By Assoc.prof.dr.mohd noor abd.

wahab

Gluconeogenesis

Synthesis of glucose from pyruvate utilizes many

of the same enzymes as Glycolysis.
Occurs in cytoplasm (when only nonsugar C
source is supplied.)
*Reversal of Glycolysis process.

Gluconeogenesis

Three Glycolysis reactions have such a large

negative G that they are essentially
irreversible.
Hexokinase (or Glucokinase)
Phosphofructokinase
Pyruvate Kinase.
Therefore,these 3 steps must be bypassed
in Gluconeogenesis. Others reactions use the
same enzyme as in Glycolysis.

Gluconeogenesis

Gluconeogenesis

glucose
Pi

Gluconeogenesis

Glucose-6-phosphatase

H2O
glucose-6-phosphate

Phosphoglucose Isomerase
fructose-6-phosphate
Pi
Fructose-1,6-bisphosphatase
H2O
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
(continued)

Glucose-6-phosphatase

6 CH OPO 2
2
3
5
O

H
4

OH

H
OH
3

H
2

OH

CH2OH
H
1

OH

glucose-6-phosphate

H2O

O
H
OH

+ Pi

H
OH

OH
H

glucose

OH

Hexokinase or Glucokinase (Glycolysis) catalyzes:

glucose + ATP glucose-6-phosphate + ADP
Glucose-6-Phosphatase (Gluconeogenesis)
catalyzes:
glucose-6-phosphate + H2O glucose + Pi

Gluconeogenesis

glucose
Pi

Gluconeogenesis

Glucose-6-phosphatase

H2O
glucose-6-phosphate

Phosphoglucose Isomerase
fructose-6-phosphate
Pi
Fructose-1,6-bisphosphatase
H2O
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
(continued)

Phosphofructokinase
6 CH OPO 2
2
3

1CH2OH

H
4

OH

ATP

HO

ADP

3 OH

fructose-6-phosphate

6 CH OPO 2
2
3

Pi

H2O

1CH2OPO32

HO

3 OH

OH

fructose-1,6-bisphosphate

Fructose-1,6-biosphosphatase

Phosphofructokinase (Glycolysis) catalyzes:

fructose-6-P + ATP fructose-1,6-bisP + ADP
Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes:
fructose-1,6-bisP + H2O fructose-6-P + Pi

Gluconeogenesis

glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
CO2 + GDP

PEP Carboxykinase

GTP
oxaloacetate
Pi + ADP
HCO3

+ ATP
pyruvate

Pyruvate Carboxylase
Gluconeogenesis

Bypass of Pyruvate Kinase:

Pyruvate Kinase (last step of Glycolysis) catalyzes:
phosphoenolpyruvate + ADP pyruvate + ATP
For bypass of the Pyruvate Kinase reaction,
cleavage of 2 ~P bonds is required.
G for cleavage of one ~P bond of ATP is
insufficient to drive synthesis of
phosphoenolpyruvate (PEP).
PEP has a higher negative G of phosphate
hydrolysis than ATP.

Gluconeogenesis

Pyruvate Carboxylase

PEP Carboxykinase

O
C

O
C

ATP ADP + Pi

C
O

CH3

GTP GDP

C
C

CO2

C
O

pyruvate

CH2

HCO3

oxaloacetate

OPO32

CH2

PEP

Bypass of Pyruvate Kinase (2 enzymes):

Pyruvate Carboxylase (Gluconeogenesis) catalyzes:
pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes:
oxaloacetate + GTP PEP + GDP + CO2

Gluconeogenesis

Pyruvate
Carboxylase
(pyruvate
oxaloactate)
is allosterically
activated by
acetyl CoA.

Glucose-6-phosphatase
glucose-6-P
glucose
Gluconeogenesis

Glycolysis
pyruvate
fatty acids
acetyl CoA

ketone bodies

oxaloacetate
citrate
[Oxaloacetate]
tends to be
Krebs Cycle
limiting for
Krebs cycle.
When gluconeogenesis is active, oxaloacetate is diverted
to form glucose. Oxaloacetate depletion hinders acetyl
CoA entry into Krebs Cycle. The increase in [acetyl CoA]
activates Pyruvate Carboxylase to make oxaloacetate.

PEP Carboxykinase Reaction

C
C

CH2
C
O

C
C
CO2

GTP GDP O

CH2

oxaloacetate

O
C
C

OPO32

CH2

PEP

PEP Carboxykinase catalyzes ATP-dependent

oxaloacetate PEP. It is thought to proceed in 2
steps:

Oxaloacetate is first decarboxylated to yield a pyruvate

enolate anion intermediate.

Phosphate transfer from ATP then yields

phosphoenolpyruvate (PEP).

In the bacterial/ fungi enzyme,

ATP is Pi donor instead of GTP.
In this crystal structure of
Saccharomyces cerevisiae PEP
Carboxykinase, pyruvate is at
the active site as an analog of
PEP/ oxaloacetate.

A metal ion such as Mn2+ is required for the PEP

Carboxykinase reaction, in addition to a Mg2+ ion that
binds with the nucleotide substrate at the active site.
Mn2+ is thought to promote Pi transfer by interacting
simultaneously with the enolate oxygen atom and an
oxygen atom of the terminal phosphate of GTP or ATP.

The source of pyruvate and

oxaloacetate for gluconeogenesis during
fasting or carbohydrate starvation is
mainly glycogen.
Some amino acids are catabolized to
pyruvate, oxaloacetate, or precursors of
these.
Glycerol, derived from hydrolysis of
triacylglycerols in fat cells, is also a
significant input to gluconeogenesis.

Summary of
Gluconeogenesis
Pathway:
Gluconeogenesis
enzyme names in
red.
Glycolysis
enzyme names in
blue.

glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
CO2 + GDP

PEP Carboxykinase

GTP
oxaloacetate
Pi + ADP
HCO3 + ATP
pyruvate

Pyruvate Carboxylase
Gluconeogenesis

glucose
Pi

Gluconeogenesis

Glucose-6-phosphatase

H2O
glucose-6-phosphate

Phosphoglucose Isomerase
fructose-6-phosphate
Pi
Fructose-1,6-bisphosphatase
H2O
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
(continued)

Glycolysis & Gluconeogenesis are both spontaneous.

If both pathways were simultaneously active in a cell, it
would constitute a "futile cycle" that would waste energy.
Glycolysis:
glucose + 2 NAD+ + 2 ADP + 2 Pi
2 pyruvate + 2 NADH + 2 ATP
Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP
glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
Questions:
1. Glycolysis yields how many ~P ? 2
2. Gluconeogenesis expends how many ~P ? 6
3. A futile cycle of both pathways would waste how many
~P per cycle ? 4

Phosphofructokinase
6 CH OPO 2
2
3

1CH2OH

H
4

OH

ATP

HO

ADP

3 OH

fructose-6-phosphate

6 CH OPO 2
2
3

Pi

H2O

1CH2OPO32

HO

3 OH

OH

fructose-1,6-bisphosphate

Fructose-1,6-biosphosphatase

To prevent the waste of a futile cycle,

Glycolysis & Gluconeogenesis are reciprocally
regulated.
Local Control includes reciprocal allosteric
regulation by adenine nucleotides.

Phosphofructokinase (Glycolysis) is inhibited by ATP and

stimulated by AMP.
Fructose-1,6-bisphosphatase (Gluconeogenesis) is
inhibited by AMP.

The opposite effects of adenine nucleotides on

Phosphofructokinase (Glycolysis)
o Fructose-1,6-bisphosphatase
(Gluconeogenesis)
o

insures that when cellular ATP is high (AMP would

then be low), glucose is not degraded to make ATP.
When ATP is high it is more useful to the cell to store
glucose as glycogen. >> Gluconeogenesis
When ATP is low (AMP would then be high), the cell
does not expend energy in synthesizing glucose.
>>

Glycolysis

GLOBAL
CONTROL

TRANSLOCATION
AND STORAGE
COMPOUND

Environment rarely contains favorable

and ideal condition of nutrients for fungal
to growth.
Storage of excess nutrients enables
fungi to survive

STORAGE COMPOUND
Storage requirements of growth takes place in all
hyphae components
Hyphae fragment function as storage
Protection from mechanism :
thick wall
protective compound
hydrophobic surface
complex structure (starvation)

STORAGE COMPOUND
Differ from those of plants or bacteria but are similar
to those of animals.
The main storage compound are :- Lipid
- Glycogen ( linked polymer of glucose)
- Trehalose (non reducing disaccharides)

Trehalose, composed of 2 glucose residue

Lipid
-

Often seen as globules in fungal cell

Up to 40% of cytoplasm made up of lipid

Oil drop can de seen in most hyphae of old

compartments
-

Glycerol is common and important in water

regulation and metabolic activity in fungi

Lipid
-Translocation of lipid was bi directional in the
same hyphae
-Lipid translocated all direction in the FMU
(Functional Myceluim Unit)
-Part of mechanism for redistributing energy in
the FMU
* FMU = integrated hyphae forms an individualistic organisms

CARBOHYDRATES
Main translocated carbohydrates
- Trehalose
- Polyols ( sugar alcohol)
Oomycetes absence of the characteristic
fungal carbohydrates

CARBOHYDRATES
TREHALOSE
- Derived from common sugars taken from the cells
- Eg :-

# Trehalose = 2 glucose compound

# Mannitol = Fructose

- Readily interconverted in hyphae

- Trehalose = mycelium
- Trehalose + arabitol = colony margin

CARBOHYDRATES
TREHALOSE
- important in resistance against adverse condition ;
cold, heat, dehydration and etc.
- synthesis and degradation highly regulated

NITROGEN
- stored as protein
- abundant at beginning of degradative cycle
- lectin found freely in cytoplasm
- lectin highly conserved nitrogen storage proteins

TRANSLOCATION OF
STORAGE COMPUND

TRANSLOCATION IN FUNGI
There are 4 different mechanisms for nutrient
translocation in fungi :# Passive
# Passive + active uptake
# Active, cytoplasmic
# Active, pressure driven bulk flow

PASSIVE
Not required extra energy
Nutrients taken by hyphae by simple diffusion
as in water phase
In non- saturated soil filled with air gaps,
hyphae forms bridges and nutrients can spread
via symplastic or apoplastic diffusion
Eg : Glucose taken at hyphae tips and trehalose
diffuse down the [ ] gradient away from hyphae
tips

PASSIVE + ACTIVE UPTAKE

Similar to passive but nutrients taken in excess
of local needs
Produced steep nutrient gradient inside hyphae
Result faster delivery of nutrients to mycelium
Requires extra energy for the excess uptake

ACTIVE, CYTOPLASMIC
Active movement in cytoplasm either through:
- movements of organelles
- peristaltic vacuole system
Mechanisms involve compartmentalization of
nutrients followed by peristaltic movements in
tubular compartment
Storage compound packaged in vesicles which
moved at hyphae
Action of cytoskeleton significant in movement
of vesicles along hyphae

ACTIVE, PRESSURE DRIVEN BULK

FLOW

If excess nutrients are taken, it result in a high

osmotic pressure inside hyphae, water will flow
through cell membrane
This will create high turgor pressure and water
& nutrient will flow inside mycelium with less
resistance
Energy are use for active uptake and overcome
water flow within hyphae
Formation of fruiting body is the evidence of
bulk flow seen as exudation of water droplet at
the surface

Bidirectional transport shown that :-

HOST

Organic carbon

HYPHAE TIPS

Mi
ner
als

ROOT

BIOSYNTHESI
S OF FUNGI

What is biosynthesis?
The production of
needed cellular
compounds, usually
from simpler
molecules

In fungi, the most important

compound to synthesis :
CHITIN

CHITIN
long-chain polymer of a N-acetylglucosamine, a
derivative of glucose, and is found in many places
throughout the natural world. It is the main
component of the cell walls of fungi.
modified polysaccharide which contains
nitrogen; it is synthesized from units of Nacetylglucosamine (more precisely, 2-(acetylamino)2-deoxy-D-glucose).
Linked by (1-4) bonds.

described as cellulose with one hydroxyl group on each

monomer substituted with an acetyl amine group.

This allows for increased hydrogen bonding between

adjacent polymers, giving the chitin-polymer matrix
increased strength.
The basic unit is derived from fructose-6-phosphate by
the addition of an amine and an acetyl group from the
amino acid glutamine and acetyl CoA, respectively.

The chitin adds rigidity and structural support to the

thin cells of the fungus, and gives protection as well.

CHITOSAN

Chitosan is a modified carbohydrate polymer

derived from the chitin component.
Chitosan is a linear polysaccharide composed of
randomly distributed -(1-4)-linked D-glucosamine
(deacetylated unit) and N-acetyl-D-glucosamine
(acetylated unit).
Poorly or non acetylated form of chitin.

 Chitosan

is obtained by removing enough

acetyl groups (CH3-CO) for the molecule
to be soluble in most diluted acids.

This

process, called deacetylation.

SYNTHESIS OF CHITIN

First combined with a donor molecule, to provide the

energy required for synthesis.

UTP + N-acetylglucosamine
acetylglucosamine + Pi

Second, UDP-N-acetylglucosamine combine with poly-Nacetylglucosamine to form UDP + chitin.

(Donor +sugar unit) + acceptor

(acceptor+ sugar unit)

UDP-N-

donor +

CHITIN SYNTHASE

Exist in zymogen form then converted into functional

enzyme by partial proteolysis with protease enzyme.
In this state, functional enzyme is formed and readily
to work.
Occur in particles, termed chitosomes.
Small spheroidal bodies, 40-70 nm
surrounded by a shell. (about 7nm thick).

diameter,

CONT

If presence of an activator (proteolytic enzyme) and

substrate N-acetylglucosamine, chitosome now can
formed chitin microfibril by breaking the shell.
As microvesicles in hyphal tips.
Extracted from members of all the main chitincontaining fungal groups:
(i) Allomyces
(ii) Mucor
(iii) Saccharomyces
(iv) Neurospora
(v) Agaricus.

SECONDARY
METABOLISM
IN FUNGI

Refers to a wide range of metabolic

reactions whose products are not directly
or obviously involved in normal growth.
Features:

Tend to produced at the end of the

exponential growth phase in batch culture or
when growth is substrate-limited in
continuous culture

Produced from common

metabolic intermediates but by
special enzymatic pathways
encoded by specific genes
Not essential for growth or
normal metabolism

Important for commercial or environmental

significance
Penicillins (from Penicillium chrysogenum)
Griseofulvin (from P.griseofulvum)-antibiotics produced
commercially from fungi
Carotenoid pigments in conidia fungi such as
Neurospora crassa
Gibberellins-plant hormone for horticulture

Many secondary metabolites have no obvious roles in

the life of the producer organism, and mutated strains
that do not produce this compound often grow as well
as the wild-type strains in culture.
Instead of that,secondary metabolism acts as an
overspill or escape valve,to remove intermediates from
the basic metabolic pathways when growth is
temporarily restricted.

The Pathway and

Precursors of
Secondary
Metabolism

A few key intermediates of the basic metabolic

pathways provide starting points for the pathways
of secondary metabolism.
The single most important secondary metabolic
pathway is the polyketide pathway.
The precursor is acetyl-Coa which is carboxylated
to form malonyl-Coa,then three or more molecules
of malonyl-Coa condense with acetyl-Coa to form
chains.

This chains undergo

cyclization,then the ring systems are
modified to give a wide range of
products such as anti biotic
griseofulvin(treat dermatophyte
infections)
Another important secondary
metabolic pathway of fungi is the
isoprenoid pathway for the synthesis
of sterols.

Acetyl-Coa is the precursor,but three

molecules of this condense to form
mevalonic acid(6-C) which is
converted to a 5-C isoprene unit.
The isoprene units condense headto-tail to form chains undergo
cyclization and further modifications.
The product in this pathway include
mycotoxins of Fusarium spp.that grow
on moist grain such as T-2 toxin and
the trichothecenes.

EXAMPLE OF SECONDARY
METABOLITES
Penicilins

Mycotoxins
Ergot

Alkaloids
Aflatoxins
Sporidesmin
Patulin
Roquefort Cheese

PENICILLINS

Was discovered by Alexander Fleming in 1929 as a metabolite

of Penicillium chrysogenum .
Most active against Gram-positive bacteria. It prevent the
cross- linking of peptides during the synthesis of
peptidoglycan layer in bacteria cell wall making it weak and
susceptible to osmotic lysis.

MYCOTOXINS
Diverse range of compounds from different
precursors and pathway.
Cause toxicity when humans ingest them over a
relatively long period of time, from low
concentrations in improperly stored food.
The problem to detect this toxins is it may take
years before the effect of exposure become evident.
So to avoid this problem we must storage our food in
its proper place.

OTHER TOXINS
Toxin

Representative

Effects

Ergot alkaloids

fungi
Aspergillus flavus and
A. parasiticus
Claviceps purpurea

Foodstuff

Nephrotoxic,
Hepatocarninomas
Nuerotoxic

Fuminosins

Fusarium moniliforme

Peanuts,
oilseeds
Cereals,
grasses
Maize

Ochratoxin A

Some Aspergillus and

Penicillium spp.

Grain crops

Patulin

Penicillium expansum
and Aspergillus
clavatus
Pithomyces chartarum

Apples

Contact edema and

hemorrhage

Grass

Aspergillus spp.

Grain, Oilseeds

Facial eczema of sheep

and cattle
Hepatocarcinogen

Aflatoxins

Sporidesmin
Sterigmatocysti
n

Human esophageal
cancer
Nephrotoxic and kidney
carcinoma

STRUCTURE OF SOME TOXINS

Ergot
alkaloid
s

Aflatoxi
ns

Sporidesmin

ROQUEFORT CHEESE
Roquefort

cheese and other blue-veined cheeses are

produced from goat milk that are inoculated with
the fungus Penicillium roqueforti.

The

cheese contain low levels of the mycotoxin

roquefortine but these levels are not considered to
be hazardous.

The end