obtained from external sources.

5.  Secondary metabolites are not essential for growth they have wide
range of chemical structures and biological activity. they are derived by
unique biosynthetic Pathways from primary metabolites and
intermediates
 That are not necessary for growth and Reproduction of organism but
which can be demonstrated genetically ,physiologically and
biochemicaliy.
 These are also known as active constituents. The quantity of active
constituents that is a secondary plant metabolites determines the
intrinsic important role in the process of biosynthesis.
Fatty acids fats
photosynthesis +carbon dioxide starch
glucose
Fructose
Erythrose Glycerate
Amino acid

6. Formation of amino acid – By Sequential-
 Photosynthesis
 Primary metabolites
 Secondary metabolites
 Phenolics
 Chalcones
 Flavones
 Dihydroflavanones
 Flavonols (yellow colour flowers)

7.  The majority of secondary metabolites belong to
one of the number of families each of which
have particular structural characteristic arising
from the way in which they are built up in nature
that is also called as the biosynthesis.
The classes of secondary metabolites are:
 Polyketides and fatty acids
 Terpenoids and steroids
 Phenyl propanoyl
 Alkaloids
 Others such as a specialised amino acids and
carbohydrates

8.  Enzyme plays a vital role as a similar to catalyst in monitoring
various cellular activity enzymes are important for both plant
and animals.
 Enzymes are very delicate sensitive and thermolabile in
nature.
 In the absence of enzyme it is impossible to carry out
reactions in the body, chemically enzymes are proteins in
nature these are colloidal catalyst which helps to increase the
speed of biochemical reactions.
 Sometimes ; only the speed of reaction is affected but it
becomes impossible for a plants to carry out the reaction in
their body.
 They are very sensitive and active on specific temperature
and pH of the medium in which the reaction is carried out the
enzymes can be destroyed and inactivated due to the
following reasons
1. due to excessive heat
2. due to excessive moisture

9.  Oxido-reductases
 Transferases
 Hydrolases
 Lyases
 Isomerases
 Ligases

10.  Co-Enzymes are the organic molecules which are present in
very small amounts their presence in highly important for normal
functioning of enzyme. It is observed that due to the absence of
the some co-enzymes, sometime enzymes are not actively
showing their potency.
 eg. Uridine phophate and Adenosine phosphate contains
Nucleotide.
 Some Co-enzymes in plant body-
Uridine triphosphate
Thiamine
Pyridixine
Nicotinamide
Uridine diphosphate
Riboflavin
Flavin. Etc.

12.  Introduction to Shikimic acid pathway-
 Shikimic acid is also known as a Sikhmate in its ionic
form. structurally it is a cyclohexane,a cyclitol and a
cyclohexane carboxylic acid.
 It is an important biochemical metabolite in plants and
microorganisms.
 Its name is derived from Japanese plant Shikimi
(Illicium anisatum) Shikimic acid is a first isolated from
this plant in 1885 by Johan fendrik Eykman.
 This pathway is not found in animals.
 This pathway utilized by bacteria, fungi, algae,
parasites and plants for biosynthesis of aromatic amino
acids like phenylalanine, tyrosine and tryptophan.

13.  AROMATIC BIOSYNTHESIS SHIKIMIC ACID PATHWAYS
 The majority of the aromatic compounds are
biosynthesized via Shikimic acid pathways. The
Shikimic acid pathway plays significantly important in
the genesis of the aromatic building blocks of lignins,
and also leads to the formation of phenyl propane
units like flavones and isoflavonone, coumarins,
tannins, vanillin and terpenoid quinones.
 The Shikimic acid pathway appears to be an
important route from carbohydrate for the
biosynthesis of Co- C1 units (phenylpropane
derivatives).
 For higher plants, the presence of enzyme system
responsible for the synthesis of Shikimic acid has
been confirmed.

14.  The important steps involved in the genesis of Shikimic
acid pathway as follows:
 Shikimic acid pathway starts with the Erythrose 4
phosphate (obtained from the pentose phosphate
pathway) and phosphoenol pyruvate (obtained from
Glycolysis pathway) on aldol condensation to yield
DHAP (2-keto-3-Deoxy-D-arabinoheptonicacid-7-
phosphate).
 DHAP on removal of phosphoric acid cyclizes to form 3-
dehydroquinic acid, which on dehydration to form 3-
dehydroshikimic acid which yields Shikimic acid by
reduction.
 Shikimic acid through phosphorylation and elimination
reactions forms a very important intermediate
compound, Chorismic acid.

15.  Chorismic acid is an important branching
point; anthranilate synthase uses chorismic
acid as substrate to give anthranilic acid
which is a precursor for Tryptophan.
 Chorismic acid via simple rearrangement
gives prephenic acid.
 Prephenic acid on dehydration and
decarboxylation yields precursor of
Phenylalanine i.e. phenylpyruvic acid.
 On dehydrogenation and decarboxylation,
prephenic acid yields p-hydroxyphenyl pyruvic
acid which is a direct precursor of Tyrosine.
The schematic representation of Biosynthesis
of aromatic compounds via Shikimic acid
pathway as follows:

17.  INTRODUCTION: Isoprenoid pathway is also known as
terpenoid pathway or acetate mevalonate pathway.
 This pathway contributes about one third of all known
secondary metabolites.
 The isoprene units from this pathway is contributed in
biosynthesis of many other metabolites such as
anthraquinones, napthaquinones, terpenoids and indole
alkaloids.
 The important steps involved in the biogenesis of
Mevalonate pathway as follows: Acetate mevalonate
pathway begins with molecule of acetyl CoA which is
produced from pyruvic acid, end product of glycolysis.
 First two molecules of acetyl CoA forms acetoacetyl CoA
through Claisen condensation.

19.  Amino acid are the compound which comprises
of amino group as well as carboxylic acid group
because protein Are linear polymer of amino
acids.
 Amino acids are amphoteric act as a acid or
base ionic electrolyte ion for light amphoteric
electrolyte that is a pH gradient under electric
field moves to its isoelectric point amino acids
can connect with the peptide Bond involving
amino and carboxylic group peptide bonds are
planar and partially ionic.

21.  Secondary plant metabolites- The secondary
metabolites are biosynthesized from primary
plant metabolites . Secondary plant
metabolites are basically potent in their
action and they are associated with marked
pharmacological actions on human body.

22.  Living plant may be considered as a
biosynthetic laboratory not only for the primary
metabolites like sugars, amino acids and fatty
acids but also for a nucleotide secondary
products of pharmaceutical significance such as
glycosides, alkaloids, flavonoids, volatile oils
etc. The various biosynthetic reaction occurring
in plant cells are enzyme dependent which are
reversible.

23.  It is through the control of enzymatic activity that plant metabolism
is directed into specific biosynthetic pathways.
 Biosynthesis: Formation of Chemical compounds by a living
organism.
 Biogenesis: Production or generation of living organisms from other
living organism.
 Primary Metabolite: Primary metabolites are required for general
growth and physiological activity of plants because of their basic
metabolism.
 Eg: Amino acid, fatty acid, Nucleic acid, Carbohydrate and protein
 Secondary Metabolite: Secondary metabolites are derived
biosynthetically are from the primary metabolites but usually
restricted to specific taxonomically group. They may represent
chemical adaptations to environmental stresses or they may serve
as defensive or protective against microorganism, insect and higher
herbivorous predators. They are present in much smaller quantities.
Eg: Alkaloids, Glycoside, Volatile oil, Flavonoid, lignin, Carotenoid
etc.

24.  Oxidation
 Reduction
 Condensation
 Amination
 Methylation
 Cyclization etc.
 Basic metabolic pathways are:
The production of secondary metabolites is mostly
dependent on genetic make -up which are
selectively excel in the fundamental process
involved in their biosynthesis. These genetically
controlled processes can be termed as basic
metabolic pathways.

25.  The understanding of biosynthetic pathways depends on various
specialized techniques. They are five major techniques which are
generally used for the synthetic study of primary and secondary
metabolites. They are as follows.
 Tracer technique
 Use of isolating organs or tissues
 Grafting method
 Use of mutant strains
 Enzymatic studies
 Tracer techniques: In tracer technique, radioisotopes are frequently used
as tracers or tagged in various fields. Radioisotope is added to the
reactant and its movement is studied by measuring radioactively in draft
plants.
 Principle: Tracer technique which utilizes a labeled compound to trace or
find out the different intermediates and various steps involved in
biosynthetic process in plant at given rate and given time. When these
labeled compounds are administered in to the plants, they become a part

26.  Introduction; The elements existed with identical chemical properties but different
atomic weights are, termed as isotopes. Isotopes may be stable.
 Eg: 2H, 13C, ISN, 180. Nucleus may be unstable
 Eg: 1H, 14C and decay with emission of radiation. It is possible to detect these
isotopes by suitable methods. These isotopes can be incorporated in to a
presumed precursor of plant constituent and used as markers in biogenetic
experiment.
 Significance of tracer techniques: Tracing of biosynthetic pathway by
incorporating radioactive isotopes in to the precursor or starting material. Eg. By
incorporation of HC to phenylalanine, the biosynthesis of cyanogenetic
glycosides, prunacin can be traced. Location and quantity of the compound can
be determined in biological system.
 Different trace elements are used for different studies:
 1. For studies on protein, alkaloid and amino acid. Nitrogen atom gives more
specific information than carbon atom.
 2. For studies on glycosidic linkage: O, N, S and C atom.
 3. For studies on Terpenoids : O atom
 Basic steps involved in Tracer techniques:
 a. Preparation labeled compound.
 b. Incorporation of labeled compound to tissue system.
 c. Separation or isolation of labeled compound from tissue system.

27.  a) Precursor-product sequence
 b) Competitive feeding
 c) Sequential analysis
 d) Use of stable isotopes

28.  Precursor-product sequences:
In this method, the constituent is labeled it is fed
to the plant for specific period of time, The
constituents produced in plant are isolated and
purified and its radioactivity is determined Further
proof is needed because the labeled precursor
fed may not be the direct precursor.
In fact, the compounds may enter the general
metabolite pathway of plant and become
distributed randomly through a whole range of
products. Further evidence can be made by
double and triple labeling experiments by using
different isotopes or by one isotope at two or
more position in the moles. Ex: In Nicotiana
glayca two double labeled lysine's to determine
which hydrogen of the lysine molecule was
involved in the formation of the piperidine ring of
anabasine.

29.  Competitive feeding: In this method, the value
in determining which of two possible
intermediates is normally used by the plant.
Competitive feeding could distinguish
whether B or B' was the normal intermediate
in the formation of C from A.
Inactive B and B' are fed with labeled A to
separate groups of plants and a control is
performed by feeding labeled A only to
another group. If the incorporation of activity
into C is inhibited in the plants receiving B but
is unaffected in the group receiving B’. May
conclude that the pathway from A to C
proceeds via in B.

30.  Sequential analysis: A second method of investigation
with 14C is to grow plants in an atmosphere of 14CO2
and by analysis of the plants at given time interval to
obtain the sequence in which various related
compound become labeled. Degradation of the isolated
radioactive compounds is important because some
units of the molecule may become labeled more rapidly
than other. It is successfully used in the elucidation of
the path of carbon in photosynthesis and also for
determining the sequential formation of the opium,
hemlock, tobacco alkaloids. Exposure period of the
plant is short as 5mins. Eg: biosynthesis sequence in
Mentha piperata.

31.  Isolated organs and tissues: The cultivation of
isolated organs and tissue of plant elimination
interference from other parts of the plant which
may produce secondary changes in the
metabolites. Used for feeding experiment in
conjunction with labeled compound. Also it is
useful for the determination of the site of
synthesis of particular compounds.

32.  Grafts: Grafting techniques have been used in
biosynthetic studies for the determination of
the sites of primary and secondary metabolism
of secondary plant products. Alkaloid formation
by grafted plants has been extensively studied
in Nicotiana and tropane alkaloids are
producing solanaceae. Eg: Tomato scions
grafted on to Datura stocks are accumulation
of tropane alkaloid. Datura scions on tomato
stocks contain only a small amount of tropane
alkaloids. The main site of alkaloid synthesis is
to be the datura species.