The PENTOSE PHOSPHATE pathway ,by carring out oxidation and decarboxylation of the 6-C sugar glucose-6-P, is basically

used for the synthesis of NADPH and 5-C sugar ribulose-5-P It plays only a minor role (compared to GLYCOLYSIS) in degradation for ATP energy

Pentose phosphate pathway

Other names: Pentose phosphate Shunt Hexose Monophosphate Shunt

Phosphogluconate pathway The pentose phosphate pathway is an alternate route for the oxidation of glucose.

Pentose phosphate pathway

Two stages: Oxidative portion (NADPH producing) Non-oxidative (carbon recycling/unit transferring) Location: cytosol Original material: glucose 6-phosphate End product: NADPH , pentose phosphate Important in adipose tissue, adrenal cortex, liver (biosynthesis)Important in red blood cells (antioxidant reasons)

The pentose pathway is a shunt.

The pathway begins with the glycolytic intermediate glucose 6-P. It reconnects with glycolysis because two of the end products of the pentose pathway are glyceraldehyde 3-P and fructose 6-P; two intermediates further down in the glycolytic pathway. It is for this reason that the pentose pathway is often referred to as a shunt.

Its a shunt

*** The significance of PPP

1. Produce ribose 5-phosphate needed for DNA and RNA synthesis 2. Generate reducing equivalents NADPH 1) Reducing power for biosynthesis of fatty acids, cholesterol, folate, and so on 2) Coenzyme of glutathione reductase to keep the normal level of reduced glutathione 3) NADPH serves as the coenzyme of mixed function oxidases (mono-oxygenases)

Pentose phosphate pathway protects cells against reactive oxygen species (ROS) Molecular oxygen and partially reduced, reactive forms of oxygen.
Reduction of molecular O2 in a series of one-electron steps yields superoxide, hydrogen peroxide, hydroxyl radical, and water. The intermediate, activated forms of oxygen are known as reactive oxygen species (ROS)

O H 3N
+

O N H CH CH2 SH C N H CH2 COO

H C

CH2

CH2

COO

-glutamyl-cysteinyl-glycine Glutathione

Glutathione is a tripeptide that includes a Glu linked by an isopeptide bond involving the side-chain carbonyl group. Its functional group is a cysteine thiol. One role of glutathione is degradation of hydroperoxides, that arise spontaneously in the oxygen-rich environment in red blood cells. Hydroperoxides can react with double bonds in fatty acids

O H 3N
+

O N H CH CH2 SH C N H CH2 COO

H C

CH2

CH2

COO

-glutamyl-cysteinyl-glycine Glutathione

Glutathione Peroxidase catalyzes degradation of organic hydroperoxides by reduction, as two glutathione molecules (represented as GSH) are oxidized to a disulfide.

2 GSH + ROOH GSSG + ROH + H2O

Glutathione Peroxidase uses the trace element selenium as functional group. The enzyme's primary structure includes an analog of cysteine, selenocysteine, with Se replacing S.

Regeneration of reduced glutathione requires NADPH, produced within erythrocytes in the Pentose Phosphate Pathway. Glutathione Reductase catalyzes: GSSG + NADPH + H+ 2 GSH + NADP+ Genetic deficiency of Glucose-6-P Dehydrogenase can lead to hemolytic anemia, due to inadequate [NADPH] within red blood cells. The effect of partial deficiency of Glucose-6-phosphate Dehydrogenase is exacerbated by substances that lead to increased production of peroxides (e.g., the antimalarial primaquine).

Role of NADPH and glutathione in protecting cells against ROS

Role of NADPH and glutathione in protecting cells against highly reactive oxygen derivatives. Reduced glutathione (GSH) protects the cell by destroying hydrogen peroxide and hydroxyl free radicals. Regeneration of GSH from it oxidized form (GSSG) requires the NADPH produced in the glucose 6phosphate dehydrogenase reaction.

Glucose-6-Phosphate Dehydrogenase Deficiency

G6PD produces NADPH, necessary to keep glutathione reduced, which detoxifies free radicals/peroxides Decreased NADPH leads to hemolytic anemia due to damage from oxidizing agents X-linked recessive, most common enzyme deficiency

Glucose-6-Phosphate Dehydrogenase Deficiency

Oxidizing agents include fava beans, sulfanamides, primaquine, anti-TB drugs Affected individuals have malarial resistance Heinz bodies
Altered precipitated hemoglobin in RBCs

Bite Cells
From phagocytic removal of Heinz bodies by spleen

Glucose-6-phosphate dehydrogenase deficiency causes hemolytic anemia

Mutations present in some populations causes a deficiency in glucose 6-phosphate dehydrogenase, with consequent impairment of NADPH production. Detoxification of H2O2 is inhibited, and cellular damage results - lipid peroxidation leads to erythrocyte membrane breakdown and hemolytic anemia. Most G6PD-deficient individuals are asymptomatic - only in combination with certain environmental factors (sulfa antibiotics, herbicides, antimalarials,) do clinical manifestations occur.

Conditions for hemolytic anemia related G6PD deficiency.

The ingestion of oxidative agents that generate peroxides or reactive oxygen species (ROS).
Antimalarials - pamaquine Sulpha drugs.

Individules with G6PD deficiency can not produce sufficient GSH to cope with the ROS. Proteins become cross linked leading to Heinz body formation and cell lysis.

Molecular Biology of G6PD

G6PD enzyme is located on sex chromosome X at q28 locus. Thus, G6PD enzyme is X-linked gene. Majority of the variants - from a single pointmutation resulting in amino acid substitution in gene encoding for G6PD located at the Xq28 region on the tip of the long arm of the Xchromosome.

Laboratory diagnosis
CBC retic profile urinalysis LDH/haptoglobin fractionated bilirubin blood smear with stains for Heinz bodies
o

G6PD fluroescent spot test

Detects deficient production of NADPH from NADP. In this test NADPH is fluorescent and its absence (due to G6PD deficiency)

results in lack of fluorescence.

G6PD spectrophotometry to detect level of activity genetic test for variants

Regulation of pentose phosphate pathway

The entry of glucose 6-phosphate into the pentose phosphate pathway is controlled by the cellular concentration of NADPH NADPH is a strong inhibitor of glucose 6-phosphate dehydrogenase As NADPH is used in various pathways, inhibition is relieved, and the enzyme is accelerated to produce more NADPH The synthesis of glucose 6-phosphate dehydrogenase is induced by the increased insulin/glucagon ratio after a high carbohydrate meal.

6CH2OPO32

Glucose-6-phosphate Dehydrogenase
OH NADP
1

H
4

O H
2

6 CH OPO 2 2 3 + NADPH + H 5 + O

6-Phosphogluconolactonase
H 2 O H+ O
1

O
1C

O OH

HC
2

H OH
3

H OH
3

H
2

HO 3CH HC OH
4

OH

OH

HC
5 6

OH

OH

OH

CH2OPO32

glucose-6-phosphate

6-phoshogluconolactone

6-phosphogluconate

Glucose-6-phosphate Dehydrogenase catalyzes oxidation of the aldehyde (hemiacetal), at C1 of glucose-6-phosphate, to a carboxylic acid, in ester linkage (lactone). NADP+ serves as electron acceptor.

6CH2OPO32

Glucose-6-phosphate Dehydrogenase
OH NADP
1

H
4

O H
2

6 CH OPO 2 2 3 + NADPH + H 5 + O

6-Phosphogluconolactonase
H 2 O H+ O
1

O
1C

O OH

HC
2

H OH
3

H OH
3

H
2

HO 3CH HC OH
4

OH

OH

HC
5 6

OH

OH

OH

CH2OPO32

glucose-6-phosphate

6-phoshogluconolactone

6-phosphogluconate

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.

O
1C

O OH

Phosphogluconate Dehydrogenase
NADP+ NADPH + H+
1CH2OH 2

HC
2

HO 3CH HC OH
4

O OH OH

HC CO2
2

HC
5 6

OH

HC
4 5

CH2OPO 3

CH2OPO 32

6-phosphogluconate

ribulose-5-phosphate

Phosphogluconate Dehydrogenase catalyzes oxidative decarboxylation of 6-phosphogluconate, to yield the 5C ketose ribulose-5-phosphate. The OH at C3 (C2 of product) is oxidized to a ketone. This promotes loss of the carboxyl at C1 as CO2. NADP+ serves as oxidant.

NAD+ & NADP+ differ only in the presence of an extra phosphate on the adenosine ribose of NADP+. This difference has little to do with redox activity, but is recognized by substrate-binding sites of enzymes. It is a mechanism for separation of catabolic and synthetic pathways.

Nicotinamide Adenine Dinucleotide

O

O C NH2

CH2 H H OH

+ N O H H OH NH2 N N

nicotinamide

P O

CH2 H H OH

N O H

adenine esterified to Pi in NADP+

H OH

Regulation of Glucose-6-phosphate Dehydrogenase: Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is 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 produce more NADPH. The rest of the pathway converts ribulose-5-P to the 5-C product ribose-5-P, or to 3-C glyceraldehyde-3-P & 6-C fructose-6-P. Additional enzymes include an Isomerase, Epimerase, Transketolase, and Transaldolase.

Epimerase inter-converts stereoisomers ribulose-5-P and xylulose-5-P. Isomerase converts the 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 are reversible. Transketolase & Transaldolase catalyze transfer of 2-C or 3-C molecular fragments respectively, in each case from a ketose donor to an aldose acceptor.

CH2OH C O H OH

Epimerase
CH2OH C H H C C O OH OH

HO H

C C

CH2OPO32

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

CH2OPO32

ribulose-5H phosphate Isomerase

H

CH2OPO32

ribose-5phosphate

Transketolase & Transaldolase catalyze transfer of 2C or 3-C molecular fragments respectively, in each case from a ketose donor to an aldose acceptor. Scientists have 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.

Transketolase
CH2OH C HO H C C O H OH H H HC C C C O OH OH OH H HC C O OH HO H H

CH2OH C C C C C O H OH OH OH

CH2OPO32

CH2OPO32

CH2OPO32

CH2OPO32

xylulose5-phosphate

riboseglyceraldehyde- sedoheptulose5-phosphate 3-phosphate 7-phosphate

Transketolase transfers a 2-C fragment from xylulose-5P to either ribose-5-P or erythrose-4-P. Transketolase utilizes as prosthetic group thiamine pyrophosphate (TPP), a derivative of vitamin B1. Pyruvate Dehydrogenase of Krebs Cycle also utilizes TPP as prosthetic group.

Transketolase
CH2OH C HO H C C O H OH H H HC C C C O OH OH OH H HC C O OH HO H H

CH2OH C C C C C O H OH OH OH

CH2OPO32

CH2OPO32

CH2OPO32

CH2OPO32

xylulose5-phosphate

riboseglyceraldehyde- sedoheptulose5-phosphate 3-phosphate 7-phosphate

Transfer of the 2-C fragment to the 5-C aldose ribose-5-phosphate yields sedoheptulose-7phosphate. Transfer of the 2-C fragment instead to the 4-C aldose erythrose-4-phosphate yields fructose-6-phosphate.

CH2OH C HO CH HC HC HC H2C OH OH OH HC O

Transaldolase
H2C C HC O OH OPO32 HC HC H2C O OH OH HO CH HC OH OH OPO32 OH O

HC H2C

HC H2C

OPO32

OPO32

sedoheptulose- glyceraldehyde- erythrose- fructose7-phosphate 3-phosphate 4-phosphate 6-phosphate

Transaldolase catalyzes transfer of a 3-C dihydroxyacetone moiety, from sedoheptulose-7phosphate to glyceraldehyde-3-phosphate.

Enz-Lys

NH2 HO

CH2OH C CH HC OH OH OH OPO32 HC O OH OH OPO32 Enz-Lys OH O Enz-Lys H N + HO

CH2OH C CH HC HC HC H2C OH OH OH OPO32 CH2OH H N + HO C C H + H+

Schiff base intermediate

sedoheptulose7-phosphate HC
H2C

HC

erythrose-4phosphate

HC HC

Transaldolase

H2C

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.

Enz-Lys

NH2 HO

CH2OH C O H Enz-Lys N + HO OH

CH2OH C CH HC HC HC H2C O OH OH OPO32 Enz-Lys H N + HO OH OH OH OPO32 CH2OH C C H + H+

Aldol cleavage releases erythrose-4phosphate.

Schiff base intermediate

CH HC OH OH OH OPO32

sedoheptulose7-phosphate HC
H2C

HC

The Schiff HC base erythrose-4phosphate HC stabilizes the HC carbanion on C3. H2C Transaldolase

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 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. IS = Isomerase EP = Epimerase TK = Transketolase TA = Transaldolase

(3) ribulose-5-P
IS EP

ribose-5-P
TK

(2) xylulose-5-P

glyceraldehyde-3-P sedoheptulose 7 P fructose-6- P erythrose-4-P

TK

TA

fructose-6-P glyceraldehyde-3-P

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-6phosphate, 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.

Regulatory enzyme

5 carbon atoms

Summary of the pentose phosphate pathway

3G6P + 6NADP+ + 3H2O

6NADPH + 6H+ + 3CO2 + 2F6P + GAP Important intermediates

Ribose-5-phosphate (nucleic acids, histidine) Erythrose-4-phosphate (aromatic amino acids)

 GLYCOLYSIS 1. Starts from glucose. 2. Breakdown of glucose to pyruvate or lactate. 3. 2 types aerobic & anaerobic. 4. 2 ATPs used. 5. ATPs are produced. 6. Produces NADH2. 7. Occurs in all cells.

HMP Shunt 1. Starts from G6P. 2. Breakdown of glucose to diff metabolites. 3. No sub type. 4. 5. 6. 7. No ATPs used. No ATP is produced. Produces NADPH2. Occurs in tissues of lipid, steroids synthesis, sex glands, adrenal cortex etc.

 GLYCOLYSIS 8. Oxidation does not occur in 1st step. 9. CO2 is not produced. 10. No pentose is produced. 11. Only 1 molecule of glucose is used. 12. End products are dependent on TCA cycle.

HMP Shunt 8. Oxidation occurs in 1st step. 9. CO2 is produced. 10. Pentoses are produced i-e ribose & ribulose. 11. 3 molecules of glucose are used. 12. End products are not dependent on TCA cycle.