04 metabolism of carbohydrates module : 21...

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Biochemistry Metabolism of Carbohydrates

Pentose Phosphate Pathway (PPP)

Dr. Vijaya Khader Dr. MC Varadaraj

Paper : 04 Metabolism of carbohydrates

Module : 21 Pentose Phosphate Pathway

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Description of Module

Subject Name Biochemistry

Paper Name 04 Metabolism of carbohydrates

Module Name/Title 21 Pentose Phosphate Pathway (PPP)

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PENTOSE PHOSPHATE PATHWAY (PPP)

Objectives

1. To understand the function of pentose phosphate pathway in production of NADPH

and ribose precursors for synthesis of nucleic acid.

2. To examine the importance of NADPH in protection of cell against highly reactive

oxygen species.

3. To relate defects in pentose phosphate pathway to disease condition.

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Introduction

Glucose is catabolised by way of glycolytic pathway in to two molecules of

pyruvate , then pyruvate is oxidized by citric acid cycle to produce ATP In

animal tissue

There is an another metabolic pathway, called as the Pentose Phosphate

Pathway (PPP), which is also termed as Hexose Monophosphate Pathway (HMP

shunt) -or Phosphogluconate pathway

The oxidation of glucose without direct consumptions occur by pentose

phosphate pathway

Thus, this pathway generate Ribose sugar, ATP, two NADPH

The pentose phosphate pathway is an optional path for the oxidation of glucose.

In mammals, NADPH acts as universal reducing agent in anabolic pathway

Mammary gland, adipose tissue, liver require large amount of NADPH for the

synthesis of fatty acid and glycerol

This pathway is absent in tissues which are less functional in fatty acid production

The generation of pentose (ribose) sugar is necessary for synthesis of nucleic acid

Erythrose -4 phosphate generated by PPP pathway is utilized for the synthesis of

aromatic amino acids

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Reactions of PPP occur in the cytosol in two phase as NADP + is used as hydrogen

acceptor

The PPP can be divided into following phases

The oxidative (non- reversible) phase

The non oxidative (reversible) phase

Phase of Pentose Phosphate Pathway (PPP)

In the first step, glucose -6-phosphate (G6P) is converted into ribulose-5-phosphate

and CO2.During this oxidation reaction, NADP+ is reduced in to NADPH

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In subsequent step of pathway, ribulose 5-phosphate (R5P) converted into other

pentose 5-phosphate (P5P) containing ribose 5-phosphates used to produced

nucleic acids

During succeeding step, a sequence of reactions occur which convert three of

pentose -5 phosphate into one molecules of triose and two molecules of hexose

During last step, some of these sugars are converted back into glucose -6-phosphate

so the cycle can be repeat again

OXIDATIVE PHASE

1. Enzyme: Glucose -6-phosphate dehydrogenase

First enzymatic step in oxidative phase reduce NADP+ to NADPH

Mechanism: Oxidation reaction of C1 position

Lactone is form by hydride transfer to the NADP+, which is an intra molecular ester

Reaction start with glucose 6- phosphate dehydrogenase, which oxidize the aldehyde

C1 of glucose 6-phosphate to a carboxylate group

Glucose aldehyde normally exists a hemicetal pyran ring, in which an aldehyde has

combined with O-5

The caboxylate anion derive from glucose is gluconate,and cyclic ester of this type

are called lactones, hence the product name is 6-phospho glucono lactone

Energetic: Very favourable reaction in the forward direction for NADPH production

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2. Enzyme: Lactonase

It is a specific enzyme that target 6- phosphoglucono-δ-lactone for hydrolysis

Mechanism

The lactoneis openedby hydrolysis, the additional water molecules to break a bond

usually a kind of amide or ester

In this case, since lactone is intra molecular, then 6- phospho-glucono-δ-lactone is

opened up to the acid form, gluconate

Energetic: Thistype of hydrolysis reactionsare favourable

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3. Enzyme: 6-Phospho gluconate dehydrogenase

It carryout the conversion of 6 carbon skeleton to a pentose with oxidative

decarboxylation

Mechanism: The C1 carboxylate is removed. The C3 position is oxidized to a ketone,

creating 5 carbon ketose, ribulose

4. Enzyme: Phosphopentoseisomarase

Mechanism: Typical ketose to aldose conversion

This is a acid base catalysed reaction

Ribulose 5-phosphate has noimmediateuse in the cell, so is rearranged in to other

form

Isomerization of ribose -5 phosphate,which can be used for RNA ,DNA and

nucleotide

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NON OXIDATIVE PHASE

Conversion of pentose phosphate to glucose -6 phosphate

In cell that require high level of NADPH for biosynthetic reaction, the ribulose -5P

produced in the oxidative phase need to be a turn back in to a glucose -6 phosphate

to maintain flux through the glucose -6P dehydrogenase reaction

The carbon shuffle reaction of non-oxidative phase which ultimately are used to

regenerate glucose -6P using transketolase and trans aldolase enzyme.

5. Enzyme: Ribulose -5 phosphate epimerase

Ribulose 5-phosphate Xylulose -5 phosphate

Mechanism: The turn of stereo centre of the substrate in the epimerization reaction. When

only one stereo centre is flipped an epimer of compound occur

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6. Enzyme: Trans ketolase and trans aldolase

This step isessential to change pentose back to glucose ,but beside to epimarase,

two enzymes namely (i) Trans ketolase and (ii) Trans aldolase are needed

Transketolase

TPP is use to stabilize a 2 carbon carbanion intermediate. TPP is a co factor of

transketolase

Trans aldolase

Protonated Schiff base intermediate with a ketose is formed by trans

aldolase,stabilizing 3 carbon carbanion intermediate, which allow an aldehyde based

sugar to react with enzyme linked ketose. Themechanism is similar to aldolase

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Reaction required to convert 5-pentose to 6- hexose

The pentose are converted in to 6and 3 carbon sugars. The way to decipher it is to

remember two key concept

1. Either 3 carbon unit (one reaction) or 2-carbon unit (two reaction) are transferred

between acceptor and donor molecules .the enzyme responsible for the 3-carbon

transfer is called trans aldolase, and the enzyme responsible for the transfer of 2-

carbon units is called trans ketolase.

2. The number of carbon involved in the reactions add up to either ten (two reaction)

or nine (one reaction).

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Trans ketolase transfer a 2- carbon fragment containing ketone group from xylulose -5 phosphate to ribose -5 phosphate.

Trans ketolse require aTPP,a derivative of vitamin B1as a co enzyme and Mg +2 as a co factor.

Transfer of 2 carbon fragment to 5-C ribose-5-P yield sedoheptulose 7-P and glyceraldehyde

3-P.

Trans aldolase catalyzes trnsfer of 3 carbon from sedohepyulose -7P to glyceraldehyde 3-Phosphste to form erythrose 4-phosphate and fructose -6 phosphate

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˟

Transketolse transfer of 2-C fragment from xylulose -5P to erythrose 4P to yield fructose -6

P and glyceraldehyde 3-P.

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SUMMARY

The balance sheet below summarizes flow of 15Catom through PPP reaction by which 5-C

sugar are converted to 3-C and 6-C .

5+5 TK 3 +7

7+3 TA 4+6

4+5 TK 3+6

-----------------------------------------

3C5 2C6 +C3

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SUMMARY OF PPP

Stage Enzyme

Reaction characteristic

Comments Non carbohydrate Irreversible?

Substrate Product

Oxidative phase

Glucose6Pdehurogenase NADP+

NADPH Yes Regulate by

NADPH/NADP+

ratio

Gluconolactonase H2O Yes

6-phosphogluconolactone dehydrogenase

NADP+ NADPH

CO2 Yes

Non oxidative

Trans ketolase -- -- No

Transfer 2C unit from ketose to

aldose

Trans aldolase -- -- No

Transfer 3C unit from ketoes to

aldose

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PPP PROTECT CELL AGAINST REACTIVE OXYGEN SPECIES

Reduction of molecular O2 in a series of one electron step yield superoxide, hydrogen

peroxide, hydroxyle redical and water.The intermediate activated form of oxygen are also

known as reactive oxygen species (ROS)

NADPH and glutathione is protect cell against ROS

RBCs lack mitochondria and thus lack the enzyme of citric acid cycle . Therefore, glucose

is metabolized exclusively by glycolytic cycle (90%) and pentose phosphate pathway

(10%)

The pentose phosphate pathway is also responsible for maintaining high leval of NADPH

in red blood cells which is used as a reductant in the glutathione redactase reaction

Glutathion is a tripeptide that has a free sulfhydryl group which function as an electron

donor in a veriety of coupled redox reaction in the cell

In erythrocyte, electrons from glutathione are used to keep cystein residue in

hemoglobin in the reduced state,and for reducing harmful reactive oxygen species and

hydroxyl free redicals that damage proteins and lipids through oxidation induced

cleavage reactions

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Glutathione reductase is flavoprotein that contain the co enzyme FAD and is releted to

ferredoxin-NADP+ reductase

To maintain the reduced state of glutathione, glutathione reductase uses two

electrons available from NADPH (GSSG--->2 GSH)

In erythrocytes to reduced hydrogen peroxide (H2O2) levals through a GSH dependent

redox reaction catalyzed by the enzyme glutathione peroxidase, High leval of GSH, and

therefor high leval of NADPH,are needed

When erythrocytes are exposed to chemicals that generates high levals of superoxide

redicals, to reduce these damaging compounds GSH require

In erythrocytes suffients levals of NADPH to maintain the GSH:GSSG ratio at about

500:1, normally provides by An active pentose phosphate pathway

Glucose-6Pdehydrogenase (G6PD) deficiency is the most comon enzyme deficiency in

the world, effecting over 400 million people

In the mid 1950 G6PD deficiency is discover came as result of observation made by 30

years earlier when it was noticed that the anti malarial drug primaquine induce acute

hemolytic anemia was found in some of people who had been treated with primaquine

prophylatically

People having deficiency of G6PD can not tolerate primaquine because their

erythrocytes do not hold enough GSH to detoxify the reactive oxygen species

produced by the compound

Primaquine work as an anti malarial drud because productive infection of the mosquito

born micro organisms, plasmodium is hinderd in erythrocytes under condition in which

NADPH level are decreased due to increased level of oxidative stress

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Glutathione and NADPH role in protecting cells against ROS

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Table show drug and chemicals that have been shown to cause clinical significant hemolytic

Anemia inG6PD deficiency

Acetenilid Niridazole Primaquine Sulfapyridine

Methylene blue Nitrofurantoin Pentaquine Sulfamethoxazole

Nalidixic acid Phenyle hydrazine Sulfanilamide Thiazolesufone

Naphthalene Toludine blue Sulfacetamide Trinitrotoluene

Favism was also caused by the same enzyme defect. I t was observed that certain

people eat food containing fava beans , a main ingradient in the mediterranean dish

falafel, they would become very sick

It is now known that same acute hemolytic anemia seen in individual with G6PD who

treated with primaquine also explaine symptoms of favism. One of the active compond

in fava beans is called vicine - a toxic glycoside that induces oxidative stress in

erythrocytes.

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REGULATION OF PPP

The initial step of pentose phosphate pathaway is irreversible commited step

This step is catalyzed by the enzyme glucose -6 phosphate dehydrogenase and

regulated allostrerically

Product of this reaction is NADPH which is a strong inhibitor of enzyme glucose 6-

phosphate dehydrogense, so, when the concentration of NADPH is high, the enzyme

glucose 6- phosphate dehydrogense activity is low. As, NADPH is used in different

pathway, inhibition is relived,and the enzyme is incresed to produce more NADPH. This

reaction is also allosterically regulated

The transcription of the gene for this enzyme is regulated by hormon

Figure show regulation of G6PD activity control flux through the glycolytic pathway and

pentose phosphate pathway

The synthesis of glucose -6 phosphate dehydrogenase is induced by the incresed

insulin/glucagne ratio after a high carbohydrate meal

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Insulin,which secreted in response to hyperglycemia, induce the synthesis of G6P

dehydrogenase and -6 phospho gluconate dehydrogenase increasing the rate of glucose

oxidation by PPP

The synthesis of glucose 6-phosphatedehydrogenase is repressed during fasting.

Importance of PPP (The main generator of NADPH)

PPP is the main generator of NADPH

NADPH is needed for reductive biosynthesis of fatty acid, cholesterol, steroid

hormon & spingoshine. Thus it is active in lactating mammary gland, liver, gonads

adipose tissue & adrenal cortex

NADPH is used in hydroxylation reaction during metabolisms of phenylalanine and

tryptophan

NADPH is used for the synthesis of Nitric Oxide (NO): Arginine + O2 + NADPH +H+

→ NADP + NO + Citruline

NO is a laughing gas, used as ansthetic, causes relaxation of vascular

smooth muscles

In macrophages, NO is effective against viral, fungal, protozoal infections.

NO potent inhibitors of platelet aggeregation

NO act as a neurotransmitter in brain

PPP provides a way for oxidation of glucose by other then TCA cycle with no production

energy

PPP provides Ribose -5 P to the cells needed for nucleoside, nucleotides, nucleic acid &

coenzyme biosynthesis

04 metabolism of carbohydrates module : 21...

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