BCHEM 253 – METABOLISM IN HEALTH AND DISEASES I

Lecture 2
Pentose Phosphate Pathway
Christopher Larbie, PhD

In most animal tissues, glucose is catabolized via the glycolytic pathway into two
molecules of pyruvate. Pyruvate is then oxidized via the citric acid cycle to generate
ATP. There is another metabolic fate for glucose used to generate NADPH and
specialized products needed by the cell. This pathway is called the pentose
phosphate pathway. Some text books call it the hexose monophosphate shunt, still
others call it the phosphogluconate pathway.

The pentose phosphate pathway produces NADPH which is the universal reductant
in anabolic pathways. In mammals the tissues requiring large amounts of NADPH
produced by this pathway are the tissues that synthesize fatty acids and steroids
such as the mammary glands, adipose tissue, adrenal cortex and the liver. Tissues
less active in fatty acid synthesis such as skeletal muscle are virtually lacking the
pentose phosphate pathway.

The second function of the pentose phosphate pathway is to generate pentoses,

particularly ribose which is necessary for the synthesis of nucleic acids.

The Pentose Phosphate Pathway (PPP)

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It is convenient to think of the pentose phosphate pathway as operating in two
phases. The first phase is the oxidative phase. Two of the first three reactions of the
first phase generate NADPH. The second phase is the nonoxidative phase.

In the first step glucose-6-phosphate is oxidized into ribulose-5-phosphate and CO2.

During the oxidation of glucose-6-phosphate, NADP+ is reduced into NADPH. The
second step of the pathway coverts the ribulose 5-phosphate into other pentose-5-
phosphates including ribose-5-phosphate used to synthesize nucleic acids. The third
step includes a series of reactions that convert three of the pentose-5-phosphates into
two molecules of hexoses and one triose. In the fourth step, some of these sugars are
converted into glucose-6-phosphate so the cycle can be repeated. The direction of the
pathway varies to meet different metabolic conditions.

Oxidative Phase: The oxidative phase of the pentose phosphate pathway is

composed of three steps.

1. Glucose-6-Phosphate dehydrogenase (G6PD) reaction: The PPP begins with

the oxidation of glucose-6-phosphate to produce a cyclic ester (the lactone of
phosphogluconic acid) and NADPH. G6PD is highly specific for NADP+. This
reaction is irreversible and highly regulated by NADPH and fatty acid esters
of coenzyme A (which are intermediates of fatty acid biosynthesis). Inhibition
by NADPH is dependent on the cytosolic NADP+/NADPH ratio (in the liver
it’s about 0.015).

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 2. Gluconolactonase reaction: The gluconolactone produced in step 1 is
hydrolytically unstable and readily undergoes a spontaneous ring-opening
hydrolysis, although the enzyme accelerates the reaction. The product is 6-
phospho-D-gluconate, a sugar acid which is further oxidized in the next step.

3. 6-phosphogluconate dehydrogenase reaction: This enzyme catalyses the

oxidative decarboxylation of 6-phosphogluconate to yield D-ribolus-5-
phosphate and NADPH. This reaction occurs in two distinct steps: the initial
NADP+-dependent dehydrogenation yields a β-keto acid, 3-ketp-6-
phosphogluconate, which is very susceptible to decarboxylation (the second
step). The product of the reaction, riboluso-5-phosphate is the substrate for
the non-oxidative reactions.

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Nonoxidative Phase: The nonoxidative phase of the pentose phosphate pathway is
composed of 5 steps but only 4 types of reactions. This portion of the pathway
begins with an isomerization and epimerization, and it leads to the formation of
either D-ribose-5-phosphate or D-xylulose5-phosphate. These intermediates can then
be converted into glycolytic intermediates or directed to biosynthetic processes.

4. Phosphopentose Isomerase Reaction: This enzyme converts ribulose-5-

phosphate and ribose-5-phosphate via an edediol intermediate. The ribose-5-
phosphate produced in this reaction is utilized in the biosynthesis of
coenzyems (including NADH, NADPH, FAD and B12), nucleatides and
nucleic acids (DNA and RNA). The net reaction to this point is

Glucose-6-phosphate + 2NADP+ → ribose-5-phosphate + CO2 + 2NADPH + 2H+

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 5. Phosphopentose Epimerase Reaction: This enzyme converts riboluse-5-
phosphate to another ketose, xylulose-5-P proceeded by an enediol
intermediate at C-3. In the reaction, an acidic proton located α- to a carbonyl
carbon is removed to generate the enediolate, but the proton is added back to
the same carbon from the opposite side. From the beginning to this point, 2
NADPH has been generated or every molecule of glucose-6-P converted to
pentose-5-P. The next three steps, there will be rearrangement to generate
three-, four-, six- and seven-carbon units, which can be utilized in various
metabolic processes. This is important because, very often the cellular need
for NADPH is considerably greater than the need for ribose-5-P. The next
three steps thus return some of the 5-C units to glyceraldehyde-3-P and
fructose-6-P, which can enter the glycolytic pathway. The advantage of this is
that the cells have met its need for NADPH and ribose-5-phosphate in a single
pathway, yet at the same time it can return excess carbon metabolites to
glycolysis.

6. Transketolase Reaction: This enzyme catalysis the transfer two carbon unit
from xylulose-5-P to ribose-5-P yielding 3-C unit glyceraldehyde-3-P and
sedoheptulose-7-P. The donor molecule is a ketone and the acceptor molecule
is an aldose. G-3-P enters the glycolytic pathway. Transketolase is dependent
on thiamine pyrophosphate as a cofactor. The mechanism involves the
abstraction of the acidic thiozole proton of TPP, attack by the resulting
carbanion at the carbonyl carbon of the ketose phosphate substrate, expulsion
of the G-3-P product, and transfer of the 2 C unit. Transketolase can process a
variety of 2-keto sugar substrates in a similar manner as in reaction 8.

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 7. Transaldolase Reaction: This enzyme catalysis the conversion of S-7-P and G-
3-P to erthrose-4-P and fructose-6-P. This reaction is similar to the aldose
reaction of glycolysis. It involves the formation of a Schiff base intermediate
between the S-7-P and an active-site lysine residue. Elimination of the E-4-P
product leaves an enamine of dihydroxyacetone, which remains stable at the
active site until the other substrate comes into position. Attack of the enamine
carbanion at the carbonyl of G-3-P is followed by hydrolysis of the Schiff base
(imine) to yield the product fructose-6-phosphate.

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 8. Transketolase Reaction: This reaction is similar to Step 6. Here the
transketolase catalysis the transfer of a 2C unit from X-5-P to E-4-P yielding
G-3-P and fructose-6-P. Bothe products enter the glycolytic pathway.

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Net Reaction
Oxidative phase:
3 Glucose-6-phosphate + 6 NADP+ → 2 Xylulose-5p + ribose-5-P + 3CO2 + 6NADPH
+ 6H+

Rearrangements of the non-oxidative phase:

2 Xylulose-5-P + ribose-5-P → 2 Frucose-6P + Glyceraldehyde-3-P

The sum of these two phases:

3 Glucose-6-phosphate + 6 NADP+ →2 Frucose-6-P + Glyceraldehyde-3-P + 3 CO2 +
6 NADPH + 6 H+

Tailoring the Pentose Phosphate Pathway to meet specific needs of the cell
1. If the cell requires both ribose-5-P and NADPH, the first four reactions of the
PPP predominate. NADPH is produced by the oxidative reactions of the
pathway, and ribose-5-P is the principal product of carbon metabolism.

2. More ribose-5 phosphate needed than NADPH: This can be can be

accomplished with the synthesis of ribose-5-P with NADPH by by-passing
the oxidative steps. This is achieved by the withdrawal of F-6P and G-3-P, but
not glucose-6-P, from glycolysis. The reaction of transketolase and
transaldolase on F-6-P and G-3-P produces three molecules of ribose-5-P from
two molecules of F-6-P and one G-3-P. In this route, no carbon metabolites
return to glycolysis.
5 G-6-P + ATP → 6 R-5-P + ADP + Pi

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 3. More NADPH needed than Ribose-5-phosphate: Large amounts of NADPH
can be supplied in the PPP without concomitant production of ribose-5-P if
ribose-5-P produced in the PPP is recycled to produce glycolytic
intermediates. This alternative involves a complex interplay between the
transketolase and transaldolase reactions to convert ribulose-5-P to F-6-P by
gluconeogenesis. The net reaction is
6 G-6-P + 12 NADP+ + 6 H2O → 6 Ribulose-5-P + 6 CO2 + 12 NADPH + 12 H+
6 Ribulose-5-P → 5 Glucose-6-P + Pi
Net: Glucose-6-P + 12 NADP+ + 6 H2O → 6 CO2 + 12 NADPH + 12 H+ + Pi

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 4. Both NADPH and ATP are needed but not ribose-5-phosphate: Under some
conditions, both NADPH and ATP must be provided in the cell. This can be
accomplished in a series of reactions similar in case 3 if the F-6-P and G-3-P
produced in this way proceed through glycolysis to produce ATP and
pyruvate, which itself can yiled even more ATP by continuing on to the TCA
cycle. The net reaction for this alternative is
3 Glucose-6-P + 5 NAD+ + 6 NADP+ + 8 ADP + 5 Pi → 5 Pyruvate + 3 CO2 + 5
NADH + 6 NADPH + 8 ATP + 2 H2O + 8 H+

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Regulation
The first step of the phosphopentose pathway is the irreversible committed step.
This reaction is catalysed by glucose-6-phosphate dehydrogenase. This step is of
course allosterically regulated. The product of this reaction NADPH is a strong
inhibitor. So when the cytosol concentration of NADPH is high, the enzyme’s
activity is low. It is also allosterically regulated by fatty acid acyl esters of coenzyme
A. The transcription of the gene for this enzyme is under hormonal regulation.

Xylulose-5-P also serves as a signalling molecule. When blood glucose levels rise,
glycolysis and PPP pathways are actvated in the liver and the X-5-P produced in the
latter pathways stimulate protein phosphate 2A (PP2A), which dephosphorylate the
bifunctional enzyme phosphofructokinase-2 or fructose-2,6-bisphosphatase.

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Increased levelf of F-2,6-BP stimulate glycolysis and inhibit gluconeogenesis. At the
same time, PP2A dephosphorylates carbohydrate responsive element –binding
protein (ChREBP), a transcription factor that activates expression of liver genes for
lipid synthesis. These effects are a powerful combination. Increased glycolysis
produces substantial amounts of acetyl-CoA, the principal substrate for lipid
synthesis. The PPP produced NADPH, the source of electrons for lipid biosynthesis.
Elevated expression of the appropriate genes sets the stage for lipid biosynthesis in
the liver, an important consequence of ingestion of carbohydrates.

Glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is an X-linked
recessive hereditary disease characterized by abnormally low levels of glucose-6-
phosphate dehydrogenase, especially important in red blood cell metabolism. G6PD
deficiency is the most common human enzyme defect. Individuals with the disease
may exhibit non-immune haemolytic anaemia in response to a number of causes,
most commonly infection or exposure to certain medications or fava beans. Glucose-
6-phosphate dehydrogenase (G6PD) is an enzyme in the pentose phosphate
pathway. G6PD converts glucose-6-phosphate into 6-phosphoglucono-δ-lactone and
is the rate-limiting enzyme of this metabolic pathway that supplies reducing energy
to cells by maintaining the level of the co-enzyme nicotinamide adenine dinucleotide
phosphate(NADPH). The NADPH in turn maintains the supply of
reduced glutathione in the cells that is used to mop up free radicals that
cause oxidative damage.

The G6PD / NADPH pathway is the only source of reduced glutathione in red blood
cells (erythrocytes). The role of red cells as oxygen carriers puts them at substantial
risk of damage from oxidizing free radicals except for the protective effect of
G6PD/NADPH/glutathione.

People with G6PD deficiency are therefore at risk of haemolytic anaemia in states
of oxidative stress. Oxidative stress can result from infection and from chemical
exposure to medication and certain foods. Broad beans, e.g., fava beans, contain high
levels of vicine, divicine, convicine and isouramil, all of which are oxidants.

When all remaining reduced glutathione is consumed, enzymes and other proteins
(including haemoglobin) are subsequently damaged by the oxidants, leading
to electrolyte imbalance, cross-bonding and protein deposition in the red cell

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membranes. Damaged red cells are phagocytosed and sequestered (taken out of
circulation) in the spleen. The haemoglobin is metabolized
to bilirubin (causing jaundice at high concentrations). The red cells rarely
disintegrate in the circulation, so haemoglobin is rarely excreted directly by
the kidney, but this can occur in severe cases, causing acute renal failure .

Deficiency of G6PD in the alternative pathway causes the build-up of glucose and
thus there is an increase of advanced glycation end-products (AGE). The deficiency
also reduces the amount of NADPH, which is required for the formation of nitric
oxide (NO). Although female carriers can have a mild form of G6PD deficiency
(dependent on the degree of inactivation of the unaffected X chromosome),
homozygous females have been described; in these females there is co-incidence of
a rare immune disorder termed chronic granulomatous disease (CGD).

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