20 Pentose Phosphate Pathway

Dr. Ramesh Kothari

epgp books

   

 

 

 

 

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.

 

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
  • 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
  • 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

 

 

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

 

 

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

   

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                           Xyluosphate

 

 

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

 

 

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
  • 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

 

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).

 

Trans catalase 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

 

Transketolse transfer of 2-C fragment from xylulose -5P to erythrose 4P to yield fructose -6 P and glyceraldehyde 3-P.

 

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 .

 

SUMMARY OF PPP

 

 

 

  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
  • 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

 

Glutathione and  NADPH role in protecting cells against ROS

 

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. It 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.

 

 

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
  • 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

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

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

Ø NO potent inhibitors of platelet aggeregation

Ø 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
you can view video on Pentose Phosphate Pathway

 

 

References

  • Kruger, nicholass;von schaewan,Antje(june 2003).the oxidative pentose phosphate pathway: structure and organisation”.currentopinion in plant biology 6(3):235-246
  • Keller, markus A;turchyn alexandra V;ralser,markus(25 april 2015).” Non enzymatic glycolysis and pentose phosphate pathway like reactionin a plausiblearchean ocean”.molecular system biology. 10(4):725-725
  • Bryant miles,(january 23 ,2013) pentose phosphate pathwayAka the hexose monophosphate shunt.

Web site

 

Books

  • Harper’sIllustrated Biochemistry by Robert K.. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
  • Lehninger’S Principle of Biochemistry David L. Nelson and Michael M. Cox,5th Edition (2008)
  • Instant Notes: Biochemistry, 2nd Edition, B.D. Hames & N. M. Hooper (2005)