Pentose Phosphate Pathway

Dr. Ahmad Ali

Univ. Dept. Life Sciences
Univ. Mumbai
Glucose
 Glycolytic pathway – major catabolic fate
 Secondary pathways of glucose metabolism –
 Pentose phosphate pathway
 Glycogen metabolism – glycogenolysis and
glycogenesis
 Gluconeogenesis
 Pentose phosphate pathway – major secondary
metabolic pathway of glucose oxidation
 Pentose Phosphate Pathway
 Phosphogluconate pathway or hexose monophosphate pathway
 Cytosolic
 Functions
 NADPH production
 Reducing power carrier
 Synthetic pathways
 Role as cellular antioxidants
 Ribose synthesis
 Nucleic acids and nucleotides
 Metabolize dietary pentose sugars derived from the digestion of
nucleic acids
 Rearrange the carbon skeletons of dietary carbohydrates into
glycolytic/gluconeogenic intermediates
Requirement of NADPH
 Biosynthetic pathways
 FA synthesis (liver, adipose, mammary)
 Cholesterol synthesis (liver)
 Steroid hormone synthesis (adrenal, ovaries,
testes)
 Detoxification (Cytochrome P-450 System) –
liver
 Reduced glutathione as an antioxidant (RBC)
 Generation of superoxide (neutrophils)
 Two Phases of PPP
 Oxidative phases
 Reactions producing NADPH & Ribose-5-P
 Irreversible
 Non-oxidative phases
 Regenerates Hexose phosphates from pentose
phosphates
 Reversible reactions feed to glycolysis
Two Phases of PPP…

(Principles of
Biochemistry, Nelson
& Cox)
 Oxidative Phase: NADPH Production
 First Step:

Glucose-6-phosphate 6-Phospho- O O
Dehydrogenase glucono- 1C
6CH OPO 2 6 CH OPO 2 lactonase
2 3 + 2 3
NADPH + H HC OH
H
5 O OH NADP
+
H
5 O H2O H+ 2
H H HO 3CH
4 OH H 1 4
OH H O
1 HC OH
4
OH H OH
3 2 3 2 HC OH
5
H OH H OH CH2OPO32
6
glucose-6-phosphate 6-phoshogluconolactone 6-phosphogluconate
Glucose-6-phosphate Dehydrogenase - oxidation of the aldehyde (hemiacetal), at C1 of
glucose-6-phosphate, to a carboxylic acid, in ester linkage (lactone)
NADP+ - electron acceptor
G6PD - highly specific for NADP+- KM for NAD+ is about a thousand times as great as that for
NADP+
Second Step:
6-Phosphogluconolactonase
catalyzes hydrolysis of the ester
linkage, resulting in ring opening.
The product is 6-
phosphogluconate.
Although ring opening occurs in the
absence of a catalyst, 6-
Phosphogluconolactonase speeds up
the reaction, decreasing the lifetime of
the highly reactive, and thus
potentially toxic, 6-
phosphogluconolactone.
Third Step: Production of NADPH
Phosphogluconate
O O Phosphogluconate
Dehydrogenase catalyzes 1C Dehydrogenase
oxidative decarboxylation of HC OH + CH OH
2 NADP + NADPH + H 1 2
6-phosphogluconate, to yield the HO 3CH
2
C O
5-C ketose ribulose-5- HC OH HC OH
4 3
phosphate. HC OH CO2 HC OH
5 4
OH at C3 (C2 of product) is CH2OPO 32 CH2OPO 32
6 5
oxidized to a ketone. 6-phosphogluconate ribulose-5-phosphate

This promotes loss of the

carboxyl at C1 as CO2.
NADP+ serves as oxidant.
 Last Step: Production of Ribose-5-P
 Phosphopentose isomerase
 Converts ribulose 5-phosphate to its aldose
isomer, ribose 5-phosphate
 In some tissues, the pentose phosphate
pathway ends at this point, and its overall
equation is
Net Result of Oxidative Phase
 Net result is the production of
 NADPH, a reductant for biosynthetic reactions
 Ribose 5-phosphate, a precursor for nucleotide synthesis
Regulation of Oxidative Phase
Regulation of Glucose-6-phosphate Dehydrogenase:
 Glucose-6-phosphate dehydrogenase - committed
step of the Pentose Phosphate Pathway
 Regulated by availability of the substrate NADP+
 As NADPH is utilized in reductive synthetic pathways,
the increasing concentration of NADP+ stimulates the
Pentose Phosphate Pathway, to replenish NADPH.
 Induced by insulin
Nonoxidative Phase
 Epimerase – interconversion of stereoisomers -
ribulose-5-P and xylulose-5-P
 Isomerase converts ketose ribulose-5-P to the
aldose ribose-5-P
 Both reactions involve deprotonation to an
endiolate intermediate followed by specific
reprotonation to yield the product
 Both reactions reversible
Generation of Xylulose-5-P and Ribose-5-P
CH2OH

C O
Epimerase
HO C H

CH2OH H C OH

C O CH2OPO32
xylulose-5-
H C OH phosphate
H C OH HC O

CH2OPO32 H C OH
ribulose-5- H C OH
phosphate Isomerase
H C OH

C H2OPO32
ribose-5-
phosphate
Transketolase & Transaldolase
 Catalyze transfer of 2-C or 3-C molecular fragments
respectively, in each case from a ketose donor to an aldose
acceptor.
 D. E. Nicholson has suggested that the names of these
enzymes should be changed, since
 Transketolase actually transfers an aldol moiety
(glycoaldehyde)
 Transaldolase actually transfers a ketol moiety
(dihydroxyacetone)
Transketolase
 Transketolase - 2-C fragment from
xylulose-5-P to either ribose-5-P or erythrose-
4-P.

 Utilizes thiamine pyrophosphate (TPP) as

prosthetic group, a derivative of vitamin B1.

• Pyruvate dehydrogenase of Krebs Cycle also

utilizes TPP as prosthetic group.
 Transketolase…
CH2OH
Transketolase
C O

CH2OH HC O HO C H

C O H C OH H C OH

HO C H H C OH HC O H C OH

H C OH +H C OH H C OH
+ H C OH

CH2OPO32 CH2OPO32 CH2OPO32 CH2OPO32

xylulose- ribose- glyceraldehyde- sedoheptulose-

5-phosphate 5-phosphate 3-phosphate 7-phosphate
 thiazolium O O
H3C ring
aminopyrimidine CH2 CH2 O P O P O
moiety
CH2
+
N O O
N S
C
H acidic H+
H3C N NH2
thiamine pyrophosphate (TPP)

 TPP binds at the active site in a “V” conformation.

 H+ dissociates from the C between N & S in the thiazolium ring.
 Aminopyrimidine amino group is near the dissociable H+, & serves as
H+ acceptor.
 This H+ transfer is promoted by a Glu residue adjacent to the pyrimidine
ring.
 H3C 2
CH2 CH2OPO2OPO 3
TPP
+
CH2 N xylulose-5-P
N S
The thiazolium C
 CH2OH

carbanion reacts H3C N NH2

C O

with the carbonyl C HO C H

of xylulose-5-P to H C OH
form an addition Transketolase CH2OPO32
compound. H3C 2
CH2 CH2OPO2OPO 3
N+ in the thiazole CH2
+
N
ring acts as an e N
C
S

sink, promoting C- HO C CH2OH

H3C N subsequent
C bond cleavage. NH2
HO C H cleavage
active site H C OH
intermediate
CH2OPO32
 2
The 3-C aldose H3C
CH2 CH2OPO2OPO 3
TPP
glyceraldehyde-3-P is CH2
+
N xylulose-5-P
released. N
C
S
 CH2OH
A 2-C fragment H3C N C O
NH2
remains on TPP. HO C H
Completion is by H C OH
reversal of these Transketolase CH2OPO32
steps. H3C 2
CH2 CH2OPO2OPO 3
The 2-C fragment +
CH2 N
condenses with one N S
C
of the aldoses
HO C CH2OH
erythrose-4-P (4-C) H3C N NH2 subsequent
HO C H cleavage
or ribose-5-P (5-C)
active site
to form a ketose-P intermediate
H C OH

product. CH2OPO32
 CH2OH
Transaldolase
C O H2C OH

HO CH C O

HC OH HC O HO CH

HC OH HC O HC OH HC OH

HC OH
+ HC OH HC OH + HC OH

H2C OPO32 H2C OPO32 H2C OPO32 H2C OPO32

sedoheptulose- glyceraldehyde- erythrose- fructose-

7-phosphate 3-phosphate 4-phosphate 6-phosphate

Transaldolase catalyzes transfer of a 3-C dihydroxyacetone

moiety, from sedoheptulose-7-phosphate to glyceraldehyde-3-
phosphate.
Transaldolase has an a,b barrel structure.
 CH2OH CH2OH
Enz-Lys NH2
H Schiff base
C O Enz-Lys N C
+ intermediate
HO CH HO CH

HC OH HC OH
OH
HC OH HC OH
sedoheptulose-
7-phosphate HC OH HC OH

H2C OPO32 H2C OPO32

HC O CH2OH
erythrose-4- H
phosphate HC OH Enz-Lys N C + H+
+
HC OH HO C H

Transaldolase H2C OPO32

In Transaldolase, the e-amino group of a lysine residue

reacts with the carbonyl C of sedoheptulose-7-P to form a
protonated Schiff base intermediate.
 CH2OH CH2OH
Enz-Lys NH2
H Schiff base
C O Enz-Lys N C
+ intermediate
Aldol HO CH HO CH
cleavage HC OH HC OH
releases HC OH

OH
HC OH
erythrose-4- sedoheptulose-
7-phosphate HC OH HC OH
phosphate.
H2C OPO32 H2C OPO32
The Schiff
base stabilizes erythrose-4-
HC O CH2OH
H
the carbanion phosphate HC OH Enz-Lys N
+
C + H+
on C3. HC OH HO C

H

Transaldolase H2C OPO32

Completion of the reaction is by reversal, as the carbanion

attacks instead the aldehyde carbon of the 3-C aldose
glyceraldehyde-3-P to yield the 6-C fructose-6-P.
The diagram at right (3) ribulose-5-P
summarizes flow of IS EP
15 C atoms through ribose-5-P (2) xylulose-5-P
Pentose Phosphate TK
Pathway reactions by glyceraldehyde-3-P
which 5-C sugars are
converted to 3-C and sedoheptulose 7 P
6-C sugars.
IS = Isomerase fructose-6- P TA
EP = Epimerase erythrose-4-P
TK = Transketolase
TK fructose-6-P
TA = Transaldolase
glyceraldehyde-3-P
Balance Sheet – Nonoxidative Phase
The balance sheet below summarizes flow of 15 C atoms through
Pentose Phosphate Pathway reactions by which 5-C sugars are
converted to 3-C and 6-C sugars.
C5 + C5  C3 + C7 (Transketolase)
C3 + C7  C6 + C4 (Transaldolase)
C5 + C4  C6 + C3 (Transketolase)
____________________________
3 C5  2 C6 + C3 (Overall)
Glucose-6-phosphate may be regenerated from either the 3-C
glyceraldehyde-3-phosphate or the 6-C fructose-6-phosphate, via
enzymes of Gluconeogenesis.
Depending on needs of a cell for ribose-5-phosphate,
NADPH, and ATP, the Pentose Phosphate Pathway can operate
in various modes, to maximize different products. There are
three major scenarios:

2 NADP+ 2 NADPH + CO2

glucose-6-P ribulose-5-P ribose-5-P

Pentose Phosphate Pathway producing

NADPH and ribose-5-phosphate

Ribulose-5-P may be converted to ribose-5-phosphate, a

substrate for synthesis of nucleotides and nucleic acids.
The pathway also produces some NADPH.
 2 NADP+ 2 NADPH + CO2
glucose-6-P ribulose-5-P ribose-5-P

fructose-6-P, &
glyceraldehyde-3-P
Pentose Phosphate Pathway producing
maximum NADPH

Glyceraldehyde-3-P and fructose-6-P may be

converted to glucose-6-P for reentry to the linear
portion of the Pentose Phosphate Pathway, maximizing
formation of NADPH.
 2 NADP+ 2 NADPH + CO2
glucose-6-P ribulose-5-P ribose-5-P

fructose-6-P, &
glyceraldehyde-3-P

to Glycolysis
for production of ATP
Pentose Phosphate Pathway producing
NADPH and ATP

Glyceraldehyde-3-P and fructose-6-P, formed from 5-C sugar

phosphates, may enter Glycolysis for ATP synthesis.
The pathway also produces some NADPH.
 2 NADP+ 2 NADPH + CO2
glucose-6-P ribulose-5-P ribose-5-P

fructose-6-P, &
glyceraldehyde-3-P

to Glycolysis
for production of ATP
Pentose Phosphate Pathway producing
NADPH and ATP
Ribose-1-phosphate generated during catabolism of
nucleosides also enters Glycolysis in this way, after first being
converted to ribose-5-phosphate.
Thus the Pentose Phosphate Pathway serves as an entry into
Glycolysis for both 5-carbon & 6-carbon sugars.
 Inborn Errors - Three
 Most common being the result of mutations in glucose-6-phosphate
dehydrogenase (G6PDH).
 Extremely rare occurrences of ribose-5-phosphate isomerase and
transaldolase deficiency
 Transaldolase deficiency - individuals liver problems were the
principal symptom in neonates.
 It should be clear that any disruption in the level of NADPH may
have a profound effect upon a cells ability to deal with oxidative
stress.
 No other cell than the erythrocyte is exposed to greater oxidizing
conditions.
 After all it is the oxygen carrier of the body
Chronic Granulomatous Disease
 Need for NADPH in phagocytic cells – NADPH oxidase
system
 Any defect in enzymes in this process can result in impaired
killing of infectious organisms
 Chronic granulomatous disease (CGD) - syndrome that
results in individuals harboring defects in the NADPH
oxidase system
 Several forms of CGD involving defects in various
components of the NADPH oxidase system
Chronic Granulomatous Disease…
 Individuals with CGD are at increased risk for specific
recurrent infections
 Most common are pneumonia, abscesses of the skin, tissues,
and organs, suppurative arthritis (invasion of the joints by
infectious agent leading to generation of pus), and
osteomyelitis (infection of the bone)
 Majority of patients with CGD harbor mutations in an X-
chromosome gene that encodes a component of the NADPH
oxidase system
 Encoded protein is the β-subunit of cytochrome b245 (gene
symbol CYBB), also called p91-PHOX or NOX2
Chronic Granulomatous Disease…
 Referred to as cytochrome b-negative X-linked CGD
 Autosomal recessive cytochrome b-negative form of CGD
due to defects in the α-subunit of cytochrome b245 (gene
symbol CYBA), also called p22-PHOX or NOX1
 Two autosomal recessive cytochrome b-positive forms of
CGD identified as cytochrome b-positive CGD type I and
type II
 Type I form is caused by mutation in the neutrophil cytosolic
factor 1 (NCF1) gene, which encodes the p47-PHOX
(phagocyte oxidase) protein.
 Type II form is caused by mutation in the NFC2 gene which
encodes the p67-PHOX (phagocyte oxidase) protein