Metabolic Pathway

1. Metabolic pathway in Higher
Plants and Their Determination
Harshita Jain
Assistant Professor
Career Point School of Pharmacy
Career Point University, Kota

2. Contents
 Introduction to metabolites
 Primary & secondary metabolite
 Metabolic pathway of Primary and secondary metabolites
 Shikimic Acid Pathway
 Acetate Pathway
 Utilization of radioisotopes
 Methods of tracer techniques

3. Introduction
Biosynthesis is a process of forming larger organic compounds
from small subunits within a living organism.
Metabolites are the organic compound synthesised by plants
using enzyme mediated chemical reactions through the
pathway known as Metabolic pathway.
These metabolites are of two types i.e., Primary metabolites
and Secondary metabolites.

4. Primary metabolites Secondary metabolites
These are synthesized through
primary metabolic pathway.
These are synthesized through
secondary metabolic pathway
Molecules that are essential for
growth and development of an
organism.
Molecules that are not essential for
growth and development of an
organism.
Therapeutically inactive Therapeutically active
Examples: Carbohydrates ,Proteins ,
Lipids, Nucleic acids , Hormones
Examples: Alkaloids, Glycosides ,
Terpenoids
Difference between primary and secondary metabolite

5. Metabolic pathways
• The series of reactions occurring with in the organism in an orderly and
regular basis known as metabolic pathways.
ABCD
(Primary metabolic pathway)
In this reaction
A – starting reactor’s
B, C – mediators reactor like different compounds, enzymes known as
metabolites
D- Product also known Metabolites.
Function
Primary metabolites are directly involved in plant metabolism, production,
growth and different physiological process.

6. Secondary metabolic pathways
• Secondary metabolic are proceed from primary Metabolites
• Production of Sec.metabolites poses different pathways for
different metabolites.
Example Shikimic acid pathway, mevalonic pathway, acetate
pathways

9. • Co2( from plants)+ H2O ( from root to plant) and leaf chlorophyll
pigments gives sun Ray’s n form photosynthesis process gives sugar
(Glucose ) form of metabolite.
• This sugar molecule to carbohydrates forms secondary Metabolites
glycosides
• From primary metabolite pentose phosphate pathways with enzyme
Erythose -4- phosphate gives Shikimic acid metabolites,phenyl propanoids,
flavonoids, tannins secondary Metabolites.
• From pyruvate to acetyl CoA gives acetate melonate pathway gives fatty
acids , lipids, waxes
• Acetyl CoA To mevalonic acid pathway forms secondary Metabolites
Terpenoids
• From primary Metabolites Tricarboxylic acid cycle with aliphatic amino
acid forms proteins alkaloids.

10. Shikimic acid pathway
The Shikimic acid pathway is a key intermediate from carbohydrate for the
biosynthesis of C6-C3 units (phenyl propane derivative).
The Shikimic acid pathway converts simple carbohydrate precursors
derived from glycolysis and the pentose phosphate pathway to the
aromatic amino acids.
The shikimate pathway is a 7 step metabolic route used by bacteria, fungi,
Algae, parasites, and plants for the biosynthesis of aromatic amino acids
(phenylalanine, tyrosine, and tryptophan).
This pathway is not found in animals; therefore, phenylalanine and
tryptophan represent essential amino acids that must be obtained from the
animal's diet.
Animals can synthesize tyrosine from phenylalanine, and therefore is not an
essential amino acid except for individuals unable to hydroxylate
phenylalanine to tyrosine).

14. Phosphoenolpyruvate and erythrose-4-phosphate react to form 2-keto3-
deoxy7phosphoglucoheptonic acid, in a reaction catalyzed by the enzyme DAHP
synthase.
2-keto3-deoxy7phosphoglucoheptonic acid is then transformed to 3-
dehydroquinate (DHQ), in a reaction catalyzed by DHQ synthase.
Although this reaction requires nicotinamide adenine dinucleotide (NAD) as a cofactor,
the enzymic mechanism regenerates it, resulting in the net use of no NAD.

15.  DHQ is dehydrated to 3-dehydroshikimic acid by the enzyme
3-dehydroquinate dehydratase, which is reduced to shikimic
acid by the enzyme shikimate dehydrogenase, which uses
nicotinamide adenine dinucleotide phosphate (NADPH) as a
cofactor.

16.  The next enzyme involved is shikimate kinase, an
enzyme that catalyzesthe ATPdependent phosphorylation
of shikimate to form shikimate 3-phosphate. Shikimate 3-
phosphate is then coupled with phosphoenol pyruvate to
give 5- enolpyruvylshikimate-3-phosphate via the enzyme
5-enolpyruvylshikimate-3- phosphate (EPSP) synthase.
Then 5-enolpyruvylshikimate-3-phosphate is transformed into
chorismate bya chorismate synthase.

17. Prephenic acid is then
synthesized by a Claisen
rearrangement of chorismate by
Chorismate mutase.
Prephenate is oxidatively
decarboxylated with retention of
the hydroxyl group by
Prephenate dehydrogenase to
give phydroxyphenylpyruvate,
which is transaminated using
glutamate as the nitrogen
source to give tyrosine and α-
ketoglutarate.

18. Role of shikimic acid pathway
Starting Point in The Biosynthesis of Some Phenolics Phenyl alanine and
tyrosine are the precursors used in the biosynthesis of phenylpropanoids.
The phenylpropanoids are then used to produce the flavonoids, coumarins,
tannins and lignin.
Gallic acid is formed from 3-dehydroshikimate by the action of the
enzyme shikimate dehydrogenase to produce 3,5- didehydroshikimate.
The latter compound spontaneously rearranges to gallic acid.
Other compounds
Shikimic acid is a precursor for:
Indole, indole derivatives and aromatic amino acid tryptophan and
tryptophan derivatives & many alkaloids and other aromatic
metabolites.

20. Acetate Pathway
• It is one of the most important metabolic pathway for synthesis of
secondary Metabolites.
• The pathways start with acetyl CoA so known as acetate pathway.
• From this acetate 2 pathways are form
(i) Acetate mevalonate pathway/ Isoprenoid pathway
(ii)Acetate melonate pathway
• This pathways are starting using end product of glycolysis cycle I.e
acetyl CoA its active form of acetate.

21. Glycolysis
Acetate mevalonate pathway
Pyruvat
e
Acetyl
CoA
Acetate melonate pathway
Two basic acetate pathways

23. Acetate Mevalonate Pathway
 The Acetate mevalonate pathway, also known as the isoprenoid
pathway or HMG-CoA reductase pathway is an essential
metabolic pathway present in eukaryotes, archaea, and some
bacteria.
 Acetate occupies central position in relation to general
metabolism, it gives straight chain compounds as well as
aromatic compounds viz., Terpenoids, steroids and fatty
acids.
 Acetyl co enzyme synthesized mevalonic acid , which is
important intermediate for synthesis of IPP-Isopentenyl
pyrophosphate, DMPP-Dimethyl allyl pyrophosphate which are
basic units for synthesis of terpenes.

24. 2 xAcetyl CoA Acetoacetyl CoA
Hydroxy methy CoA
(HMG CoA)
Mevalonate
5-
Phosphomevalon
ate
5- pyrophospho
mevalonate
Isopentyl pyrophosphate
(IPP) {C5 }
Dimethyl allyl pyrophosphate
(DMAPP) {C5 }
Geranyl pyrophosphate {C10 }
Farnesyl pyrophosphate {C15}
(FPP)
Geranyl geranyl pyrophospate {
C20 }
Squalene {C30 }
3
45
7
8
9
10
1
AcetylCoA
thiolase
2
HMG CoA
synthase
HMG CoA
reductase
Mevalonate
kinase
ATP -->ADP
phosphorylatio
n
5-pyrophosphate
mevalonate
decarboxylase
6
11
IPP
Isomerase
Condensatio
n
+IPP (C5)
+IPP (C5) +FPP (C15)

25. Steps involved in pathway

27. Products ofpathway

28. Mevalonic Acid
DMAPP IPP
Geranyl pyrophospate (C10)
Farnesyl pyrophosphate
(FPP) (C15)
Squalene (C30) Geranyl geranyl pyrophosphate
(C20)
Monoterpenes
(menthol, citral)
Sesquiterpenes
(Santelene,
Zingiberene)
+FPP +IPP
Steroids
(Diosgenin)
Triterpenes
Diterpenes(
Taxol)
Polyterpene(rubber)
Tetraterpene (C40)
(carotenoids)
Gibberellins
phytol
+IPP
x2

29. ROLE OF MEVALONIC ACID PATHWAY
Biosynthesis of farnesyl pyrophosphate.
Biosynthesis of Psoralens.
Biosynthesis of rubber.
Biosynthesis of diterpenoids.
Biosynthesis of Gernylgeranyl pyrophosphate.
Biosynthesis of cholesterol.
Biosynthesis if Gibberellin.

30. Acetate Malonate Pathway
Acetate
Malonate
Pathway
Acetyl co enzyme
synthesized melonic
acid , which is
important intermediate
for synthesis of Fatty
acids

33. Study of utilization of Radioactive Isotopes in
the Investigation of Biogenetic studies

34. Introduction
 Living plants considered as biosynthetic laboratory primary as well
as secondary metabolite.
 Different biosynthetic pathway:
» Shikmic acid pathway: Aromatic amino acids
» Mevalonic acid pathway: Terpenes
» Acetate Malonate pathway: Fatty acids
Biosynthesis: Formation of a chemical compound by a living organisms
Biogenesis: Production or generation of living organisms from other living
organisms

35. Isotopes-
Iso= Same (Equal), Topes=Place
• They occupy same place in periodic table. “Elements with same atomic number but different
atomic weight (Same number of protons but differ in neutrons).
13C6
• Example- 12C6
14C6 (Isotopes)
Atomic mass = No. of Protons+ No. of Neutrons
Atomic Number = No. of protons
Hydrogen Isotopes: 1H1
2H1
3H
1
[1P] [1P, 1N] [1P, 2
N]
Hydrogen Deuterium Tritium

36. Two types of Isotopes
1. Radioactive Isotopes (Radioisotopes)- Radio (Radiation ) + Isotopes Unstable Isotopes
The isotopes which emits the radiation are called Radioisotopes. Decay with the
emission of radiation (α, β, γ radiation).
Example - 3H, 14C, 35S, 131I, 24Na, 42K, 35S, 35P, 131I
- For biological Investigation- Carbon and Hydrogen
- For Metabolic studies- S, P and alkali and alkaline earth metals are used.
- For studies on proteins, alkaloids and amino acids-labelled nitrogen atom give more specific
information.
- 3H compound is commercially available.
2. Stable Isotopes:
• Stable isotopes are non-radioactive forms of atoms (they do not emit radiations).
• Although they do not emit radiations, their unique properties enable them to be used in a broad variety
of applications, including water and soil management, environmental studies, nutrition assessment
studies and forensics.
Examples- 2H, 13C, 15N, 18O
- Used for labelled compounds as possible intermediates in biosynthetic pathways
- Usual method of detection are: - MASS Spectroscopy [15N, 18O]
- NMR Spectroscopy [2H, 13Cl]

37. Radiolabelled Tracers (Radiolabelled compound)
• When one or more atom of chemical compound replaced by radioisotopes used-
for the study of the biosynthetic pathway, is known as Radiotracers.
Radiotracer Technique: The technique which utilises radioactive labelled compound
to find out or to trace various precursors and intermediates involved at different
stages of biosynthetic pathway at given rate and time.
In this technique, different isotope, mainly the radioactive isotopes which are
incorporated into the presumed precursor of plant metabolites and used as
marker in the biogenic studies.

38. Steps in Tracer Technique
1. Selection of Radioisotopes
2. Preparation of Radioisotopes
3. Introduction/Insertion of Radiolabelled compound in
biological system (Plant part)
4. Seperation and determination of labelled compound in
various biochemical reaction

39. (a) Growing chlorella in atmosphere of 14CO2
(b) Nuclear Reactor/Acceralator
14N7 + 1n0
14C6 + 1P1
c) Tritium gas: Tritium labelled compound (3H1) are commercially available.
Tritium labelling is effected by catallytic exchange in aqueous media by
hydrogenation of unsaturated compound with tritium gas.
d) by the use of Organic Synthesis:
CH3MgBr + 14CO2 CH3
14COOHMgBr + H2O
Preparation of labelled compound
CH3
14COOH+ MgOHBr
Radiolabelled Acetic acid

40. Insertion of Radiolabelled compound in plant part
• The precursor should react at necessary site of synthesis in plants and also on correct time
• Plant at the experiment time should synthesize the compound under
investigation.
• The dose given is for short period.
1. Root feeding: When the root is biosynthetic site (Ex- Tobacco and Datura alkaloid).
2. Stem Feeding: Cut end of stem immersed in water, nutrient and
radiolabelled compound.
3. Direct Injection: Which have hollow stem (Umbelliferous fruits).
4. Infilteration: It is also called Wick Feeding method.
5. Floating method: Substrate solution which contain Radioactive compound.
6. Spray technique

41. Separation or Isolation of Radiolabelled compound and
detection of radioisotope labelled compound
Depends on Nature of drugs and Sources of drugs-
Soft tissue- Infusion, Maceration
Hard Tissue- Decoction, hot percolation
Unorganised drugs- Maceration
Alkaloid, Glycoside, Flavonoids- Slightly polar solvent

42. (a) Geiger –Muller Counter- Detection and measurement of all types of radiation
(b) Liquid Scintillation Counter – Scintillators are used (For β emitting radioisotopes)
(c) Gas Ionization Chamber
(d) Bernstein-Bellentine Counter
(e) Mass spectroscopy
(f) NMR Electrodemeter
(g) Autoradiography – to trace the location of radioactive isotope in biological
system
(h) Radio paper chromatography
Detection and assay of Radioactive labelled compound

43. Geiger – Muller counter
 A Geiger counter (Geiger-Muller tube) is a device used for the
detection and measurement of all types of radiation: alpha, beta
and gamma radiation.
 Basically it consists of a pair of electrodes surrounded by a gas. The
electrodes have a high voltage across them. The gas used is usually
Helium or Argon. When radiation enters the tube it can ionize the
gas. The ions (and electrons) are attracted to the electrodes and an
electric current is produced.
 A scaler counts the current pulses, and one obtains a "count"
whenever radiation ionizes the gas.

44. Liquid Scintillation
counter
Scintillations literal meaning is luminescence
and scintillators are material which have
property to luminate.
When ionizing particles colloids on scintillating
material they absorbs its energy and exhibits
scintillation ( i.e. re-emit the absorbed energy in
the form of light)
Light strikes on photosensitive surface, leads to
release of photoelectron and multiplication
through the series of photo multiplier
tubes(PMT).
Light converts into an electrical signal.

45. Autoradiography
Autoradiography is the bio-analytical technique used to visualize the distribution radioactive
labelled substance in a biological sample. It is a method by which a radioactive material can be
localized within a particular tissue, cell, cell organelles or even biomolecules.
In this method, the specimen under study is kept in contact with a suitable photographic emulsion
such as silver bromide gel or X-ray sensitive film for suitable time period.
After the film is developed in the usual manner and results are correlated with darkening of
the film.

46. Methods in Tracer Technique
1. Precursor – Product sequence
In this technique, the presumed precursor of the constituent under
investigation on a labelled form is fed into the plant and after a
suitable time the constituent is isolated, purified and radioactivity
is determined.
Disadvantages:
The radioactivity of isolated compound alone is not usually
sufficient evidence that the particular compound fed is direct
precursor, because substance may enter the general metabolic
pathway and from there may become randomly distributed
through a whole range of product.

47. 2. Double and Multiple Labelling:
This method give the evidence for nature of biochemical
incorporation of precursor arises double and triple labelling. In
this method specifically labelled precursor and their subsequent
degradation of recover product are more employed.
Application:
1. This method is extensively applied to study the biogenesis of
plant secondary metabolite.
2. Used for study of morpheine alkaloid.

49. 3. Competitive Feeding:
If incorporation is obtained it is necessary to consider whether
this infact, the normal route of synthesis in plant not the
subsidary pathway. Competitive feeding can distinguish whether
B and B’ is the normal intermediate for the formation of C from
A.
Application:
This method is used for the biogenesis of propane alkaloids.
Biosynthesis of hemlock alkaloids (conline, conhhydrine, etc) e.g.
biosynthesis of alkaloids of Conium manuculatum (Hemlock)
using 14C labelled compounds.

50. Sequential Analysis
The principle of this method of investigation is to grow plant in
atmosphere of 14CO2 & then analyze the plant at given time
interval to obtain the sequence in which various correlated
compound become labelled.
Application: -
14CO2 & sequential analysis has been very successfully used
in elucidation of carbon in photosynthesis.
Determination of sequential formation of opium hemlock and
tobacco alkaloids.
Exposure as less as 5 min. 14CO2, is used in detecting
biosynthetic sequence as –
Piperitone --------- (-) Menthone ---------- (-) Menthol in Mentha
piperita.

52. 1. Study of squalene cyclization by use of 14C, 3H labelled mevalonic acid.
2. Interrelationship among 4-methyl sterols and 4,4-dimethyl sterols, by use of 14C
acetate.
3. Terpenoid biosynthesis by chloroplast isolated in organic solvent by use of 2-14C
mevalonate.
4. Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin.
5. Origin of carbon and nitrogen atoms of purine ring system by use of 14C or 15N labelled
precursor.
6. Study of formation of scopoletin by use of labelled phenylalanine.
7. By use of 45Ca as tracer, found that the uptake of calcium by plants from the soil (CaO
and CaCO2).
8. By adding ammonium phosphate labelled with 32P of known specific activity the uptake
of phosphorus is followed by measuring the radioactivity as label reaches first in lower
part of the plant, than the upper part i.e. branches, leaves, etc.
Applications of Tracer Technique