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UNIT II:
Intermediary Metabolism
Pentose phosphate pathway
and NADPH
Figure 13.1. Hexose monophosphate pathway shown as a component of the
metabolic map
Overview
• The pentose phosphate pathway (a.k.a, hexose
monophosphate shunt, or 6-phosphogluconate pathway)
occurs in cytosol of the cell
• It consists of two, irreversible oxidative reactions, followed by
a series of reversible sugar-phosphate interconversions.
• No ATP is directly consumed or produced in the cycle.
Carbon one of G-6-P is released as CO2, & 2 NADPH are
produced for each G-6-P molecule entering the oxidative
part of the pathway.
• The rate & direction of the reversible reactions of the
pathway are determined by the supply of & demand for
intermediates of the cycle.
• The pathway provides a major portion of the body’s NADPH,
which functions as a biochemical reductant.
• It also produces ribose-5-P required for biosynthesis of
nucleotides, & provides a mechanism for metabolic use of
five-carbon sugars obtained from diet or degradation of
structural CHOs in the body.
II. Irreversible oxidative reactions
-
-
The oxidative portion of pentose phosphate
pathway consists of 3 reactions that lead to
formation of ribulose 5-P, CO2, & 2 molecules
of NADPH for each molecule of G-6-P
oxidized.
This portion of the pathway is particularly
important in the liver & lactating mammary
glands, which are active in the biosynthesis of
fatty acids, in adrenal cortex, which is active in
the NADPH-dependent synthesis of steroids, &
in RBCs, which require NADPH to keep
glutathione reduced
A. Dehyerogenation of glucose-6-phosphate
- Glucose 6-phosphate dehydrogenase (G6PD) catalyzes
an irreversible oxidation of G-6-P to 6phosphogluconolactone in a reaction that is specific for
NADP+ as its coenz.
- The pentose phosphate pathway is regulated primarily at
the G6PD reaction. NADPH is a potent competitive
inhibitor of the enz, &, under metabolic conditions, the
ratio of NADPH/NADP+ is sufficiently high to
substantially inhibit enz activity.
- However, with increased demand for NADPH, the ratio of
NADPH/NADP+ decreases & flux through the cycle
increases in response to the enhanced activity of G6PD.
- Insulin enhances G6PD gene expression, & flux through
the pathway increases in the well-fed state
B. Formation of ribulose-5-phosphate
- 6-phosphogluconolactone is hydrolyzed by 6phosphogluconolactone hydrolase. The reaction is
irreversible & not rate-limiting.
- The subsequent oxidative decarboxylation of 6phosphogluconate is catalyzed by 6-phosphogluconate
dehydrogenase. This irreversible reaction produces a
pentose sugar-phosphate (ribulose 5-P), CO2 (from C-1 of
glucose), & a 2nd molecule of NADPH.
Figure 13.2. Reactions of the hexose monophosphate pathway. Enzymes numbered
above are 1) glucose 6-phosphate dehydrogenase and 6-phosphogluconolactone
hydrolase, 2) 6-phosphogluconate dehydrogenase, 3) ribose 5-phosphate isomerase,
4) phosphopentose epimerase, 5) and 7) transketolase (coenzyme: thiamine
pyrophosphate), and 6) transaldolase.
III. Reversible nonoxidative reactions
- The nonoxidative reactions of pentose
phosphate pathway occur in all cell types
synthesizing nucleotides & nucleic acids.
- These reactions catalyze the interconversion of
3-, 4-, 5-, 6-, & 7-carbon sugars. These
reversible reactions permit ribulose-5-P
(produced by oxidative portion of pathway) to be
converted either to ribose 5-P (needed for
nucleotide synthesis) or to intermediates of
glycolysis, F-6-P & glyceraldehyde 3-P.
- E.g., many cells that carry out reductive
biosynthetic reactions have a greater need for
NADPH than for ribose-5-P. In this case,
transketolase (which transfers 2-C units) &
transaldolase (which transfers 3-C units) convert
ribulose 5-P produced as an end-product of the
oxidative reactions to glyceraldehyde 3-P & F-6P, which are intermediates of glycolysis.
- In contrast, under conditions in which the
demand for ribose for incorporation into
nucleotides & nucleic acids is greater than the
need for NADPH, the nonoxidative reactions can
provide the biosynthesis of ribose-5-P from
glyceraldehyde 3-P & F-6-P in the absence of
the oxidative steps
Figure 13.3. Formation of ribose 5-phosphate from intermediates of glycolysis.
IV. Uses of NADPH
- The coenz NADP+ differs from NAD+ only by
the presence of a P-group (-PO4=) on one of the
ribose units.
- This seemingly small change in structure allows
NADP+ to interact with NADP+-specific enz’s
that have unique roles in the cell. E.g., the
steady-state ratio of NADP+/NADPH in the
cytosol of hepatocytes is ~ 0.1, which favors the
use of NADPH in reductive biosynthetic
reactions.
- This contrasts with the high ratio of NAD+/NADH
(~ 1000 in the cytosol of hepatocytes), which
favors an oxidative role of NAD+.
Figure 13.4. Structure of NADPH.
A. Reductive biosynthesis
- NADPH can be thought of as a highenergy molecule, much in the same way
as NADH. However, the e’s of NADPH are
destined for use in reductive biosynthesis,
rather than for transfer to oxygen as is the
case with NADH.
- Thus, in the metabolic transformations of
the pentose phosphate pathway, part of
the energy of G-6-P is conserved in
NADPH, a molecule that can be used in
reactions requiring a high electronpotential e-donor.
B. Reduction of hydrogen peroxide
- Hydrogen peroxide is one of a family of reactive oxygen
species that are formed from the partial reduction of
molecular oxygen.
- These cpds are formed continuously as by-products of
aerobic metabolism, through reactions with drugs &
environmental toxins, or when level of antioxidants is
diminished, all creating the condition “oxidative stress”.
- The highly reactive oxygen intermediates can cause
serious chemical damage to DNA, proteins, &
unsaturated lipids, and can lead to cell death.
- These reactive oxygen species have been implicated in
a number of pathologic processes including reperfusion
injury, cancer, inflammatory disease, and aging.
- The cell has several protective mechanisms that
minimize the toxic potential of these cpds.
1. Enzymes that catalyze antioxidant reactions:
- A tripeptide-thiol (γ-glutamylcysteinylglycine) present in
most cells, can chemically detoxify hydrogen peroxide
- This reaction, catalyzed by the selenium-requiring
glutathione peroxidase, forms oxidized glutathione, which
no longer has protective property
- The cell regenerates reduced glutathione in a reaction
catalyzed by glutathione reductase, using NADPH as a
source of reducing electrons
- Thus, NADPH indirectly provides e’s for the reduction of
hydrogen peroxide
- Additional enz’s, such as superoxide dismutase &
catalase, catalyze the conversion of other toxic oxygen
intermediates to harmless products
- As a group, these enz’s serve as a defense system to
guard against the toxic effects of reactive oxygen species
Figure 13.5
A. Formation of reactive intermediates from molecular oxygen. B. Actions of
antioxidant enzymes. G-SH = reduced
glutathione; G-S-S-G = oxidized glutathione.
Figure 13.6. A. Structure of glutathione (G-SH). [Note:
Glutamate is linked to cysteine through a γ-carboxyl, rather
than an α-carboxyl.] B. Glutathione-mediated reduction of
hydrogen peroxide by NADPH.
Note:
- RBCs are totally dependent on the pentose phosphate
pathway for their supply of NADPH because, unlike other
cell types, RBCs do not have an alternate source for this
essential coenz.
- If G6PD is compromised in some way, NADPH levels will
fall, & oxidized glutathione can’t be reduced
- As a result, hydrogen peroxide will accumulate,
threatening memb stability & causing RBC lysis
2. Antioxidant chemicals
- A number of intracellular reducing agents such as ascorbate, vitamin
E, & β-carotene, are able to reduce &, thus, detoxify oxygen
intermediates in the lab.
- Consumption of foods rich in these antioxidant cpds has been
correlated with a reduced risk for certain types of cancers, as well as
decreased frequency of certain other chronic health problems
- Thus, it is tempting to speculate that the effects of these cpds are, in
part, an expression of their ability to quench the toxic effect of
oxygen intermediates
- However, clinical trials with antioxidants as dietary supplements
have failed to show clear beneficial effects.
- In the case of dietary supplementation with β-carotene, the rate of
lung cancer in smokers increased rather than decreased.
- Thus, the health-promoting effects of dietary fruits & vegetables
probably reflects a complex interaction among many naturally
occurring cpds, which has not been duplicated by consumption of
isolated antioxidant cpds
C. Cytochrome P450 monooxygenase system
- Monooxygenases (mixed function oxidases) incorporate
one atom from molecular oxygen into a substrate
(creating a hydroxyl group), with the other atom being
reduced to water
- In the cytochrome P450 monooxygenase system,
NADPH provides the reducing equivalents required by
this series of reactions
- This system performs different functions in two separate
locations in cells. The overall reaction catalyzed by CytP450 enz is:
R-H + O2 + NADPH + H+ → R-OH + H2O + NADP+
Where R may be a steroid, drug, or other chemical
Note: Cyt-P450s (CYPs) are actually a superfamily
comprised of 100s of genes, coding for related, hemecontaining enz’s that participate in a broad variety of
reactions
Figure 13.7
Cytochrome P450
monooxygenase cycle.
1. Mitochondrial system:
- The function of the mitoch Cyt-P450 monooxygenase
system is to participate in the hydroxylation of steroids, a
process that makes these hydrophobic cpds more water
soluble
- E.g., in the steroid hormone-producing tissues, e.g.,
placenta, ovaries, testes, & adrenal cortex, it is used to
hydroxylate intermediates in the conversion of
cholesterol to steroid hormones
- The liver uses this system in bile acid synthesis, & the
kidney uses it to hydroxylate vitamin 25hydroxycholecalciferol (vitamin D) to its biologically
active 1,25-hydroxylated form
2. Microsomal system:
- An extremely important function of the microsomal Cyt-P450
monooxygenase system found associated with memb’s of
sER (particularly in liver) is the detoxification foreign cpds
(xenobiotics)
- Xenobiotics include numerous drugs & such varied
pollutants as petroleum products, carcinogens, & pesticides
- The Cyt-P450 monooxygenase system can be used to
hydroxylate these toxins, again using NADPH as source of
reducing equivalents
- The purpose of these modifications is 2-fold. 1st, it may itself
activate or inactivate a drug or 2nd, make a toxic cpd more
soluble, thus facilitating its excretion in the urine or feces.
- Frequently, however, the new hydroxyl group will serve as a
site for conjugation with a polar cpd, such as glucuronic
acid, which will significantly increase the cpd’s solubility
D. Phagocytosis by white blood cells
- Phagocytosis is the ingestion by receptor-mediated
endocytosis of m/o’s, foreign particles, & cellular debris
by cells such as neutrophils & macrophages
(monocytes)
- It is an important body defense mechanism, particularly
in bacterial infections.
- Neutrophils & monocytes are armed with both oxygenindependent & oxygen-dependent mechanisms for killing
bacteria.
- The oxygen-dependent mechanisms include the
myeloperoxidase (MPO) system & a system that
generates oxygen-derived free radicals
- Oxygen-independent systems use pH changes in
phagolysosomes & lysosomal enz’s to destroy
pathogens
• Overall, the MPO system is the most potent of the
bactericidal mechanisms.
• An invading bacterium is recognized by the immune
system & attacked by antibodies that bind it to a receptor
on a phagocytic cell
• After internalization of the m/o has occurred, NADPH
oxidase, located in the leukocyte CM, converts molecular
oxygen from the surrounding tissue into superoxide
• The rapid consumption of molecular oxygen that
accompanies formation of superoxide is referred to as
the respiratory burst
Note:
- NADPH oxidase is a complex enz, with subunits
containing a cytochrome & a flavin coenz group
- Genetic deficiencies in this enz cause chronic
granulomatosis, a disease characterized by severe,
persistent, chronic pyogenic infections
- Next, superoxide is spontaneously converted into
hydrogen peroxide. Any superoxide that escapes the
phagolysosome is converted to hydrogen peroxide by
superoxide dismutase (SOD).
- This product is then neutralized by catalase or
glutathione peroxidase
- In the presence of MPO, a lysosomal enz present within
the phagolysosome, peroxide plus chloride ions are
converted into hypochorous acid (HOCl, the major
component of household bleach), which kills the
bacteria.
- Excess peroxide is either neutralized by catalase or by
glutathione peroxidase
Figure 13.8
Phagocytosis and the oxygen
dependent pathway of microbial
killing. IgG = the antibody
immunoglobulin G.
E. Synthesis of nitric oxide
- Nitric oxide (NO) is recognized as a mediator in a broad
array of biologic systems
- NO is the endothelium-derived relaxing factor, which
causes vasodilation by relaxing vascular smooth muscle.
- NO also acts as a neurotransmitter, prevents platelet
aggregation, & plays an essential role in macrophage
function.
- NO has a very short half-life in tissues (3-10 seconds)
because it reacts with oxygen & superoxide, & then is
converted into nitrates & nitrites.
Note:
- NO is a free radical gas that is confused with nitrous oxide
(N2O), the “laughing gas” that is used as an anesthetic &
is chemically stable
1. Synthesis of NO:
- Arg, O2, & NADPH are substrates for cytosolic NO synthase.
- Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD),
heme, & tetrahydropterin are coenz’s for the enz, & NO & citrulline
are products of the reaction
- Three NO synthases have been identified. Two are constitutive
(synthesized at a constant rate regardless of physiologic demand),
Ca2+-calmodulin-dependent enzymes. They are found primarily in
endothelium (eNOS), & neural tissue (nNOS), & constantly produce
low levels of NO
- An inducible, Ca2+-independent enz. (iNOS) can be expressed in
many cells, including hepatocytes, macrophages, monocytes, &
neutrophils
- The specific inducers for NO synthase vary with cell type, & include
tumor necrosis factor-α, bacterial endotoxins, & inflammatory
cytokines. These cpds have been shown to promote synthesis of
iNOS, which can result in large amounts of NO being produced over
hours or even days.
Figure 13.9
Synthesis and some of the
actions of nitric oxide.
2. Action of NO on vascular endothelium:
- NO is an important mediator in the control of vascular smooth
muscle tone
- NO is synthesized by eNOS in endothelial cells & diffuses to
vascular smooth muscle, where it activates the cytosolic form of
guanylate cyclase
Note: this reaction is analogous to the formation of cAMP by adenylate
cyclase, except that this guanylate cyclase is not membraneassociated
- The resultant rise in cGMP causes muscle relaxation through
activation of protein kinase G, which phospho. myosin light-chain
kinase & renders it inactive, thereby decreasing smooth muscle
contraction
Note: vasodilator nitrates, such as nitroglycerin & nitroprusside, are
metabolized to nitric oxide, which causes relaxation of vascular
smooth muscle &, therefore, lowers blood pressure. Thus, NO can
be envisioned as an endogenous nitrovasodilator
3. Role of NO in mediating macrophage bactericidal
activity:
- In macrophages, iNOS activity is normally low, but
synthesis of the enz is significantly stimulated by
bacterial LPS & γ-IFN release in response to infection.
- Activated macrophages form superoxide radicals that
combine with NO to form intermediates that decompose,
forming the highly bactericidal OH•- radical
Note: NO production in macrophages is also effective
against viral, fungal, helmintic, & protozoan infections
4. Other functions of NO:
- NO is a potent inhibitor of platelet aggregation (by
activating cGMP pathway). It is characterized as a
neurotransmitter in the brain
V. Glucose 6-P dehydrogenase deficiency
- G6PD deficiency is an inherited disease characterized
by hemolytic anemia caused by the inability to detoxify
oxidizing agent
- G6PD deficiency is the most common disease-producing
enz abnormality in humans, affecting > 200 million
individuals worldwide.
- This deficiency has the highest prevalence in the Middle
East, tropical Africa & Asia, & parts of the Mediterranean
- G6PD deficiency is X-linked, & is in fact, a family of
deficiencies caused by > 400 different mutations in the
gene coding for G6PD. Only some of these mutations
cause clinical symptoms
• The lifespan of many individuals with G6PD deficiency is
somewhat shortened as a result of complications arising
from chronic hemolysis
• This slightly negative effect of G6PD deficiency has been
balanced in evolution by an advantage in survival, an
increased resistance to falciparum malaria shown by
female carrier of the mutation
Note: sickle cell trait & β-thalassemia minor also confer
resistance
A. Role of G6PD in RBCs
- Diminished G6PD activity impairs the ability of the cell to
form NADPH that is essential for the maintenance of
reduced glutathione pool. This results in decrease in the
cellular detoxification of free radicals & peroxides formed
within cell
- Glutathione also helps maintain the reduced states of
sulfhydryl groups in proteins, including Hb. Oxidation of
those sulfhydryl groups leads to formation of denatured
proteins that form insoluble masses (called Heinz
bodies) that attach to the red cell memb’s.
- Additional oxidation of memb proteins causes the red
cells to be rigid & non-deformable, & they are removed
from the circulation by macrophages in spleen & liver
Figure 13.10. Pathways of G-6-P mtabolism in the erythrocyte
Figure 13.11
Heinz bodies in erythrocytes of patient with G6PD deficiency.
- Although G6PD deficiency occurs in all cells of the
affected individual, it is most severe in erythrocytes,
where the pentose phosphate pathway provides the only
means of generating NADPH
- Other tissues have alternative sources for NADPH
production (e.g., NADP+-dependent malate
dehydrogenase) that keep glutathione reduced
- The erythrocyte has no nucleus or ribosomes & can’t
renew its supply of the enz. Thus, RBCs are particularly
vulnerable to enz variants with diminished stability
B. Precipitating factors in G6PD deficiency
Most individuals who have inherited one of the many
G6PD mutations do not show clinical manifestations.
However, some patients with G6PD deficiency develop
hemolytic anemia if they are treated with an oxidant
drug, ingest fava beans, or contract a severe infection
1. Oxidant drugs: commonly used drugs that produce
hemolytic anemia in patients with G6PD deficiency are
best remembered from the mnemonic AAA =
Antibiotics (e.g., sulfa, methoxazole &
chloramphenicol), Antimalarials (e.g., primaquine but
not quinine), and Antipyretics (e.g., acetanilid but not
acetaminophen)
2.
3.
4.
Favism: some forms of G6PD deficiency, e.g., the
Mediterranean variant, are particularly susceptible to
the hemolytic effect of the fava bean, a dietary staple
in Mediterranean region. Favism, the hemolytic effect
of ingesting fava beans, is not observed in all
individuals with G6PD deficiency, but all patients with
favism have G6PD deficiency
Infection: infection is the most common precipitating
factor of hemolysis in G6PD deficiency. The
inflammatory response to infection results in the
generation of free radicals in macrophages, which can
diffuse into the RBCs & cause oxidative damage
Neonatal jaundice: babies with G6PD deficiency may
experience neonatal jaundice appearing 1-4 days after
birth. The jaundice, which may be severe, results from
impaired hepatic catabolism of heme or increased
production of bilirubin
C. Properties of the variant enzymes
- Almost all G6PD variants are caused by point mutations
in the G6PD gene.
- Some mutations do not disrupt the structure of the enz’s
active site &, hence, do not affect enzymic activity
- However, many mutant enz’s show altered kinetic
properties. E.g., variant enz’s may show decreased
catalytic activity, decreased stability, or an alteration of
binding affinity for NADP+, NADPH, or G-6-P
- Severity of disease usually correlates with amount of
residual enz activity in patients’ RBCs. E.g., variants can
be classified as:
Figure 13.12
Classification of G6PD deficiency variants.
• G6PD A- is the prototype of the moderate (class III) form
of disease. The RBCs contain an unstable, but kinetically
normal G6PD, with most of the enz activity present in the
reticulocytes & younger erythrocytes
• The oldest cells, therefore, have the lowest level of enz
activity, & are preferentially removed in a hemolytic
episode.
• G6PD Mediterranean is the prototype of a more severe
(class II) deficiency in which the enz shows normal
stability but scarcely detectable activity in all RBCs.
• Class I mutations are often associated with chronic nonspherocytic anemia, which occurs even in absence of
oxidative stress
Figure 13.13
Decline of erythrocyte G6PD
activity with cell
age for the three most
commonly encountered
forms of the enzyme.
D. Molecular biology of G6PD
- Cloning of G6PD gene & the sequencing of its cDNA
have permitted identification of mutations that cause
G6PD deficiency
- More than 300 different mutations or mutation
combinations have been identified in this gene, a finding
that explains the numerous biochemical variants
- Most of these DNA changes are missense, point
mutations. Both G6PD A- & G6PD Mediterranean
represent mutant enz’s that differ from the respective
normal variants by a single aa
- Large deletions or frameshifts mutations have not been
identified, suggesting that complete absence of G6PD
activity is probably lethal
5 yr old boy presents to the emergency room:
febrile, pale, tachycardic, tachypneic and minimally responsive
AM: good health
PM: abdominal pain, headache, fever
by late evening: tachypneic and incoherent
Lab tests: massive nonimmune intravasuclar hemolysis and
hemoglobinurea
The patient is of Greek ethnicity.
Mother notes that although there is no family history of
hemolysis, she has some European cousins with a ‘blood
problem’
She later recalls that her son had been eating fava beans in
the garden while she worked in the yard
Summary
• The pentose phosphate pathway consists of 2 irreversible oxidative
reactions followed by a series of reversible sugar-phosphate
interconversions
• No ATP is directly consumed or produced in the cycle
• The oxidative portion is particularly important in liver & mammary
glands, which are active in biosynthesis of fatty acids, in adrenal
cortex, which is active in NADPH-dependent synthesis of steroids, &
in erythrocytes, which require NADPH to keep glutathione reduced
• G-6-P is irreversibly converted to ribulose-5-P, & 2 NADPH are
produced.
• The regulated step is G6PD, which is strongly inhibited by NADPH
• Reversible nonoxidative reactions interconvert sugars. This part of
pathway is the source of ribose 5-P required for nt & nucleic acid
synthesis
• Because reactions are reversible, they can be entered from F-6-P &
GA 3P (glycolytic intermediates) if ribose is needed & G6PD is
inhibited
• NADPH is a source of reducing equivalents in reductive
biosynthesis, such as production of fatty acids &
steroids. It is also required for reduction of hydrogen
peroxide, providing the reducing equivalents required by
glutathione (GSH).
• GSH is used by glutathione peroxidase to reduce
peroxide to water. The oxidized glutathione is reduced by
glutathione reductase, using NADPH as the source of e’s
• NADPH provides reducing equivalents for cyt-P450
monooxygenase system, which is used in hydroxylation
of steroids to produce steroid hormones, bile acid
synthesis by liver, & activation of vitamin D. the system
also detoxify foreign cpds, e.g., drugs & varied
pollutants, including carcinogens, pesticides, &
petroleum products
• NADPH provides reducing equivalents for phagocytes in
the process of eliminating invading m/o’s
• NADPH oxidase uses molecular oxygen & NADPH e’s to
produce superoxide radicals, which, in turn, can be
converted to peroxide, hypochlorous acid, & hydroxyl
radicals. Myeloperoxidase is an important enz in this
pathway
• A genetic defect in NADPH oxidase causes chronic
granulomatosis, a disease characterized by severe,
persistent, chronic pyogenic infections
• NADPH is required for synthesis of nitric oxide (NO), an
important molecule that causes vasodilation by relaxing
vascular smooth muscle, acts as a kind of
neurotransmitter, prevents platelet aggregation, & helps
mediate macrophage bactericidal activity
• G6PD deficiency is a genetic disease characterized by hemolytic
anemia. It impairs ability of cell to form NADPH that is essential for
maintenance of reduced glutathione pool.
• Cells most affected are RBCs because they do not have additional
sources of NADPH
• Free radicals & peroxides formed within the cells can’t be
neutralized, causing denaturation of protein (e.g., Hb, forming Heinz
bodies) & memb proteins. Cells become rigid, & they are removed
by reticuloendothelial system of spleen & liver
• Hemolytic anemia can be caused by production of free radicals &
peroxides following the taking of oxidant drugs, ingestion of fava
beans, or severe infections
• Babies with G6PD deficiency may experience neonatal jaundice
appearing 1-4 days after birth
• Degree of severity of anemia depends on location of mutation in
G6PD gene. Class I mutations are the most severe (e.g., G6PD
Mediterranean). They are often associated with chronic nonspherocytic anemia. Class III mutations (e.g., G6PD A-) cause a
more moderate form of the disease