HMP( PPP) Pathway
The hexose monophspate or the pentose phosphate pathway is an active
pathway in:
1- Fat synthesizing tissues such as: liver & adipose tissue.
2- Steroids synthesizing tissues such as: gonads (testis & ovaries) &
Adrenal gland.
3- RBCs (for fighting oxidants).
The pathway is composed of two phases:
1- Oxidative phase (irreversible)
2- Non-oxidative phase (reversible)
-Oxidative phase:
Glucose-6-phosphate (g-6-p) undergoes oxidation by glucose-6phosphate dehydrogenase (G6PD) & the first NADPH is produced + a
compound named 6-phosphogluconate.
Then 6-phosphogluconate undergoes further oxidation by
6-phosphogluconate DH where we have the second NADPH being
produced, CO2 is released & we end up with a 5C sugar Ribulose-5-p (a
pentose).
At the end of this phase we achieved the production of 2 NADPH & a
pentose.
Note: G6PD is the rate limiting enzyme for the entire pathway .its
inhibited by excess NADPH.
-Non-Oxidative phase:
in this phase we'll see how the pentose Ribulose-5-p will be converted
back to a hexose . The ribulose-5-phosphate acts as a substrate for two
different enzymes an epimerase and an Isomerase.
1- Ribulose-5-phosphate isomerase
The isomerase converts the ketopentose ribulose-5-phosphate to the
aldopentose ribose-5-phosphate which is the precursor for all
Nucleotide synthesis.
2- Ribulose-5-phosphate epimerase
Ribulose-5-phosphate epimerase converts ribulose-5-phosphate to its
epimer xylulose-5-phosphate.
Then the 2 products Ribose-5-p & xylulose-5-p go interconversion
reaction by a Transketolase ( an enzyme that catalyzes the transfer of
2 carbons reactions , needs TPP as a cofactor) it transfers two carbons
from xylulose-5-phosphate to ribulose-5 phosphate to release the
Seven-carbon sedoheptulose-7-phosphate. The remaining carbons from
the xylulose-5-phosphate are released as glyceraldehyde-3-phosphate.
Transaldolase
Transaldolase catalyzes a three-carbon transfer from sedoheptulose-7phosphate to glyceraldehyde-3-phosphate, leaving erythrose-4-phosphate,
And forming fructose-6-phosphate.
After that erythrose-4-phosphate reacts with ANOTHER molecule of
xylulose-5-p by a Transketolase to produce fructose-6-phosphate &
glyceraldehyde-3-phosphate.
Which means we needed 3 molecules of Ribulose-5-p (one to produce the
xylulose-5-p, a second to produce Ribose-5-p and a third to produce a
second xylulose-5-p).
The net reaction of the reversible phase :
3 ribulose-5-phosphate → 1 ribose-5-phosphate + 2 xylulose-5phosphate → 2 fructose-6-phosphate + glyceraldehyde-3-phosphate .
The whole pathway net reaction:
3glucose-6-phosphate + 6 NADP→ 3 CO2 + 2 glucose-6-phosphate
Glyceraldehyde-3-phosphate + 6 NADPH
Notes:
-This pathway could be used to produce a pentose from a hexose that’s
why it's called (PPP) or to produce hexoses (that’s why it's named HMP
shunt pathway) what really directs the starting point is the cellular need.
- The main function of this pathway is the production of NADPH and
pentoses.
-NADPH:
NAD and NADP are very similar molecules. The only difference is the
Phosphate on the 2´-position of the adenosine ribose of NADP instead of
the free hydroxyl at this position in NAD. The cellular concentration of
NADPH is much higher than the concentration of NADP. NADPH gets
oxidized (it donates electrons for synthetic reactions)
While NADP really stands by to accept electrons in catabolic processes
for the production of energy.
NADPH is used for the reduction of oxidizing substances that we are
always exposed to & these are:
1-environmental toxins.
2-smoking.
3-drugs.
4-oxidants that are produced by aerobic metabolism / reactive oxygen
intermediates or species (ROS) which include:
1) Hydrogen peroxide (H2O2).
2) Superoxide ( O2− ).
3) Hydroxyl free radical OH •
Note:
a free radical is any atom with at least one unpaired electron in the
outermost orbit , these are formed when an atom donates a single electron
to another atom producing 2 atoms each one of them will be considered
as a free radical that are highly reactive.
ROS are damaging compounds. They damage the DNA, proteins &
unsaturated lipids producing variety of diseases that affect all of the body
systems such as the nervous system, the GI & the respiratory system , and
they (ROS) contribute to complication of many other diseases.
Defensive mechanisms against ROS:
-SOD (superoxide dismutase ) is an enzyme that converts superoxide ion
to H2O2 & O2 , then H2O2 is neutralized by 2 other enzymes Catalase
and Glutathione peroxidase .
Glutathione peroxidase oxidizes GSH to neutralize the superoxide since
GSH (Glutathione, reduced form) is considered a major scavenger
(Antioxidant) compound against oxidant, it needs to be regenerated /
reduced back by an enzyme GSH reductase.
Other Antioxidants are Vitamins like E, C and carotenes (the precursors
of vitamin A).
NADPH Functions:
1- Reductive biosynthesis like steroids synthesis in testis, ovaries placenta
& adrenal cortex in addition to fat synthesis in liver, lactating mammary
glands and adipose tissue.
2-detoxification and fighting oxidants (required by RBCs for maintenance
of GSH).
3-synthesis of neurotransmitters.
4-fat chains elongation.
5-used by Cytochrome p450 mono-oxygenase for hydrolyzation of
steroids & detoxification of foreign compounds.
6- Used by PPP/HMP pathway for the production of Ribose-5-p the
precursor for all nucleotides synthesis.
Glucose-6-phosphate dehydrogenase deficiency
G6PD is the rate limiting enzyme that catalyzes the irreversible step in
PPP/HMP pathway. It's inhibited by an excess of NADPH.
Prevalence & geographic distribution
G6PD enzyme has the highest incidence of deficiency in the world (200400) million individuals are deficient in this enzyme. The gene of this
enzyme is an X-linked which means that males are more affected.
Middle East, tropical Africa & southern Asia people along with
Mediterranean have the highest incidence of the deficiency.
-A G6PD deficient will live normally until he or she is exposed to
oxidants (drugs, toxins, certain food like Fava beans (fool a59'ar) ) then
he or she will suffer from hemolytic anemia .
Note: every favism (someone who has an allergy to fava beans) is a
G6PD deficient but not every G6PD deficient is favism .
-NEVER describe an oxidant drug for a favism or G6PD deficient, but
Why?
It's well known that G6PD enzyme produces NADPH that will be utilized
to reduce glutathione that was oxidized to neutralize the oxidizing
substances that resulted from oxidizing stresses such as: drugs, ingesting
of certain food like fava beans or the process of fighting an infection.
a deficiency of the G6PD will result in reduced NADPH which will affect
the body as following:
- The Macrophages cellular defense line is responsible for ingulfing
foreign antigens & producing ROS to degrade the foreign substances. But
if the body lacked a good mechanism to neutralize these macrophageal
ROS (because of reduced NADPH, which is needed for regeneration of
GSH that neutralizes these ROS's) the surrounding tissues and cells will
be affected and among these cells is the RBC in which hemoglobin will
be oxidized and polymerized in the form of Heinz bodies which will lead
eventually to shortening of RBCs half life followed by its hemolysis.
- People who are G6PD deficient are provided with natural resistance to
malaria parasite which lives inside the RBC. Because the life span of the
RBC in those people is reduced (less than 120 days) which affects the life
cycle of this parasite. This leads to a high incidence of G6PD deficiency
in areas where malaria is common, because those people are naturally
protected from malaria which helps to preserve the gene that’s G6PD
deficient but malaria resistant.
G6PD Variants:
The normal G6PD enzyme is designated with the letter B. This enzyme
has a variety of variants which indicates that the gene could be affected in
different locations and differs from one family to another.
Mediterranean variant (classII) is the most prevalent among G6PD
variants it’s a polymorphic variant that has a remaining residual activity
less than 10%. Nucleotides that are affected here are located on the long
arm of x chromosome specifically on position 563 (Exon 6) and here
cytosine is converted into thiamine so we have single base substitution
that converts a serine into phenylalanine.
- Mediterranean variant is designated (B-) because in electrophoretic
migration it migrates to the same extant as the normal B designated
variant, the minus indicates its deficiency
Africans / black people have a variant known as A2 variant (classIII)
G6PD was classified into many classes because of the many variants that
it has, these are classes I-IV
Class I has the least incidence (rare) with a remaining residual activity
less than 2%, people who are class I deficient suffer from a severe
chronic hemolysis.
Class II has the highest incidence among our population with a residual
activity less than 10% still there is enough activity if the individual was
kept away from oxidants.
Class III high incidence in Africa , with a remaining residual activity
ranges from 10%-20% it requires a 2 point mutations the first substitutes
Asparagine with Asparatate & the second substitutes Valine with
methionine ( this type is designated A-) . If only one of these mutations
occurred and the other didn’t we'll end up with a variant named The
Normal African Variant with a remaining residual activity reaches 80%
(Normal, Asymptomatic).
The book illustrates the differences in heat stability of the Enzyme
Variants. As we learned from the previous Biochemistry course enzyme
deficiency is expressed by
1- Insufficient amounts of the enzyme.
2-abnormal properties (a very high Km, a low Vmax or heat instability)
Both the A2 & the Mediterranean variants have low heat stability.
Comparing the activity profile with the age of the RBC has shown that
young RBCs have high activity of (A-) that reaches 80% (they are
deficient but active). that’s why sometimes an A- variant with hemolytic
anemia could be misdiagnosed if the blood sample was full of young
RBCs ( because old RBCs are already dead because of hemolysis) that
will show a normal activity of the enzyme that’s why the test should be
repeated after 2 weeks for a better diagnose .
Role of G6PD in RBC's
as we mentioned earlier G6PD is used to produce NADPH which is
needed for regeneration of GSH (important antioxidant, reduces organic
& inorganic peroxides) and it’s the only enzyme in RBC's that produces
NADPH so its deficiency effect on RBC's is massive (hemolysis).
Another enzyme that’s used in lipid metabolism also produces NADPH
(but not found in RBC's) is Malate dehydrogenase.
precipitation factor in G6PD deficiency
Few individuals show chronic hemolysis (residual activity is less than
2%) clinical manifestations shows that most individuals develop
hemolytic anemia if the were treated with oxidant drugs, ingested
fava beans because it contains a glycosylated glycine oxidant in
addition to severe infection that might cause hemolysis.
G6PD & Neonatal Jaundice
It has been found that children who have G6PD6 deficiency have a higher
incidence of getting neonatal jaundice than normal children.
Molecular biology of G6PD deficiency
More than 300 different locations for the mutations that cause the
deficiency have been found.
- the deficiency is caused by Point Mutation not a frame shift or a large
deletion mutations , because it has been found that frame shift
mutations completely inactivate the enzyme and it seems like life
isn’t compatible with the complete absence of G6PD activity.
"If you can't fly then run, if you can't run then walk , if you can't walk
then crawl, but whatever you do you have to keep moving forward"
:P ‫–الحج مارتن لوثر‬
If you have any question regarding this sheet don’t hesitate to ask me, but
don’t expect an answer :P