Pentose Phosphate Pathway (PPP)

Transcription

1 Paper : 04 Metabolism of carbohydrates Module : 21 Pentose Phosphate Pathway Dr. Vijaya Khader Dr. MC Varadaraj 1

2 Description of Module Subject Name Paper Name Module Name/Title 04 Metabolism of carbohydrates 21 2

3 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. 3

4 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 4

5 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 In the first step, glucose -6-phosphate (G6P) is converted into ribulose-5-phosphate and CO 2. During this oxidation reaction, NADP + is reduced in to NADPH 5

6 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 6

7 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 7

8 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 creating 5 carbon ketose, ribulose C3 position is oxidized to a ketone, 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 8

9 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 9

10 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 10

11 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). 11

12 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 12

13 Transketolse transfer of 2-C fragment from xylulose -5P to erythrose 4P to yield fructose -6 P and glyceraldehyde 3-P. 13

14 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 TA TK C 5 2C 6 +C 3 14

15 SUMMARY OF PPP Stage Oxidative phase Non oxidative Enzyme Glucose6Pdehurogenase Reaction characteristic Non carbohydrate Substrate Product Irreversible? NADP + NADPH Yes Gluconolactonase H 2 O Yes 6-phosphogluconolactone dehydrogenase NADP + NADPH CO 2 Trans ketolase No Trans aldolase No Yes Comments Regulate by NADPH/NADP + ratio Transfer 2C unit from ketose to aldose Transfer 3C unit from ketoes to aldose 15

16 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 16

17 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 (H 2 O 2 ) 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 17

18 Glutathione and NADPH role in protecting cells against ROS 18

19 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. 19

20 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 20

21 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 + O 2 + 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 21