Pentose Phosphate Pathway

2010. dr. waseem kausar

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

OBJECTIVESTo understand the function of the pentose

phosphate pathway in production of NADPH and ribose precursors for nucleic acid synthesis.

To examine the importance and uses of NADPH in protection of cells against highly reactive oxygen species.

To relate defects in the pentose phosphate pathway to disease conditions.

Other names: Phosphogluconate Pathway

Hexose Monophosphate Shunt- active in liver, adipose tissue, adrenal cortex, thyroid,

erythrocytes, testes, and lactating mammary gland not active in non-lactating mammary gland and has

low activity in skeletal muscle. . Generation of NADPH

- mainly used for reductive syntheses of fatty acids, steroids, amino acids via glutamate dehydrogenase; and production of reduced glutathione in erythrocytes and other cells.

Production of ribose residues for nucleotide and nucleic acid synthesis.

No ATP generation 3 molecules of Glu-6-phos gives = 3Co2 & 3 five C

sugar.In tissues requiring primarily NADPH rather than ribose 5-phosphate, these pentose phosphates can be recycled into glucose 6-phosphate. Overall, 6 five-carbon sugars are converted to 5 six-carbon sugars

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.

H

CNH2

O

CH2

H

N

HOH OH

H HOOP

O

HHOH OH

H HO

CH2

N

N

N

NH2

OP

O

O

O+

N O

nicotinamide

adenine

esterified to Pi in NADP+

Nicotinamide Adenine Dinucleotide

Reactions of the pentose phosphate pathway occur in the cytosol in two phases

Oxidative non-reversible phaseNon-oxidative reversible phaseNADP+, not NAD +, is used as hydrogen acceptor1st phase1st phase

- Glucose 6-phosphate undergoes dehydrogenation and decarboxylation to give a pentose, ribulose 5-phosphate, which is converted to its isomer, D-ribose 5-phosphate.- Overall equation of 1st phase:Glucose 6-phosphate + 2 NADP++ H2O

ribose 5-phosphate + CO2 + 2 NADPH + 2 H+

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.6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage, resulting in ring opening. The product is 6-phosphogluconate

H O

O H

H

O HH

O H

CH 2O PO 32

H

H

O H H O

O H

H

O HH

O H

CH 2O PO 32

HO

23

4

5

6

11

6

5

4

3 2

C

HC

CH

HC

HC

CH 2O PO 32

O O

O H

HO

O H

O H

NAD PH + H +

NADP + H 2O H +

1

2

3

4

5

6

G lucose-6-phosphate D ehydrogenase

6-Phospho- glucono-lactonase

glucose-6-phosphate 6-phoshogluconolactone 6-phosphogluconate

Phosphogluconate Dehydrogenase catalyzes oxidative decarboxylation of 6-phosphogluconate, to yield the 5-C 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.

C

HC

CH

HC

HC

CH 2OPO 32

O O

OH

HO

OH

OH

1

2

3

4

5

6

CH 2OH

C

HC

HC

CH 2OPO 32

OH

OH

1

2

3

4

5

ONADP + NADPH + H+

CO 2

Phosphogluconate Dehydrogenase

6-phosphogluconate ribulose-5-phosphate

NON-OXIDATIVE PHASE, Reversible reactions

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.

C

C

C

CH2OPO32

O

OHH

OHH

CH2OH

C

C

C

CH2OPO32

O

HHO

OHH

CH2OH

C

C

C

CH2OPO32

OH

OHH

OHH

HC O

H

ribulose-5- phosphate

xylulose-5- phosphate

ribose-5- phosphate

Epimerase

Isomerase

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

Transketolase actually transfers an aldol moiety (glycoaldehyde), and

Transaldolase actually transfers a ketol moiety (dihydroxyacetone).

Transketolase transfers a 2-C fragment from xylulose-5-P 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.

C

C

C

CH 2O P O 32

O

HHO

OHH

CH 2O H

C

C

C

CH 2 O P O 32

OH

OHH

OHH

HC O

H C

C

C

CH 2O P O 32

OH

OHH

OHH

C H

H

HC

C

CH 2O P O 32

O

OHH

C

CH 2O H

O

HO

+ +

x y lu lo se - r ib o se - g ly c e ra ld e h y d e - se d o h e p tu lo se - 5 -p h o sp h a te 5 -p h o sp h a te 3 -p h o sp h a te 7 -p h o sp h a te

T ran sk e to la se

Transfer of the 2-C fragment to the 5-C aldose ribose-5-phosphate yields sedoheptulose-7-phosphate.

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

C

C

C

CH 2O P O 32

O

HHO

OHH

CH 2O H

C

C

C

CH 2O P O 32

OH

OHH

OHH

HC O

H C

C

C

CH 2O P O 32

OH

OHH

OHH

C H

H

HC

C

CH 2O P O 32

O

OHH

C

CH 2O H

O

HO

+ +

x y lu lo se - r ib o se - g ly c e ra ld e h y d e - se d o h e p tu lo se - 5 -p h o sp h a te 5 -p h o sp h a te 3 -p h o sp h a te 7 -p h o sp h a te

T ran sk e to la se

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

C H 2 O H

C

C H

HC

HC

HC

H 2 C

O H

O H

O PO 32

O H

H O

O

HC

HC

HC

H 2 C

O

O H

O PO 32

O H

HC

HC

H 2 C

O

O PO 32

O H

H 2 C

C

C H

HC

HC

H 2 C

O H

O PO 32

O H

O H

H O

O

se d o h e p tu lo se - g ly c e ra ld e h y d e - e ry th ro se - f ru c to se - 7 -p h o sp h a te 3 -p h o sp h a te 4 -p h o sp h a te 6 -p h o sp h a te

T ran sa ld o la se

+ +

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-6-phosphate, via enzymes of Gluconeogenesis.

Regulation of pentose phosphate pathway

G 6PD , rate limiting step.controlled by the cytoplasmic conc- of NADP/NADPHNADPH 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.

Thyroid hormone ↑ activity of G6PD

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.

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

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

Pentose Phosphate Pathway producing maximum NADPH

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

Uses of NADPH

deNOVO F.A synt-.Cholesterol synt-.Steroids synt-.Sphingolipid synt-.Conversion of phenylalanine to tyrosineAs a coenzyme for meth-hb reductase.In uronic acid pathwayConversion of oxi-glutathion to red- glutathion.

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)

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 of membrane lipids, making membranes leaky.

H3N+HC CH2 CH2

COO

C

O

NH

CH

CH2

SH

C

O

NH

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 + H2OGlutathione Peroxidase uses the trace element selenium as functional group.

H3N+HC CH2 CH2

COO

C

O

NH

CH

CH2

SH

C

O

NH

CH2 COO

-glutamyl-cysteinyl-glycine Glutathione

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 (GS-SG) requires the NADPH produced in the glucose 6-phosphate dehydrogenase reaction.

Glucose-6-phosphate dehydrogenase deficiency causes hemolytic anemia

Generally sex linked, homozygous female.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,acetyl salicylic acid)

Favism and wernicke-KorsakoffIn G-6-PD deficient. On ingestion of fava beans(cooked or lightly cooked)or inhalation of pollens

from plant. Acute hemolytic anemia of sudden onset.

A genetically variant form of transketolase.Cannot bind TPP so affecting transketolase reaction.

also seen in ch- thiamine deficiency(alcoholism) characterized by lesions and hemorrhages near 3rd ventricles, impaired mental function, depression, loss of memory, weakness, paralysis of eye movement, abnormal gait.

Diagnosis= measurement of transketolase in RBCsЯ =

Similarities & differences ё EMSITEOrgansCyclesSubstrateProductsATP, CO2,NAD,NADH,NADPH generationRIBOSE CAN B SYNTHESIZED IN ALL TISSUES No or little ribose in circulation, must synthesis for nuc- .In muscles low activity of g-6PD & 6-phos-gluco dehydrog-.

Capable of synth- by reversal way (non-oxidative) utilizing fructose 6-phosphate.