Hexose Monophosphate Shunt 56

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PENTOSE-PHOSPHATE PATHWAY

The Pentose phosphate pathway is also called the Hexose Monophosphate

Shunt and Phosphogluconate Pathway. The pathway is active in cells that are
actively converting glucose and storing it as fats, as well as actively dividing cells
where the demand for ribose-5-phosphate is high. The pathway produces
reducing equivalents (NADPH) for reductive synthesis and 5-carbon precursors for
the synthesis of nucleotides.

The pathway has two phases: oxidative and non-oxidative phase. The
oxidative phase is a 3-step process that generates 2 NADPH/ carbon oxidized or
CO2 produced. This also produces the five-carbon pentoses that are precursors of
nucleotide synthesis. The non-oxidative phase (produced in cells actively
synthesizing fats) recycles the 5-carbon sugars to synthesize glyceraldehyde-3-
phosphate and/or fructose-6-phosphate.

Oxidative phase:

The pathway reroutes glucose-6-phosphate from cellular respiration.

1. Glucose-6-phosphate is acted upon by Glucose-6-phosphate

Dehydrogenase oxidizing C1 (aldehyde or hemiacetal, to a carboxylic acid
in ester linkage (lactone). The oxidation uses NADP+ as electron acceptor.
2. 6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage
(lactone) 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.

3. Phosphogluconate Dehydrogenase catalyzes oxidative decarboxylation of

6-phosphogluconate, to yield the 5-C ketose ribulose-5-phosphate. The
hydroxyl at C3 (C2 of the product) is oxidized to a ketone. This promotes
loss of the carboxyl at C1 as CO2. NADP+ again serves as oxidant (electron
acceptor).
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Reduction of NADP+ (as with NAD+) involves transfer of 2e- plus 1H+ to the
nicotinamide moiety. The reaction is shown below:

The product of the oxidative phase, ribulose-5-phosphate can undergo

isomerization. Additional enzyme involved include
Ribulose-5-phosphate Epimerase, and Ribulose-5-phosphate Isomerase. Their
actions are given below:
Epimerase interconverts the stereoisomers ribulose-5-phosphate and
xylulose-5-phosphate.
Isomerase converts the ketose ribulose-5-phosphate to the aldose ribose-5-
phosphate.
Both reactions involve deprotonation to form an enediolate intermediate, followed
by specific reprotonation to yield the product.
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1. Why is the synthesis of phosphogluconolactone a slow step, while the

subsequent oxidation to phosphogluconate is a very fast step?
2. What are the sources of NADPH in the oxidative phase?
3. Show the mechanism and the Cleland diagram for the reactions of the
oxidative phase of the pentose phosphate pathway. How is the pathway
regulated?
4. How is NADPH synthesis regulated? Explain briefly.

Non-oxidative phase
The reactions involve transfer of 2-C (transketolation) and 3-C
(transaldolation) fragments from donors to receivers.
Transketolase and Transaldolase catalyzes these reactions. The donor is usually
a ketose and acceptor is an aldose. D. E. Nicholson has suggested that the names
of these enzymes should be changed, since Transketolase actually transfers an
aldol moiety (glycoaldehyde) and Transaldolase actually transfers a ketol moiety
(dihydroxyacetone). However the traditional enzyme names are used here.

Mechanism:
1. The first transketolation reaction involves transfer of a 2-C fragment from
xylulose-5-phosphate to ribose-5-phosphate. The positively charged N in
the thiazole ring acts as an electron sink, promoting C-C bond cleavage.
The 3-C aldose glyceraldehyde-3-phosphate is released. A 2-C fragment
remains on TPP.
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Transketolase utilizes the coenzyme thiamine pyrophosphate (TPP), a derivative of

vitamin B1. H+ readily dissociates from the C between N and S in the thiazolium
ring, this being an acidic hydrogen. The positive charge at N and the
electronegativity of S contributes to lowering of the electron density around C. TPP
binds at the active sites of enzymes in a "V" conformation. The amino group of the
aminopyrimidine moiety is close to the dissociable proton, and serves as the proton
acceptor. This proton transfer is promoted by a glutamate residue adjacent to the
pyrimidine ring.

The thiazolium carbanion that results from proton dissociation reacts with the
carbonyl C of xylulose-5-P to form an addition compound.

Overall reaction:

2. The 2-C fragment remaining with TPP condenses with ribose-5-phosphate

(5-C) to form a 7-C ketose-phosphate product, sedoheptulose.
3. The second process is catalyzed by transaldolase that transfers a 3-C
dihydroxyacetone from sedoheptulose-7-phosphate to glyceraldehydes-3-
phosphate to yield fructose-6-phosphate regenerating the 6-carbon sugar
that was oxidized after rerouting to the oxidative phase.
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The e-amino group of an active site lysine residue reacts with the carbonyl C of
sedoheptulose-7-phosphate to form a protonated Schiff base intermediate. Aldol
cleavage results in release of erythrose-4-phosphate. The Schiff base stabilizes
the carbanion on C3. Completion of the reaction occurs by reversal, as the
carbanion attacks instead the aldehyde carbon of the 3-carbon aldose
glyceraldehyde-3-phosphate to yield the 6-carbon fructose-6-phosphate.

4. The 3rd step is the second transketolation reaction which transfers 2-C
fragments to the 4-C aldose erythrose-4-phosphate yielding fructose-6-
phosphate and glyceraldehydre-3-phosphate.
5. Glyceraldehyde-3-phosphate can condense with DHAP (isomerization
product of glyceraldehydes-3-phosphate) to form again, fructose-6-
phosphate.
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Ribulose-5-phosphate may be converted to ribose-5-phosphate, a substrate for

synthesis of nucleotides and nucleic acids. The pathway also produces some
NADPH.

Glyceraldehyde-3-phosphate and fructose-6-phosphate, formed from the 5-carbon

sugar phosphates, may be converted to glucose-6-phosphate for re-entry into the
oxidative phase of the Pentose Phosphate Pathway, maximizing formation of
NADPH
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5. Both TPP and lysine (of the transketolase) bind substrate for the reaction.
What effect will this binding have on the substrates of the enzymes?
6. What advantage(s) will the reactions of the non-oxidative phase of the
hexose monosphosphate shunt have in terms of energy utilization?
7. When does the process not proceed to the non-oxidative phase? Explain
briefly.
8. What is/are the function(s) of the pentose phosphate pathway? Explain
briefly.
9. The hexose monophosphate shunt also protects cells such as RBC from
oxidative stress. How? Explain briefly.

References:

1. http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/pentose.htm
2. Mathews, C.K., Van Holde, K.E., and Ahern, K.G. (2000). Biochemistry, 3rd
Ed. San Francisco: Addison-Wesley Publishing Company.
3. Horton, R.H., Moran, L.A., Ochs, R.S., Rawn, J.D. and Scrimgeour, K.G.
(1996) Principles of Biochemistry, 2nd ed. New Jersey: Prentice-Hall
International, Inc.